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Sample records for prostaglandin synthase inhibitor

  1. High-quality crystals of human haematopoietic prostaglandin D synthase with novel inhibitors.

    PubMed

    Takahashi, Sachiko; Tsurumura, Toshiharu; Aritake, Kosuke; Furubayashi, Naoki; Sato, Masaru; Yamanaka, Mari; Hirota, Erika; Sano, Satoshi; Kobayashi, Tomoyuki; Tanaka, Tetsuo; Inaka, Koji; Tanaka, Hiroaki; Urade, Yoshihiro

    2010-07-01

    Human haematopoietic prostaglandin D synthase (H-PGDS; EC 5.3.99.2) produces prostaglandin D(2), an allergic and inflammatory mediator, in mast cells and Th2 cells. H-PGDS has been crystallized with novel inhibitors with half-maximal inhibitory concentrations (IC(50)) in the low nanomolar range by the counter-diffusion method onboard the Russian Service Module on the International Space Station. The X-ray diffraction of a microgravity-grown crystal of H-PGDS complexed with an inhibitor with an IC(50) value of 50 nM extended to 1.1 A resolution at 100 K using SPring-8 synchrotron radiation, which is one of the highest resolutions obtained to date for this protein.

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

    PubMed

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

    2011-09-01

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

  3. Discovery and Characterization of 2-Acylaminoimidazole Microsomal Prostaglandin E Synthase-1 Inhibitors.

    PubMed

    Schiffler, Matthew A; Antonysamy, Stephen; Bhattachar, Shobha N; Campanale, Kristina M; Chandrasekhar, Srinivasan; Condon, Bradley; Desai, Prashant V; Fisher, Matthew J; Groshong, Christopher; Harvey, Anita; Hickey, Michael J; Hughes, Norman E; Jones, Scott A; Kim, Euibong J; Kuklish, Steven L; Luz, John G; Norman, Bryan H; Rathmell, Richard E; Rizzo, John R; Seng, Thomas W; Thibodeaux, Stefan J; Woods, Timothy A; York, Jeremy S; Yu, Xiao-Peng

    2016-01-14

    As part of a program aimed at the discovery of antinociceptive therapy for inflammatory conditions, a screening hit was found to inhibit microsomal prostaglandin E synthase-1 (mPGES-1) with an IC50 of 17.4 μM. Structural information was used to improve enzyme potency by over 1000-fold. Addition of an appropriate substituent alleviated time-dependent cytochrome P450 3A4 (CYP3A4) inhibition. Further structure-activity relationship (SAR) studies led to 8, which had desirable potency (IC50 = 12 nM in an ex vivo human whole blood (HWB) assay) and absorption, distribution, metabolism, and excretion (ADME) properties. Studies on the formulation of 8 identified 8·H3PO4 as suitable for clinical development. Omission of a lipophilic portion of the compound led to 26, a readily orally bioavailable inhibitor with potency in HWB comparable to celecoxib. Furthermore, 26 was selective for mPGES-1 inhibition versus other mechanisms in the prostanoid pathway. These factors led to the selection of 26 as a second clinical candidate. PMID:26653180

  4. Molecular docking and competitive binding study discovered different binding modes of microsomal prostaglandin E synthase-1 inhibitors.

    PubMed

    He, Shan; Lai, Luhua

    2011-12-27

    Microsomal prostaglandin E synthase-1 (mPGES-1) is a newly recognized therapeutic target for the treatment of inflammation, pain, cancer, atherosclerosis, and stroke. Many mPGES-1 inhibitors have been discovered. However, as the structure of the binding site is not well-characterized, none of these inhibitors was designed based on the mPGES-1 structure, and their inhibition mechanism remains to be fully disclosed. Recently, we built a new structural model of mPGES-1 which was well supported by experimental data. Based on this model, molecular docking and competition experiments were used to investigate the binding modes of four representive mPGES-1 inhibitors. As the inhibitor binding sites predicted by docking overlapped with both the substrate and the cofactor binding sites, mPGES-1 inhibitors might act as dual-site inhibitors. This inhibitory mechanism was further verified by inhibitor-cofactor and inhibitor-substrate competition experiments. To investigate the potency-binding site relationships of mPGES-1 inhibitors, we also carried out molecular docking studies for another series of compounds. The docking results correlated well with the different inhibitory effects observed experimentally. Our data revealed that mPGES-1 inhibitors could bind to the substrate and the cofactor binding sites simultaneously, and this dual-site binding mode improved their potency. Future rational design and optimization of mPGES-1 inhibitors can be carried out based on this binding mechanism.

  5. Pharmacophore Modeling and Virtual Screening for Novel Acidic Inhibitors of Microsomal Prostaglandin E2 Synthase-1 (mPGES-1)

    PubMed Central

    2011-01-01

    Microsomal prostaglandin E2 synthase-1 (mPGES-1) catalyzes prostaglandin E2 formation and is considered as a potential anti-inflammatory pharmacological target. To identify novel chemical scaffolds active on this enzyme, two pharmacophore models for acidic mPGES-1 inhibitors were developed and theoretically validated using information on mPGES-1 inhibitors from literature. The models were used to screen chemical databases supplied from the National Cancer Institute (NCI) and the Specs. Out of 29 compounds selected for biological evaluation, nine chemically diverse compounds caused concentration-dependent inhibition of mPGES-1 activity in a cell-free assay with IC50 values between 0.4 and 7.9 μM, respectively. Further pharmacological characterization revealed that also 5-lipoxygenase (5-LO) was inhibited by most of these active compounds in cell-free and cell-based assays with IC50 values in the low micromolar range. Together, nine novel chemical scaffolds inhibiting mPGES-1 are presented that may possess anti-inflammatory properties based on the interference with eicosanoid biosynthesis. PMID:21466167

  6. Characterization of 3,3-dimethyl substituted N-aryl piperidines as potent microsomal prostaglandin E synthase-1 inhibitors.

    PubMed

    Kuklish, Steven L; Antonysamy, Stephen; Bhattachar, Shobha N; Chandrasekhar, Srinivasan; Fisher, Matthew J; Fretland, Adrian J; Gooding, Karen; Harvey, Anita; Hughes, Norman E; Luz, John G; Manninen, Peter R; McGee, James E; Navarro, Antonio; Norman, Bryan H; Partridge, Katherine M; Quimby, Steven J; Schiffler, Matthew A; Sloan, Ashley V; Warshawsky, Alan M; York, Jeremy S; Yu, Xiao-Peng

    2016-10-01

    Here we report on novel, potent 3,3-dimethyl substituted N-aryl piperidine inhibitors of microsomal prostaglandin E synthases-1(mPGES-1). Example 14 potently inhibited PGE2 synthesis in an ex vivo human whole blood (HWB) assay with an IC50 of 7nM. In addition, 14 had no activity in human COX-1 or COX-2 assays at 30μM, and failed to inhibit human mPGES-2 at 62.5μM in a microsomal prep assay. These data are consistent with selective mPGES-1-mediated reduction of PGE2. In dog, 14 had oral bioavailability (74%), clearance (3.62mL/(min*kg)) and volume of distribution (Vd,ss=1.6L/kg) values within our target ranges. For these reasons, 14 was selected for further study. PMID:27554445

  7. Inhibition of rabbit erythroid 15-lipoxygenase and sheep vesicular gland prostaglandin H synthase by gallic esters.

    PubMed

    Luther, H; Jordanov, D; Ludwig, P; Schewe, T

    1991-02-01

    Gallic acid esters possessing a varying chain length of their alcohol moiety were tested for their inhibitory potencies on 15-lipoxygenase from rabbit reticulocytes and prostaglandin H synthase from sheep vesicular glands. Octyl gallate and decyl gallate proved to be the most powerful inhibitors of both enzymes showing concentrations of half-inhibition of about 0.25 mumol/l for the reticulocyte lipoxygenase and of about 25 mumol/l for the prostaglandin H synthase.

  8. Improvement in the quality of hematopoietic prostaglandin D synthase crystals in a microgravity environment.

    PubMed

    Tanaka, Hiroaki; Tsurumura, Toshiharu; Aritake, Kosuke; Furubayashi, Naoki; Takahashi, Sachiko; Yamanaka, Mari; Hirota, Erika; Sano, Satoshi; Sato, Masaru; Kobayashi, Tomoyuki; Tanaka, Tetsuo; Inaka, Koji; Urade, Yoshihiro

    2011-01-01

    Human hematopoietic prostaglandin synthase, one of the better therapeutic target enzymes for allergy and inflammation, was crystallized with 22 inhibitors and in three inhibitor-free conditions in microgravity. Most of the space-grown crystals showed better X-ray diffraction patterns than the terrestrially grown ones, indicating the advantage of a microgravity environment on protein crystallization, especially in the case of this protein. PMID:21169700

  9. UVB light upregulates prostaglandin synthases and prostaglandin receptors in mouse keratinocytes

    SciTech Connect

    Black, Adrienne T.; Gray, Joshua P.; Shakarjian, Michael P.; Mishin, Vladimir; Laskin, Debra L.; Heck, Diane E.; Laskin, Jeffrey D.

    2008-10-01

    Prostaglandins belong to a class of cyclic lipid-derived mediators synthesized from arachidonic acid via COX-1, COX-2 and various prostaglandin synthases. Members of this family include prostaglandins such as PGE{sub 2}, PGF{sub 2{alpha}}, PGD{sub 2} and PGI{sub 2} (prostacyclin) as well as thromboxane. In the present studies we analyzed the effects of UVB on prostaglandin production and prostaglandin synthase expression in primary cultures of undifferentiated and calcium-differentiated mouse keratinocytes. Both cell types were found to constitutively synthesize PGE{sub 2}, PGD{sub 2} and the PGD{sub 2} metabolite PGJ{sub 2}. Twenty-four hours after treatment with UVB (25 mJ/cm{sup 2}), production of PGE{sub 2} and PGJ{sub 2} increased, while PGD{sub 2} production decreased. This was associated with increased expression of COX-2 mRNA and protein. UVB (2.5-25 mJ/cm{sup 2}) also caused marked increases in mRNA expression for the prostanoid synthases PGDS, mPGES-1, mPGES-2, PGFS and PGIS, as well as expression of receptors for PGE{sub 2} (EP1 and EP2), PGD{sub 2} (DP and CRTH2) and prostacyclin (IP). UVB was more effective in inducing COX-2 and DP in differentiated cells and EP1 and IP in undifferentiated cells. UVB readily activated keratinocyte PI-3-kinase (PI3K)/Akt, JNK and p38 MAP signaling pathways which are known to regulate COX-2 expression. While inhibition of PI3K suppressed UVB-induced mPGES-1 and CRTH2 expression, JNK inhibition suppressed mPGES-1, PGIS, EP2 and CRTH2, and p38 kinase inhibition only suppressed EP1 and EP2. These data indicate that UVB modulates expression of prostaglandin synthases and receptors by distinct mechanisms. Moreover, both the capacity of keratinocytes to generate prostaglandins and their ability to respond to these lipid mediators are stimulated by exposure to UVB.

  10. Biochemistry of prostaglandin endoperoxide H synthase-1 and synthase-2 and their differential susceptibility to nonsteroidal anti-inflammatory drugs.

    PubMed

    Smith, W L; DeWitt, D L

    1995-05-01

    The principal pharmacological effects of nonsteroidal anti-inflammatory drugs (NSAIDs) are due to their ability to inhibit prostaglandin synthesis. NSAIDs block the cyclooxygenase activities of the closely related PGH synthase-1 and PGH synthase-2 (PGHS-1 and PGHS-2) isozymes. NSAIDs are therapeutically useful due to their analgesic, anti-pyretic, anti-inflammatory, and anti-thrombogenic properties. Major side-effects of NSAIDs include their ulcerogenic and nephrotoxic activities. All clinically approved NSAIDs in general use today inhibit both PGHS-1 and PGHS-2. Recently, inhibitors have been identified that are selective toward PGHS-2 and that have potent analgesic and anti-inflammatory activities with minimal ulcerogenic activity. If the new PGHS-2 selective NSAIDs can effectively inhibit inflammatory prostaglandin synthesis by PGHS-2, without inhibiting PGHS-1 prostaglandin synthesis required to regulate sodium and water resorption, and renal blood flow, it is likely that these new drugs will also have significantly less renal toxicity than present-day NSAIDs. In this article, the mechanisms of actions of NSAIDs primarily at the biochemical level, including the reactions catalyzed by PGHSs, will be discussed. In addition, the biochemical properties of these isozymes, and the differential regulation of the PGHS-1 and PGHS-2 genes, will be examined. PMID:7631045

  11. Mechanical stimulation of skeletal muscle cells mitigates glucocorticoid-induced decreases in prostaglandin production and prostaglandin synthase activity

    NASA Technical Reports Server (NTRS)

    Chromiak, J. A.; Vandenburgh, H. H.

    1994-01-01

    The glucocorticoid dexamethasone (Dex) induces a decline in protein synthesis and protein content in tissue cultured, avian skeletal muscle cells, and this atrophy is attenuated by repetitive mechanical stretch. Since the prostaglandin synthesis inhibitor indomethacin mitigated this stretch attenuation of muscle atrophy, the effects of Dex and mechanical stretch on prostaglandin production and prostaglandin H synthase (PGHS) activity were examined. In static cultures, 10(-8) M Dex reduced PGF2 alpha production 55-65% and PGE2 production 84-90% after 24-72 h of incubation. Repetitive 10% stretch-relaxations of non-Dex-treated cultures increased PGF2 alpha efflux 41% at 24 h and 276% at 72 h, and increased PGE2 production 51% at 24 h and 236% at 72 h. Mechanical stimulation of Dex-treated cultures increased PGF2 alpha production 162% after 24 h, returning PGF2 alpha efflux to the level of non-Dex-treated cultures. At 72 h, stretch increased PGF2 alpha efflux 65% in Dex-treated cultures. Mechanical stimulation of Dex-treated cultures also increased PGE2 production at 24 h, but not at 72 h. Dex reduced PGHS activity in the muscle cultures by 70% after 8-24 h of incubation, and mechanical stimulation of the Dex-treated cultures increased PGHS activity by 98% after 24 h. Repetitive mechanical stimulation attenuates the catabolic effects of Dex on cultured skeletal muscle cells in part by mitigating the Dex-induced declines in PGHS activity and prostaglandin production.

  12. Development of a scintillation proximity binding assay for high-throughput screening of hematopoietic prostaglandin D2 synthase.

    PubMed

    Meleza, Cesar; Thomasson, Bobbie; Ramachandran, Chidambaram; O'Neill, Jason W; Michelsen, Klaus; Lo, Mei-Chu

    2016-10-15

    Prostaglandin D2 synthase (PGDS) catalyzes the isomerization of prostaglandin H2 (PGH2) to prostaglandin D2 (PGD2). PGD2 produced by hematopoietic prostaglandin D2 synthase (H-PGDS) in mast cells and Th2 cells is proposed to be a mediator of allergic and inflammatory responses. Consequently, inhibitors of H-PGDS represent potential therapeutic agents for the treatment of inflammatory diseases such as asthma. Due to the instability of the PGDS substrate PGH2, an in-vitro enzymatic assay is not feasible for large-scale screening of H-PGDS inhibitors. Herein, we report the development of a competition binding assay amenable to high-throughput screening (HTS) in a scintillation proximity assay (SPA) format. This assay was used to screen an in-house compound library of approximately 280,000 compounds for novel H-PGDS inhibitors. The hit rate of the H-PGDS primary screen was found to be 4%. This high hit rate suggests that the active site of H-PGDS can accommodate a large diversity of chemical scaffolds. For hit prioritization, these initial hits were rescreened at a lower concentration in SPA and tested in the LAD2 cell assay. 116 compounds were active in both assays with IC50s ranging from 6 to 807 nM in SPA and 82 nM to 10 μM in the LAD2 cell assay. PMID:27485270

  13. Sulforaphane Inhibits Prostaglandin E2 Synthesis by Suppressing Microsomal Prostaglandin E Synthase 1

    PubMed Central

    Zhou, Jiping; Joplin, Denise G.; Cross, Janet V.; Templeton, Dennis J.

    2012-01-01

    Sulforaphane (SFN) is a dietary cancer preventive with incompletely characterized mechanism(s) of cancer prevention. Since prostaglandin E2 (PGE2) promotes cancer progression, we hypothesized that SFN may block PGE2 synthesis in cancer cells. We found that SFN indeed blocked PGE2 production in human A549 cancer cells not by inhibiting COX-2, but rather by suppressing the expression of microsomal prostaglandin E synthase (mPGES-1), the enzyme that directly synthesizes PGE2. We identified the Hypoxia Inducible Factor 1 alpha (HIF-1α) as the target of SFN-mediated mPGES-1 suppression. SFN suppressed HIF-1α protein expression and the presence of HIF-1α at the mPGES-1 promoter, resulting in reduced transcription of mPGES-1. Finally, SFN also reduced expression of mPGES-1 and PGE2 production in A549 xenograft tumors in mice. Together, these results point to the HIF-1α, mPGES-1 and PGE2 axis as a potential mediator of the anti-cancer effects of SFN, and illustrate the potential of SFN for therapeutic control of cancer and inflammation. Harmful side effects in patients taking agents that target the more upstream COX-2 enzyme render the downstream target mPGES-1 a significant target for anti-inflammatory therapy. Thus, SFN could prove to be an important therapeutic approach to both cancer and inflammation. PMID:23166763

  14. Prostaglandin endoperoxide H synthases: peroxidase hydroperoxide specificity and cyclooxygenase activation.

    PubMed

    Liu, Jiayan; Seibold, Steve A; Rieke, Caroline J; Song, Inseok; Cukier, Robert I; Smith, William L

    2007-06-22

    The cyclooxygenase (COX) activity of prostaglandin endoperoxide H synthases (PGHSs) converts arachidonic acid and O2 to prostaglandin G2 (PGG2). PGHS peroxidase (POX) activity reduces PGG2 to PGH2. The first step in POX catalysis is formation of an oxyferryl heme radical cation (Compound I), which undergoes intramolecular electron transfer forming Intermediate II having an oxyferryl heme and a Tyr-385 radical required for COX catalysis. PGHS POX catalyzes heterolytic cleavage of primary and secondary hydroperoxides much more readily than H2O2, but the basis for this specificity has been unresolved. Several large amino acids form a hydrophobic "dome" over part of the heme, but when these residues were mutated to alanines there was little effect on Compound I formation from H2O2 or 15-hydroperoxyeicosatetraenoic acid, a surrogate substrate for PGG2. Ab initio calculations of heterolytic bond dissociation energies of the peroxyl groups of small peroxides indicated that they are almost the same. Molecular Dynamics simulations suggest that PGG2 binds the POX site through a peroxyl-iron bond, a hydrogen bond with His-207 and van der Waals interactions involving methylene groups adjoining the carbon bearing the peroxyl group and the protoporphyrin IX. We speculate that these latter interactions, which are not possible with H2O2, are major contributors to PGHS POX specificity. The distal Gln-203 four residues removed from His-207 have been thought to be essential for Compound I formation. However, Q203V PGHS-1 and PGHS-2 mutants catalyzed heterolytic cleavage of peroxides and exhibited native COX activity. PGHSs are homodimers with each monomer having a POX site and COX site. Cross-talk occurs between the COX sites of adjoining monomers. However, no cross-talk between the POX and COX sites of monomers was detected in a PGHS-2 heterodimer comprised of a Q203R monomer having an inactive POX site and a G533A monomer with an inactive COX site.

  15. Sequential induction of prostaglandin E and D synthases in inflammation

    SciTech Connect

    Schuligoi, Rufina . E-mail: rufina.schuligoi@meduni-graz.at; Grill, Magdalena; Heinemann, Akos; Peskar, Bernhard A.; Amann, Rainer

    2005-09-30

    Enhanced biosynthesis of prostaglandin (PG)D{sub 2} and subsequent formation of 15-deoxy-{delta}{sup 12,14}-PGJ{sub 2} has been suggested to contribute to resolution of inflammation. The primary aim of the present study in mouse heart was, therefore, to determine at the transcriptional level if there is sequential induction of PGE and PGD synthases (S) during inflammation. Expression of interleukin (IL)-1{beta} in heart was enhanced 4 h after systemic inflammation and declined thereafter within 3-5 days to basal levels. In contrast to cyclooxygenase-2 and membrane-bound (m)-PGES-1, which both peaked 4 h after endotoxin administration, hematopoietic (H)-PGDS expression was enhanced only 48 h after endotoxin. The expression of lipocalin-type (L)-PGDS was not significantly influenced. mRNA encoding the putative target of 15-deoxy-{delta}{sup 12,14}-PGJ{sub 2}, peroxisome proliferator-activated receptor {gamma}, was enhanced between 4 and 24 h after induction of inflammation. Treatment of mice with acetylsalicylic acid or indomethacin at doses effective to cause near-complete inhibition of PGE{sub 2} and PGD{sub 2} biosynthesis in heart ex vivo resulted in enhanced expression of IL-1{beta} 24 h after endotoxin administration. These results provide additional support for the hypothesis of a shift towards PGD{sub 2} biosynthesis during resolution of inflammation.

  16. Perspective of microsomal prostaglandin E2 synthase-1 as drug target in inflammation-related disorders.

    PubMed

    Koeberle, Andreas; Werz, Oliver

    2015-11-01

    Prostaglandin (PG)E2 encompasses crucial roles in pain, fever, inflammation and diseases with inflammatory component, such as cancer, but is also essential for gastric, renal, cardiovascular and immune homeostasis. Cyclooxygenases (COX) convert arachidonic acid to the intermediate PGH2 which is isomerized to PGE2 by at least three different PGE2 synthases. Inhibitors of COX - non-steroidal anti-inflammatory drugs (NSAIDs) - are currently the only available therapeutics that target PGE2 biosynthesis. Due to adverse effects of COX inhibitors on the cardiovascular system (COX-2-selective), stomach and kidney (COX-1/2-unselective), novel pharmacological strategies are in demand. The inducible microsomal PGE2 synthase (mPGES)-1 is considered mainly responsible for the excessive PGE2 synthesis during inflammation and was suggested as promising drug target for suppressing PGE2 biosynthesis. However, 15 years after intensive research on the biology and pharmacology of mPGES-1, the therapeutic value of mPGES-1 as drug target is still vague and mPGES-1 inhibitors did not enter the market so far. This commentary will first shed light on the structure, mechanism and regulation of mPGES-1 and will then discuss its biological function and the consequence of its inhibition for the dynamic network of eicosanoids. Moreover, we (i) present current strategies for interfering with mPGES-1-mediated PGE2 synthesis, (ii) summarize bioanalytical approaches for mPGES-1 drug discovery and (iii) describe preclinical test systems for the characterization of mPGES-1 inhibitors. The pharmacological potential of selective mPGES-1 inhibitor classes as well as dual mPGES-1/5-lipoxygenase inhibitors is reviewed and pitfalls in their development, including species discrepancies and loss of in vivo activity, are discussed.

  17. Metabolism of aromatic amines by prostaglandin H synthase.

    PubMed Central

    Boyd, J A; Eling, T E

    1985-01-01

    The metabolism of aromatic amines by the peroxidase activity of prostaglandin H synthase (PHS) has been studied in this laboratory by use of two model compounds, the carcinogenic primary amine 2-aminofluorene (2-AF) and the substituted amine aminopyrine (AP). 2-AF is oxidized by PHS to 2, 2-azobisfluorene, 2-aminodifluorenylamine, 2-nitrofluorene, polymeric material, and products covalently bound to macromolecules. In the presence of phenolic compounds, 2-AF oxidation results in the formation of amine/phenol adducts. The data are consistent with a one-electron mechanism of 2-AF oxidation by PHS; furthermore, an N-hydroxy intermediate is not involved in 2-AF metabolism by PHS. PHS also catalyzes the binding of 2-AF to DNA in vitro. Unique 2-AF/DNA adducts were isolated and are distinct from the N-(deoxyguanosin-8-yl)-2-AF adduct formed from the reaction of N-hydroxy-2-AF with DNA. These new adducts represent a marker unique to peroxidative activation of 2-AF. AP is oxidized by the peroxidase activity of PHS to the cation radical, with one molecule of hydroperoxy fatty acid reduced for every two molecules of AP free radical formed. The decay of the AP radical follows second order kinetics, supporting the proposed mechanism in which the AP radical disproportionates to an iminium cation, followed by hydrolysis of this species to the demethylated amine and formaldehyde. In the presence of glutathione, the cation radical is reduced to the parent amine, resulting in the formation of the glutathione thiyl radical. It thus appears that both primary and substituted aromatic amines may undergo one-electron oxidation by PHS. PMID:3938394

  18. Metabolism of aromatic amines by prostaglandin H synthase

    SciTech Connect

    Boyd, J.A.; Eling, T.E.

    1985-12-01

    The metabolism of aromatic amines by the peroxidase activity of prostaglandin H synthase (PHS) has been studied in this laboratory by use of two model compounds, the carcinogenic primary amine 2-aminofluorene (2-AF) and the substituted amine aminopyrine (AP). 2-AF is oxidized by PHS to 2, 2-azobisfluorene, 2-aminodifluorenylamine, 2-nitrofluorene, polymeric material, and products covalently bound to macromolecules. In the presence of phenolic compounds, 2-AF oxidation results in the formation of amine-phenol adducts. The data are consistent with a one-electron mechanism of 2-AF oxidation by PHS; furthermore, an N-hydroxy intermediate is not involved in 2-AF metabolism by PHS. PHS also catalyzes the binding of 2-AF to DNA in vitro. Unique 2-AF/DNA adducts were isolated and are distinct from the N-(deoxyguanosin-8-yl)-2-AF adduct formed from the reaction of N-hydroxy-2-AF with DNA. These new adducts represent a marker unique to peroxidative activation of 2-AF. AP is oxidized by the peroxidase activity of PHS to the cation radical, with one molecule of hydroperoxy fatty acid reduced for every two molecules of AP free radical formed. The decay of the AP radical follows second order kinetics, supporting the proposed mechanism in which the AP radical disproportionates to an iminium cation, followed by hydrolysis of this species to the demethylated amine and formaldehyde. In the presence of glutathione, the cation radical is reduced to the parent amine, resulting in the formation of the glutathione thiyl radical. It thus appears that both primary and substituted aromatic amines may undergo one-electron oxidation by PHS. 19 references.

  19. A dynamic Asp–Arg interaction is essential for catalysis in microsomal prostaglandin E2 synthase

    PubMed Central

    Brock, Joseph S.; Hamberg, Mats; Balagunaseelan, Navisraj; Goodman, Michael; Morgenstern, Ralf; Strandback, Emilia; Samuelsson, Bengt; Rinaldo-Matthis, Agnes; Haeggström, Jesper Z.

    2016-01-01

    Microsomal prostaglandin E2 synthase type 1 (mPGES-1) is responsible for the formation of the potent lipid mediator prostaglandin E2 under proinflammatory conditions, and this enzyme has received considerable attention as a drug target. Recently, a high-resolution crystal structure of human mPGES-1 was presented, with Ser-127 being proposed as the hydrogen-bond donor stabilizing thiolate anion formation within the cofactor, glutathione (GSH). We have combined site-directed mutagenesis and activity assays with a structural dynamics analysis to probe the functional roles of such putative catalytic residues. We found that Ser-127 is not required for activity, whereas an interaction between Arg-126 and Asp-49 is essential for catalysis. We postulate that both residues, in addition to a crystallographic water, serve critical roles within the enzymatic mechanism. After characterizing the size or charge conservative mutations Arg-126–Gln, Asp-49–Asn, and Arg-126–Lys, we inferred that a crystallographic water acts as a general base during GSH thiolate formation, stabilized by interaction with Arg-126, which is itself modulated by its respective interaction with Asp-49. We subsequently found hidden conformational ensembles within the crystal structure that correlate well with our biochemical data. The resulting contact signaling network connects Asp-49 to distal residues involved in GSH binding and is ligand dependent. Our work has broad implications for development of efficient mPGES-1 inhibitors, potential anti-inflammatory and anticancer agents. PMID:26755582

  20. A dynamic Asp-Arg interaction is essential for catalysis in microsomal prostaglandin E2 synthase.

    PubMed

    Brock, Joseph S; Hamberg, Mats; Balagunaseelan, Navisraj; Goodman, Michael; Morgenstern, Ralf; Strandback, Emilia; Samuelsson, Bengt; Rinaldo-Matthis, Agnes; Haeggström, Jesper Z

    2016-01-26

    Microsomal prostaglandin E2 synthase type 1 (mPGES-1) is responsible for the formation of the potent lipid mediator prostaglandin E2 under proinflammatory conditions, and this enzyme has received considerable attention as a drug target. Recently, a high-resolution crystal structure of human mPGES-1 was presented, with Ser-127 being proposed as the hydrogen-bond donor stabilizing thiolate anion formation within the cofactor, glutathione (GSH). We have combined site-directed mutagenesis and activity assays with a structural dynamics analysis to probe the functional roles of such putative catalytic residues. We found that Ser-127 is not required for activity, whereas an interaction between Arg-126 and Asp-49 is essential for catalysis. We postulate that both residues, in addition to a crystallographic water, serve critical roles within the enzymatic mechanism. After characterizing the size or charge conservative mutations Arg-126-Gln, Asp-49-Asn, and Arg-126-Lys, we inferred that a crystallographic water acts as a general base during GSH thiolate formation, stabilized by interaction with Arg-126, which is itself modulated by its respective interaction with Asp-49. We subsequently found hidden conformational ensembles within the crystal structure that correlate well with our biochemical data. The resulting contact signaling network connects Asp-49 to distal residues involved in GSH binding and is ligand dependent. Our work has broad implications for development of efficient mPGES-1 inhibitors, potential anti-inflammatory and anticancer agents.

  1. Novel membrane-associated prostaglandin E synthase-2 from crustacean arthropods.

    PubMed

    Hansen, Kristella; Varvas, Külliki; Järving, Ivar; Samel, Nigulas

    2014-08-01

    Prostaglandins (PG) have been shown to play important physiological roles in insects and marine invertebrates, yet the knowledge of their biosynthetic pathways is often lacking. Recently, we described cyclooxygenases in two amphipod crustaceans, Gammarus sp. and Caprella sp. In the present study, we report the cloning and characterization of prostaglandin E synthases (PGES) from the same organisms. The amphipod membrane-bound PGES-2-type enzymes share about 40% of the amino acid sequence identity with human mPGES-2, contain a conserved Cys110-x-x-Cys113 motif and have very low heme-binding affinity. The recombinant enzymes purified in the absence of dithiothreitol specifically catalyze the isomerization of PGH2 into PGE2. The PGES activity is increased in the presence of reduced glutathione and inhibited with a sulfhydryl group inhibitor. We assume that the amphipod mPGES-2, unlike in their mammalian counterparts, is responsible for PGE2 synthesis, not only in vitro but also in vivo.

  2. Bromoenol Lactone, an Inhibitor of Calcium-Independent Phospholipase A2, Suppresses Carrageenan-Induced Prostaglandin Production and Hyperalgesia in Rat Hind Paw

    PubMed Central

    Tsuchida, Keiichiro; Ibuki, Takae; Matsumura, Kiyoshi

    2015-01-01

    Prostaglandin (PG) E2 and PGI2 are essential to hyperalgesia in inflammatory tissues. These prostaglandins are produced from arachidonic acid, which is cleaved from membrane phospholipids by the action of phospholipase A2 (PLA2). Which isozyme of PLA2 is responsible for the cleavage of arachidonic acid and the production of prostaglandins essential to inflammation-induced hyperalgesia is not clear. In this study, we examined the effects of two PLA2 isozyme-specific inhibitors on carrageenan-induced production of PGE2 and PGI2 in rat hind paw and behavioral nociceptive response to radiant heat. Local administration of bromoenol lactone (BEL), an inhibitor of calcium-independent PLA2 (iPLA2), significantly reduced carrageenan-induced elevation of prostaglandins in the inflamed foot pad 3 h after injection. It also ameliorated the hyperalgesic response between 1 h and 3 h after carrageenan injection. On the other hand, AACOCF3, an inhibitor of cytosolic PLA2, suppressed neither prostaglandin production nor the hyperalgesic response. BEL did not suppress the mRNA levels of iPLA2β, iPLA2γ, cyclooxygenase-2, microsomal prostaglandin E synthase, prostaglandin I synthase, or proinflammatory cytokines in the inflamed foot pad, indicating that BEL did not suppress inflammation itself. These results suggest that iPLA2 is involved in the production of prostaglandins and hyperalgesia at the inflammatory loci. PMID:26063975

  3. Inhibition of microsomal prostaglandin E synthase-1 by aminothiazoles decreases prostaglandin E2 synthesis in vitro and ameliorates experimental periodontitis in vivo

    PubMed Central

    Kats, Anna; Båge, Tove; Georgsson, Pierre; Jönsson, Jörgen; Quezada, Hernán Concha; Gustafsson, Anders; Jansson, Leif; Lindberg, Claes; Näsström, Karin; Yucel-Lindberg, Tülay

    2013-01-01

    The potent inflammatory mediator prostaglandin E2 (PGE2) is implicated in the pathogenesis of several chronic inflammatory conditions, including periodontitis. The inducible enzyme microsomal prostaglandin E synthase-1 (mPGES-1), catalyzing the terminal step of PGE2 biosynthesis, is an attractive target for selective PGE2 inhibition. To identify mPGES-1 inhibitors, we investigated the effect of aminothiazoles on inflammation-induced PGE2 synthesis in vitro, using human gingival fibroblasts stimulated with the cytokine IL-1β and a cell-free mPGES-1 activity assay, as well as on inflammation-induced bone resorption in vivo, using ligature-induced experimental periodontitis in Sprague-Dawley rats. Aminothiazoles 4-([4-(2-naphthyl)-1,3-thiazol-2-yl]amino)phenol (TH-848) and 4-(3-fluoro-4-methoxyphenyl)-N-(4-phenoxyphenyl)-1,3-thiazol-2-amine (TH-644) reduced IL-1β-induced PGE2 production in fibroblasts (IC50 1.1 and 1.5 μM, respectively) as well as recombinant mPGES-1 activity, without affecting activity or expression of the upstream enzyme cyclooxygenase-2. In ligature-induced experimental periodontitis, alveolar bone loss, assessed by X-ray imaging, was reduced by 46% by local treatment with TH-848, compared to vehicle, without any systemic effects on PGE2, 6-keto PGF1α, LTB4 or cytokine levels. In summary, these results demonstrate that the aminothiazoles represent novel mPGES-1 inhibitors for inhibition of PGE2 production and reduction of bone resorption in experimental periodontitis, and may be used as potential anti-inflammatory drugs for treatment of chronic inflammatory diseases, including periodontitis.—Kats, A., Båge, T., Georgsson, P., Jönsson, J., Quezada, H. C., Gustafsson, A., Jansson, L., Lindberg, C., Näsström, K., Yucel-Lindberg, T. Inhibition of microsomal prostaglandin E synthase-1 by aminothiazoles decreases prostaglandin E2 synthesis in vitro and ameliorates experimental periodontitis in vivo. PMID:23447581

  4. [Effect of prostaglandin synthesis inhibitors of diabetic cystoid macular edema].

    PubMed

    Kieselbach, G; Juen, S

    1990-01-01

    In most cases, diabetic macular edema is treated successfully with central laser photocoagulation. However, only few studies report such favorable results in cystoid macular edema, which has a poor visual prognosis. In the present prospective study on diabetics with cystoid macular edema, aged less than 40 years, a better visual outcome was obtained in patients treated with prostaglandin synthesis inhibitors than in an untreated group. PMID:2345629

  5. Curcumin blocks prostaglandin E2 biosynthesis through direct inhibition of the microsomal prostaglandin E2 synthase-1.

    PubMed

    Koeberle, Andreas; Northoff, Hinnak; Werz, Oliver

    2009-08-01

    Prostaglandin E(2) (PGE(2)) plays a crucial role in the apparent link between tumor growth and chronic inflammation. Cyclooxygenase (COX)-2 and microsomal PGE(2) synthase-1, which are overexpressed in many cancers, are functionally coupled and thus produce massive PGE(2) in various tumors. Curcumin, a polyphenolic beta-diketone from tumeric with anti-carcinogenic and anti-inflammatory activities, was shown to suppress PGE(2) formation and to block the expression of COX-2 and of microsomal PGE(2) synthase-1. Here, we identified microsomal PGE(2) synthase-1 as a molecular target of curcumin and we show that inhibition of microsomal PGE(2) synthase-1 activity is the predominant mechanism of curcumin to suppress PGE(2) biosynthesis. Curcumin reversibly inhibited the conversion of PGH(2) to PGE(2) by microsomal PGE(2) synthase-1 in microsomes of interleukin-1beta-stimulated A549 lung carcinoma cells with an IC(50) of 0.2 to 0.3 micromol/L. Closely related polyphenols (e.g., resveratrol, coniferyl alcohol, eugenol, rosmarinic acid) failed in this respect, and isolated ovine COX-1 and human recombinant COX-2 were not inhibited by curcumin up to 30 micromol/L. In lipopolysaccharide-stimulated human whole blood, curcumin inhibited COX-2-derived PGE(2) formation from endogenous or from exogenous arachidonic acid, whereas the concomitant formation of COX-2-mediated 6-keto PGF(1)alpha and COX-1-derived 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid was suppressed only at significant higher concentrations. Based on the key function of PGE(2) in inflammation and carcinogenesis, inhibition of microsomal PGE(2) synthase-1 by curcumin provides a molecular basis for its anticarcinogenic and anti-inflammatory activities.

  6. Aspirin inhibits interleukin 1-induced prostaglandin H synthase expression in cultured endothelial cells

    SciTech Connect

    Wu, K.K.; Sanduja, R.; Tsai, A.L.; Ferhanoglu, B.; Loose-Mitchell, D.S. )

    1991-03-15

    Prostaglandin H (PGH) synthase is a key enzyme in the biosynthesis of prostaglandins, thromboxane, and prostacyclin. In cultured human umbilical vein endothelial cells, interleukin 1 (IL-1) is known to induce the synthesis of this enzyme, thereby raising the level of PGH synthase protein severalfold over the basal level. Pretreatment with aspirin at low concentrations inhibited more than 60% of the enzyme mass and also the cyclooxygenase activity in IL-1-induced cells with only minimal effects on the basal level of the synthase enzyme in cells without IL-1. Sodium salicylate exhibited a similar inhibitory action whereas indomethacin had no apparent effect. Similarly low levels of aspirin inhibited the increased L-({sup 35}S)methionine incorporation into PGH synthase that was induced by IL0-1 and also suppressed expression of the 2.7-kilobase PGH synthase mRNA. These results suggest that in cultured endothelial cells a potent inhibition of eicosanoid biosynthetic capacity can be effected by aspirin or salicylate at the level of PGH synthase gene expression. The aspirin effect may well be due to degradation of salicylate.

  7. An Update of Microsomal Prostaglandin E Synthase-1 and PGE2 Receptors in Cardiovascular Health and Diseases

    PubMed Central

    2016-01-01

    Nonsteroidal anti-inflammatory drugs (NSAIDs), especially cyclooxygenase-2 (COX-2) selective inhibitors, are among the most widely used drugs to treat pain and inflammation. However, clinical trials have revealed that these inhibitors predisposed patients to a significantly increased cardiovascular risk, consisting of thrombosis, hypertension, myocardial infarction, heart failure, and sudden cardiac death. Thus, microsomal prostaglandin E (PGE) synthase-1 (mPGES-1), the key terminal enzyme involved in the synthesis of inflammatory prostaglandin E2 (PGE2), and the four PGE2 receptors (EP1–4) have gained much attention as alternative targets for the development of novel analgesics. The cardiovascular consequences of targeting mPGES-1 and the PGE2 receptors are substantially studied. Inhibition of mPGES-1 has displayed a relatively innocuous or preferable cardiovascular profile. The modulation of the four EP receptors in cardiovascular system is diversely reported as well. In this review, we highlight the most recent advances from our and other studies on the regulation of PGE2, particularly mPGES-1 and the four PGE2 receptors, in cardiovascular function, with a particular emphasis on blood pressure regulation, atherosclerosis, thrombosis, and myocardial infarction. This might lead to new avenues to improve cardiovascular disease management strategies and to seek optimized anti-inflammatory therapeutic options.

  8. An Update of Microsomal Prostaglandin E Synthase-1 and PGE2 Receptors in Cardiovascular Health and Diseases

    PubMed Central

    2016-01-01

    Nonsteroidal anti-inflammatory drugs (NSAIDs), especially cyclooxygenase-2 (COX-2) selective inhibitors, are among the most widely used drugs to treat pain and inflammation. However, clinical trials have revealed that these inhibitors predisposed patients to a significantly increased cardiovascular risk, consisting of thrombosis, hypertension, myocardial infarction, heart failure, and sudden cardiac death. Thus, microsomal prostaglandin E (PGE) synthase-1 (mPGES-1), the key terminal enzyme involved in the synthesis of inflammatory prostaglandin E2 (PGE2), and the four PGE2 receptors (EP1–4) have gained much attention as alternative targets for the development of novel analgesics. The cardiovascular consequences of targeting mPGES-1 and the PGE2 receptors are substantially studied. Inhibition of mPGES-1 has displayed a relatively innocuous or preferable cardiovascular profile. The modulation of the four EP receptors in cardiovascular system is diversely reported as well. In this review, we highlight the most recent advances from our and other studies on the regulation of PGE2, particularly mPGES-1 and the four PGE2 receptors, in cardiovascular function, with a particular emphasis on blood pressure regulation, atherosclerosis, thrombosis, and myocardial infarction. This might lead to new avenues to improve cardiovascular disease management strategies and to seek optimized anti-inflammatory therapeutic options. PMID:27594972

  9. Bacterial lipopolysaccharide induction of the prostaglandin G/H synthase 2 gene causes thromboxane-dependent pulmonary hypertension in rabbits.

    PubMed

    Delong, P; O'Sullivan, M G; Huggins, E; Hubbard, C L; McCall, C

    1999-03-01

    Two genes encode proteins with prostaglandin G/H synthase (PGHS) activity. PGHS-1 is primarily a constitutively expressed gene, whereas inflammatory agents such as bacterial lipopolysaccharide (LPS) endotoxin rapidly induce the PGHS-2 gene in leukocytes. Both PGHS-1 and PGHS-2 are rate-limiting enzymes for the production of prostaglandins and thromboxane following release of arachidonic acid by phospholipases. We previously reported that LPS perfusion into the circulation of isolated perfused rabbit lung (IPL) results in thromboxane-dependent pulmonary hypertension and lung edema when the LPS-primed lung is subsequently stimulated with platelet activating factor (PAF) (J. Clin. Invest. 1990;85:1135). In this study, we showed that the mechanism by which LPS primes IPL for enhanced production of thromboxane and pulmonary hypertension in response to PAF depends on specific upregulation of the PGHS-2 gene in the rabbit lung. LPS perfusion of IPL induced PGHS-2 gene expression, which correlated with the conversion of free arachidonic acid to thromboxane-B2 (TXB2) and the onset of pulmonary hypertension. LPS-induced PGHS-2 expression, TXB2 release, and pulmonary hypertension were inhibited by actinomycin D (an inhibitor of transcription) and cycloheximide (an inhibitor of protein synthesis). The constitutively expressed PGHS-1 remained unchanged with LPS perfusion, and did not convert free arachidonic acid to TXB2, suggesting that PGHS-1 does not contribute to the induction of pulmonary hypertension by LPS. These studies reveal a pathogenic role for induction of PGHS-2 in lung injury.

  10. An Update of Microsomal Prostaglandin E Synthase-1 and PGE2 Receptors in Cardiovascular Health and Diseases.

    PubMed

    Yang, Guangrui; Chen, Lihong

    2016-01-01

    Nonsteroidal anti-inflammatory drugs (NSAIDs), especially cyclooxygenase-2 (COX-2) selective inhibitors, are among the most widely used drugs to treat pain and inflammation. However, clinical trials have revealed that these inhibitors predisposed patients to a significantly increased cardiovascular risk, consisting of thrombosis, hypertension, myocardial infarction, heart failure, and sudden cardiac death. Thus, microsomal prostaglandin E (PGE) synthase-1 (mPGES-1), the key terminal enzyme involved in the synthesis of inflammatory prostaglandin E2 (PGE2), and the four PGE2 receptors (EP1-4) have gained much attention as alternative targets for the development of novel analgesics. The cardiovascular consequences of targeting mPGES-1 and the PGE2 receptors are substantially studied. Inhibition of mPGES-1 has displayed a relatively innocuous or preferable cardiovascular profile. The modulation of the four EP receptors in cardiovascular system is diversely reported as well. In this review, we highlight the most recent advances from our and other studies on the regulation of PGE2, particularly mPGES-1 and the four PGE2 receptors, in cardiovascular function, with a particular emphasis on blood pressure regulation, atherosclerosis, thrombosis, and myocardial infarction. This might lead to new avenues to improve cardiovascular disease management strategies and to seek optimized anti-inflammatory therapeutic options. PMID:27594972

  11. Cellular localization of ovarian prostaglandin endoperoxide synthase during pseudopregnancy in the rat.

    PubMed

    Curry, T E; Bryant, C; Clark, M R

    1991-05-01

    The specific cellular localization of prostaglandin endoperoxide (PGH) synthase, the enzyme responsible for initiating the conversion of arachidonic acid to prostaglandins, was studied throughout pseudopregnancy in the rat. Pseudopregnancy was induced by administration of eCG (20 IU) to immature, 27-day-old rats followed by hCG injection (10 IU) on Day 29. Animals were necropsied on Days 1 (Day 1 = 1 day post hCG), 5, 9, and 13 of pseudopregnancy. Ovaries were removed and processed for cellular identification of PGH synthase by immunohistochemistry. Immunoreactive PGH synthase was distributed throughout the CL at each of the 4 different days of pseudopregnancy, with the majority of the luteal cells exhibiting varying degrees of staining. The connective tissue centrum of the CL, however, lacked PGH synthase immunoreactivity. Quantitative assessment of the immunostaining distribution was accomplished with a computer-based image analysis program (Kontron IBAS). Results are expressed as the percentage of a digitized luteal area that contained intense immunoreactivity between Day 1 (0.6 +/- 0.2% immunoreactive area) and Day 5 (16.8 +/- 2.7%) of pseudopregnancy. The area of luteal immunostaining was similar on Day 5 and Day 9 (16.8 +/- 2.7% and 14.7 +/- 2.0%, respectively) followed by a decrease (p less than 0.05) in immunoreactivity on Day 13 (9.1 +/- 2.2%). The region of the CL adjacent to the germinal epithelium had an increase (p less than 0.01) in PG synthase staining distribution compared to the region of the CL adjacent to the ovarian medulla on all days of pseudopregnancy except Day 1. These findings demonstrate that PGH synthase is present in the rat CL throughout pseudopregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)

  12. Structures of prostaglandin F synthase from the protozoa Leishmania major and Trypanosoma cruzi with NADP.

    PubMed

    Moen, Spencer O; Fairman, James W; Barnes, Steve R; Sullivan, Amy; Nakazawa-Hewitt, Stephen; Van Voorhis, Wesley C; Staker, Bart L; Lorimer, Donald D; Myler, Peter J; Edwards, Thomas E

    2015-05-01

    The crystal structures of prostaglandin F synthase (PGF) from both Leishmania major and Trypanosoma cruzi with and without their cofactor NADP have been determined to resolutions of 2.6 Å for T. cruzi PGF, 1.25 Å for T. cruzi PGF with NADP, 1.6 Å for L. major PGF and 1.8 Å for L. major PGF with NADP. These structures were determined by molecular replacement to a final R factor of less than 18.6% (Rfree of less than 22.9%). PGF in the infectious protozoa L. major and T. cruzi is a potential therapeutic target.

  13. Pharmacological Inhibition of Microsomal Prostaglandin E Synthase-1 Suppresses Epidermal Growth Factor Receptor-Mediated Tumor Growth and Angiogenesis

    PubMed Central

    Bocci, Elena; Coletta, Isabella; Polenzani, Lorenzo; Mangano, Giorgina; Alisi, Maria Alessandra; Cazzolla, Nicola; Giachetti, Antonio; Ziche, Marina; Donnini, Sandra

    2012-01-01

    Background Blockade of Prostaglandin (PG) E2 production via deletion of microsomal Prostaglandin E synthase-1 (mPGES-1) gene reduces tumor cell proliferation in vitro and in vivo on xenograft tumors. So far the therapeutic potential of the pharmacological inhibition of mPGES-1 has not been elucidated. PGE2 promotes epithelial tumor progression via multiple signaling pathways including the epidermal growth factor receptor (EGFR) signaling pathway. Methodology/Principal Findings Here we evaluated the antitumor activity of AF3485, a compound of a novel family of human mPGES-1 inhibitors, in vitro and in vivo, in mice bearing human A431 xenografts overexpressing EGFR. Treatment of the human cell line A431 with interleukin-1beta (IL-1β) increased mPGES-1 expression, PGE2 production and induced EGFR phosphorylation, and vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) expression. AF3485 reduced PGE2 production, both in quiescent and in cells stimulated by IL-1β. AF3485 abolished IL-1β-induced activation of the EGFR, decreasing VEGF and FGF-2 expression, and tumor-mediated endothelial tube formation. In vivo, in A431 xenograft, AF3485, administered sub-chronically, decreased tumor growth, an effect related to inhibition of EGFR signalling, and to tumor microvessel rarefaction. In fact, we observed a decrease of EGFR phosphorylation, and VEGF and FGF-2 expression in tumours explanted from treated mice. Conclusion Our work demonstrates that the pharmacological inhibition of mPGES-1 reduces squamous carcinoma growth by suppressing PGE2 mediated-EGFR signalling and by impairing tumor associated angiogenesis. These results underscore the potential of mPGES-1 inhibitors as agents capable of controlling tumor growth. PMID:22815767

  14. Dendritic cells express hematopoietic prostaglandin D synthase and function as a source of prostaglandin D2 in the skin.

    PubMed

    Shimura, Chieko; Satoh, Takahiro; Igawa, Ken; Aritake, Kosuke; Urade, Yoshihiro; Nakamura, Masataka; Yokozeki, Hiroo

    2010-01-01

    Prostaglandin D2 (PGD2), an arachidonic acid metabolite, has been implicated in allergic responses. A major source of PGD2 in the skin is mast cells that express hematopoietic PGD synthase (H-PGDS). In this study, we show the expression of H-PGDS in human dendritic cells (DCs) and the regulatory mechanisms by which DCs produce PGD2. We detected H-PGDS in epidermal Langerhans cells, dermal DCs, plasmacytoid DCs, and myeloid DCs. Monocyte-derived DCs rapidly secreted PGD2 when stimulated with the calcium ionophore A23187. More importantly, pretreatment of monocyte-derived DCs with PMA (phorbol 12-myrisate 13-acetate) synergistically enhanced the rapid PGD2 secretion induced by A23187, whereas PMA alone did not induce PGD2 secretion. Lipopolysaccharide (LPS) reduced H-PGDS expression, but interferon-gamma followed by LPS induced significant PGD2 production in a delayed time course at 6 hours. This effect was associated with inhibition of LPS-induced H-PGDS reduction. Interestingly, an irritant compound, SDS, also induced a rapid PGD2 release. PGD2 synergistically enhanced CCL22/macrophage-derived chemokine synthesis in interferon-gamma-treated human keratinocytes. In addition, bone marrow-derived DCs from wild-type mice stimulated lymph node cells to produce higher amounts of interleukin-17 than did DCs from mice lacking the H-PGDS gene. Thus, DCs could be an important source of skin PGD2 and may mediate or regulate skin inflammation by releasing PGD2 in response to various stimuli, contributing to the innate and/or acquired immune responses.

  15. Leptin protection of salivary gland acinar cells against ethanol cytotoxicity involves Src kinase-mediated parallel activation of prostaglandin and constitutive nitric oxide synthase pathways.

    PubMed

    Slomiany, B L; Slomiany, A

    2008-04-01

    Leptin, a pleiotropic cytokine secreted by adipocytes but also identified in salivary glands and saliva, is recognized as an important element of oral mucosal defense. Here, we report that in sublingual salivary glands leptin protects the acinar cells of against ethanol cytotoxicity. We show that ethanol- induced cytotoxicity, characterized by a marked drop in the acinar cell capacity for NO production, arachidonic acid release and prostaglandin generation, was subject to suppression by leptin. The loss in countering capacity of leptin on the ethanol-induced cytotoxicity was attained with cyclooxygenase inhibitor, indomethacin and nitric oxide synthase (cNOS) inhibitor, L-NAME, as well as PP2, an inhibitor of Src kinase. Indomethacin, while not affecting leptin-induced arachidonic acid release, caused the inhibition in PGE2 generation, pretreatment with L-NAME led to the inhibition in NO production, whereas PP2 exerted the inhibitory effect on leptin-induced changes in NO, arachidonic acid, and PGE2. The leptin-induced changes in arachidonic acid release and PGE2 generation were blocked by ERK inhibitor, PD98059, but not by PI3K inhibitor, wortmannin. Further, leptin suppression of ethanol cytotoxicity was reflected in the increased Akt and cNOS phosphorylation that was sensitive to PP2. Moreover, the stimulatory effect of leptin on the acinar cell cNOS activity was inhibited not only by PP2, but also by Akt inhibitor, SH-5, while wortmannin had no effect. Our findings demonstrate that leptin protection of salivary gland acinar cells against ethanol cytotoxicity involves Src kinase-mediated parallel activation of MAPK/ERK and Akt that result in up-regulation of the respective prostaglandin and nitric oxide synthase pathways.

  16. Undecaprenyl diphosphate synthase inhibitors: antibacterial drug leads.

    PubMed

    Sinko, William; Wang, Yang; Zhu, Wei; Zhang, Yonghui; Feixas, Ferran; Cox, Courtney L; Mitchell, Douglas A; Oldfield, Eric; McCammon, J Andrew

    2014-07-10

    There is a significant need for new antibiotics due to the rise in drug resistance. Drugs such as methicillin and vancomycin target bacterial cell wall biosynthesis, but methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) have now arisen and are of major concern. Inhibitors acting on new targets in cell wall biosynthesis are thus of particular interest since they might also restore sensitivity to existing drugs, and the cis-prenyl transferase undecaprenyl diphosphate synthase (UPPS), essential for lipid I, lipid II, and thus, peptidoglycan biosynthesis, is one such target. We used 12 UPPS crystal structures to validate virtual screening models and then assayed 100 virtual hits (from 450,000 compounds) against UPPS from S. aureus and Escherichia coli. The most promising inhibitors (IC50 ∼2 μM, Ki ∼300 nM) had activity against MRSA, Listeria monocytogenes, Bacillus anthracis, and a vancomycin-resistant Enterococcus sp. with MIC or IC50 values in the 0.25-4 μg/mL range. Moreover, one compound (1), a rhodanine with close structural similarity to the commercial diabetes drug epalrestat, exhibited good activity as well as a fractional inhibitory concentration index (FICI) of 0.1 with methicillin against the community-acquired MRSA USA300 strain, indicating strong synergism. PMID:24827744

  17. Prostaglandin E synthase interacts with inducible heat shock protein 70 after heat stress in bovine primary dermal fibroblast cells.

    PubMed

    Richter, Constanze; Viergutz, Torsten; Schwerin, Manfred; Weitzel, Joachim M

    2015-01-01

    Exposure to heat stress in dairy cows leads to undesired side effects that are reflected by complex alterations in endocrine parameters, such as reduced progesterone, estradiol, and thyroid hormone concentrations. These endocrine maladaptation leads to failure to resume cyclicity, a poor uterine environment and inappropriate immune responses in postpartum dairy cows. Prostaglandins (PG's) are lipid mediators, which serve as signal molecules in response to various external stimuli as well as to cell-specific internal signal molecules. A central role in PG synthesis plays prostaglandin E synthase (PGES) that catalyzes the isomerization of PGH2 to PGE2 .The present study was conducted to investigate heat stress associated PGES expression. Expression of PGES and inducible heat shock protein 70 (HSP70), as a putative chaperonic protein, was studied in bovine primary fibroblasts under different heat shock conditions. Bovine primary fibroblasts produce PGE2 at homoiothermical norm temperature (38.5°C in bovine), but reduce PGE2 production rates under extreme heat stress (at 45°C for 6 h). By contrast, PGE2 production rates are maintained after a milder heat stress (at 41.5°C for 6 h). PGE2 synthesis is abolished by application of cyclooxygenase inhibitor indomethacin, indicating de novo synthesis. Heat stress increases HSP70 but not PGES protein concentrations. HSP70 physically interacts with PGES and the PGES-HSP70 complex did not dissociate upon heat stress at 45°C even after returning the cells to 37°C. The PGE2 production negatively correlates with the portion of PGES-HSP70 complex. These results suggest a protein interaction between HSP70 and PGES in dermal fibroblast cells. Blockage of PGES protein by HSP70 seems to interfere with the regulatory processes essential for cellular adaptive protection. © 2014 International Society for Advancement of Cytometry.

  18. Prostaglandins induce early growth response 1 transcription factor mediated microsomal prostaglandin E2 synthase up-regulation for colorectal cancer progression

    PubMed Central

    Stamatakis, Konstantinos; Jimenez-Martinez, Marta; Jimenez-Segovia, Alba; Chico-Calero, Isabel; Conde, Elisa; Galán-Martínez, Javier; Ruiz, Julia; Pascual, Alejandro; Barrocal, Beatriz; López-Pérez, Ricardo; García-Bermejo, María Laura; Fresno, Manuel

    2015-01-01

    Cyclooxygenase2 (COX2) has been associated with cell growth, invasiveness, tumor progression and metastasis of colorectal carcinomas. However, the downstream prostaglandin (PG)-PG receptor pathway involved in these effects is poorly characterized. We studied the PG-pathway in gene expression databases and we found that PTGS2 (prostaglandin G/H synthase and cyclooxygenase) and PTGES (prostaglandin E synthase) are co-expressed in human colorectal tumors. Moreover, we detected that COX2 and microsomal Prostaglandin E2 synthase 1 (mPGES1) proteins are both up-regulated in colorectal human tumor biopsies. Using colon carcinoma cell cultures we found that COX2 overexpression significantly increased mPGES1 mRNA and protein. This up-regulation was due to an increase in early growth response 1 (EGR1) levels and its transcriptional activity. EGR1 was induced by COX2-generated PGF2α. A PGF2α receptor antagonist, or EGR1 silencing, inhibited the mPGES1 induction by COX2 overexpression. Moreover, using immunodeficient mice, we also demonstrated that both COX2- and mPGES1-overexpressing carcinoma cells were more efficient forming tumors. Our results describe for the first time the molecular pathway correlating PTGS2 and PTGES in colon cancer progression. We demonstrated that in this pathway mPGES1 is induced by COX2 overexpression, via autocrine PGs release, likely PGF2α, through an EGR1-dependent mechanism. This signaling provides a molecular explanation to PTGS2 and PTGES association and contribute to colon cancer advance, pointing out novel potential therapeutic targets in this oncological context. PMID:26498686

  19. Hypoxia activates the cyclooxygenase-2–prostaglandin E synthase axis

    PubMed Central

    Lee, James J.; Natsuizaka, Mitsuteru; Ohashi, Shinya; Wong, Gabrielle S.; Takaoka, Munenori; Michaylira, Carmen Z.; Budo, Daniela; Tobias, John W.; Kanai, Michiyuki; Shirakawa, Yasuhiro; Naomoto, Yoshio; Klein-Szanto, Andres J.P.; Haase, Volker H.; Nakagawa, Hiroshi

    2010-01-01

    Hypoxia-inducible factors (HIFs), in particular HIF-1α, have been implicated in tumor biology. However, HIF target genes in the esophageal tumor microenvironment remain elusive. Gene expression profiling was performed upon hypoxia-exposed non-transformed immortalized human esophageal epithelial cells, EPC2-hTERT, and comparing with a gene signature of esophageal squamous cell carcinoma (ESCC). In addition to known HIF-1α target genes such as carbonic anhydrase 9, insulin-like growth factor binding protein-3 (IGFBP3) and cyclooxygenase (COX)-2, prostaglandin E synthase (PTGES) was identified as a novel target gene among the commonly upregulated genes in ESCC as well as the cells exposed to hypoxia. The PTGES induction was augmented upon stabilization of HIF-1α by hypoxia or cobalt chloride under normoxic conditions and suppressed by dominant-negative HIF-1α. Whereas PTGES messenger RNA (mRNA) was negatively regulated by normoxia, PTGES protein remained stable upon reoxygenation. Prostaglandin E2 (PGE2) biosynthesis was documented in transformed human esophageal cells by ectopic expression of PTGES as well as RNA interference directed against PTGES. Moreover, hypoxia stimulated PGE2 production in a HIF-1α-dependent manner. In ESCC, PTGES was overexpressed frequently at the mRNA and protein levels. Finally, COX-2 and PTGES were colocalized in primary tumors along with HIF-1α and IGFBP3. Activation of the COX-2–PTGES axis in primary tumors was further corroborated by concomitant upregulation of interleukin-1β and downregulation of hydroxylprostaglandin dehydrogenase. Thus, PTGES is a novel HIF-1α target gene, involved in prostaglandin E biosynthesis in the esophageal tumor hypoxic microenvironment, and this has implications in diverse tumors types, especially of squamous origin. PMID:20042640

  20. Structures of prostaglandin F synthase from the protozoa Leishmania major and Trypanosoma cruzi with NADP

    PubMed Central

    Moen, Spencer O.; Fairman, James W.; Barnes, Steve R.; Sullivan, Amy; Nakazawa-Hewitt, Stephen; Van Voorhis, Wesley C.; Staker, Bart L.; Lorimer, Donald D.; Myler, Peter J.; Edwards, Thomas E.

    2015-01-01

    The crystal structures of prostaglandin F synthase (PGF) from both Leishmania major and Trypanosoma cruzi with and without their cofactor NADP have been determined to resolutions of 2.6 Å for T. cruzi PGF, 1.25 Å for T. cruzi PGF with NADP, 1.6 Å for L. major PGF and 1.8 Å for L. major PGF with NADP. These structures were determined by molecular replacement to a final R factor of less than 18.6% (R free of less than 22.9%). PGF in the infectious protozoa L. major and T. cruzi is a potential therapeutic target. PMID:25945716

  1. Prostaglandin E synthase is upregulated by Gas6 during cancer-induced venous thrombosis.

    PubMed

    Aghourian, Meghedi N; Lemarié, Catherine A; Bertin, Francois-René; Blostein, Mark D

    2016-02-11

    Venous thromboembolism is a common complication of cancer. Based on recent evidence that (1) growth arrest-specific 6 (Gas6) regulates the expression of tissue factor during venous thrombosis, and (2) cancer promotes a procoagulant milieu, we hypothesize that Gas6 may be involved in cancer-induced coagulopathy. Venous thrombi were induced in both wild-type (WT) and Gas6-deficient ((-/-)) mice with cancer. WT mice with cancer developed larger thrombi than their healthy counterparts; these larger thrombi induced by cancer were not seen in Gas6(-/-) mice. Whole genome microarray analysis of differential gene expression in WT and Gas6(-/-) endothelial cells exposed to M27 murine lung carcinoma cells reveal that Gas6 increases prostaglandin E synthase (Ptges) expression in endothelial cells. This was confirmed using real-time polymerase chain reaction and immunofluorescence staining. Culture of WT endothelial cells with M27 increases the secretion of prostaglandin E2 (PGE2), the enzymatic product of Ptges, in WT but not in Gas6(-/-) endothelial cells. In WT endothelial cells, Ptges expression was regulated through extracellular signal-regulated kinase 1/2 phosphorylation (ERK1/2). In vitro, PGE2 activates platelets after binding to its receptor, EP3. In vivo, EP3 receptor antagonism reversed the effect of cancer-induced thrombosis in WT mice. These results show that Gas6, through upregulation of PGE2, contributes to cancer-induced venous thrombosis. PMID:26585956

  2. Human brain prostaglandin D synthase has been evolutionarily differentiated from lipophilic-ligand carrier proteins.

    PubMed Central

    Nagata, A; Suzuki, Y; Igarashi, M; Eguchi, N; Toh, H; Urade, Y; Hayaishi, O

    1991-01-01

    cDNAs for glutathione-independent prostaglandin D synthase were isolated from cDNA libraries of human brain. The longest cDNA insert was 837 base pairs long and contained a coding region of 570 base pairs corresponding to 190 amino acid residues with a calculated Mr of 21,016. Between two cDNA inserts isolated from the two different libraries, nucleotide substitutions were observed at 16 positions, including conservative amino acid substitutions at 2 positions and nonconservative substitutions at 5 positions, indicating genetic heterogeneity of this enzyme in humans. The computer-assisted homology search revealed that the enzyme is a member of the lipocalin superfamily, comprising secretory hydrophobic molecule transporters, showing the greatest homology (28.8-29.4% identity; 51.3-53.1% similarity) to alpha 1-microglobulin among the members of this superfamily. In a phylogenetic tree of the superfamily, this enzyme, alpha 1-microglobulin, and the gamma chain of the complement component C8 form a cluster separate from the other 14 members. The two distinctive characteristics of glutathione-independent prostaglandin D synthase, as compared to the other members of this superfamily, are its enzymatic properties and its association with membranes that were probably acquired after evolutionary divergence of the two lipocalins. Based on the observed sequence homology, the tertiary structure of the enzyme was deduced to consist of an eight-stranded anti-parallel beta-barrel forming a hydrophobic pocket. Furthermore, the Cys-65 residue in the pocket, which is conserved only in the human and rat enzymes but not in other lipocalins, was considered to be a putative active site of the enzyme. Images PMID:1902577

  3. Hematopoietic prostaglandin D synthase suppresses intestinal adenomas in ApcMin/+ mice.

    PubMed

    Park, Jae Man; Kanaoka, Yoshihide; Eguchi, Naomi; Aritake, Kosuke; Grujic, Sava; Materi, Alicia M; Buslon, Virgilio S; Tippin, Brigette L; Kwong, Alan M; Salido, Eduardo; French, Samuel W; Urade, Yoshihiro; Lin, Henry J

    2007-02-01

    Aspirin and other nonsteroidal anti-inflammatory drugs prevent some cases of colon cancer by inhibiting prostaglandin (PG) synthesis. PGE(2) promotes colon neoplasia, as shown by knockout mouse studies on enzymes and receptors in the PG cascade. A few experiments 20 to 30 years ago suggested that PGD(2) may suppress tumors, but a role for biosynthetic enzymes for PGD(2) in tumor development has not been studied. We report here that disruption of the gene for hematopoietic PGD synthase in Apc(Min/+) mice led to approximately 50% more intestinal adenomas compared with controls. Tumor size was not affected. By immunohistochemistry, we detected hematopoietic PGD synthase mainly in macrophages and monocytes of the gut mucosa. The mean number of tumors did not increase with knockout of the gene for the lipocalin type of the enzyme, which is not produced in the intestine. On the other hand, Apc(Min/+) mice with transgenic human hematopoietic PGD synthase tended to have 80% fewer intestinal adenomas. The transgene produced high mRNA levels (375-fold over endogenous). There was a suggestion of higher urinary excretion of 11beta-PGF(2alpha) and a lower excretion of a PGE(2) metabolite in transgenic mice, but differences (30-40%) were not statistically significant. The results support an interpretation that hematopoietic PGD synthase controls an inhibitory effect on intestinal tumors. Further studies will be needed to prove possible mechanisms, such as routing of PG production away from protumorigenic PGE(2) or inhibition of the nuclear factor-kappaB cascade by PGD(2) metabolites. PMID:17283118

  4. Essential role for hematopoietic prostaglandin D2 synthase in the control of delayed type hypersensitivity.

    PubMed

    Trivedi, Seema G; Newson, Justine; Rajakariar, Ravindra; Jacques, Thomas S; Hannon, Robert; Kanaoka, Yoshihide; Eguchi, Naomi; Colville-Nash, Paul; Gilroy, Derek W

    2006-03-28

    Hematopoietic prostaglandin D(2) synthase (hPGD(2)S) metabolizes cyclooxygenase-derived prostaglandin (PG) H(2) to PGD(2), which is dehydrated to cyclopentenone PGs, including 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)). PGD(2) acts through two receptors (DP1 and DP2/CRTH2), whereas 15d-PGJ(2) can activate peroxisome proliferator-activated receptors or inhibit a range of proinflammatory signaling pathways, including NF-kappaB. Despite eliciting asthmatic and allergic reactions through the generation of PGD(2), it is not known what role hPGD(2)S plays in T helper (Th)1-driven adaptive immunity. To investigate this question, the severity and duration of a delayed type hypersensitivity reaction was examined in hPGD(2)S knockout and transgenic mice. Compared with their respective controls, knockouts displayed a more severe inflammatory response that failed to resolve, characterized histologically as persistent acute inflammation, whereas transgenic mice had little detectable inflammation. Lymphocytes isolated from inguinal lymph nodes of hPGD(2)S(-/-) animals showed hyperproliferation and increased IL-2 synthesis effects that were rescued by 15d-PGJ(2), but not PGD(2), working through either of its receptors. Crucially, 15d-PGJ(2) exerted its suppressive effects through the inhibition of NF-kappaB activation and not through peroxisome proliferator-activated receptor signaling. In contrast, lymph node cultures from transgenics proliferated more slowly and synthesized significantly less IL-2 than controls. Therefore, contrary to its role in driving Th2-like responses, this report shows that hPGD(2)S may act as an internal braking signal essential for bringing about the resolution of Th1-driven delayed type hypersensitivity reactions. Consequently, hPGD(2)S-derived cyclopentenone PGs may protect against inflammatory diseases, where T lymphocytes play a pathogenic role, as in rheumatoid arthritis, atopic eczema, and chronic rejection. PMID:16547141

  5. Human platelet/erythroleukemia cell prostaglandin G/H synthase: cDNA cloning, expression, and gene chromosomal assignment

    SciTech Connect

    Funk, C.D.; Funk, L.B.; Kennedy, M.E.; Pong, A.S.; Fitzgerald, G.A. )

    1991-06-01

    Platelets metabolize arachidonic acid to thromboxane A{sub 2}, a potent platelet aggregator and vasoconstrictor compound. The first step of this transformation is catalyzed by prostaglandin (PG) G/H synthase, a target site for nonsteroidal antiinflammatory drugs. We have isolated the cDNA for both human platelet and human erythroleukemia cell PGG/H synthase using the polymerase chain reaction and conventional screening procedures. The cDNA encoding the full-length protein was expressed in COS-M6 cells. Microsomal fractions from transfected cells produced prostaglandin endoperoxide derived products which were inhibited by indomethacin and aspirin. Mutagenesis of the serine residue at position 529, the putative aspirin acetylation site, to an asparagine reduced cyclooxygenase activity to barely detectable levels, an effect observed previously with the expressed sheep vesicular gland enzyme. Platelet-derived growth factor and phorbol ester differentially regulated the expression of PGG/H synthase mRNA levels in the megakaryocytic/platelet-like HEL cell line. The PGG/H synthase gene was assigned to chromosome 9 by analysis of a human-hamster somatic hybrid DNA panel. The availability of platelet PGG/H synthase cDNA should enhance our understanding of the important structure/function domains of this protein and it gene regulation.

  6. Higher oxidation states of prostaglandin H synthase. EPR study of a transient tyrosyl radical in the enzyme during the peroxidase reaction.

    PubMed

    Karthein, R; Dietz, R; Nastainczyk, W; Ruf, H H

    1988-01-15

    Purified prostaglandin H synthase (EC 1.14.99.1), reconstituted with hemin, was reacted with substrates of the cyclooxygenase and peroxidase reaction. The resulting EPR spectra were measured below 90 K. Arachidonic acid, added under anaerobic conditions, did not change the EPR spectrum of the native enzyme due to high-spin ferric heme. Arachidonic acid with O2, as well as prostaglandin G2 or H2O2, decreased the spectrum of the native enzyme and concomitantly a doublet signal at g = 2.005 was formed with maximal intensity of 0.35 spins/enzyme and a half-life of less than 20 s at -12 degrees C. From the conditions for the formation and the effect of inhibitors, this doublet signal was assigned to an enzyme intermediate of the peroxidase reaction, namely a higher oxidation state. The doublet signal with characteristic hyperfine structure was nearly identical to the signal of the tyrosyl radical in ribonucleotide reductase (EC 1.17.4.1). Hence the signal of prostaglandin H synthase was assigned to a tyrosyl radical. Electronic spectra as well as decreased power saturation of the tyrosyl radical signal indicated heme in its ferryl state which coupled to the tyrosyl radical weakly. [FeIVO(protoporphyrin IX)]...Tyr+. was suggested as the structure of this two-electron oxidized state of the enzyme. A hypothetical role for the tyrosyl radical could be the abstraction of a hydrogen at C-13 of arachidonic acid which is assumed to be the initial step of the cyclooxygenase reaction.

  7. Induction of human microsomal prostaglandin E synthase 1 by activated oncogene RhoA GTPase in A549 human epithelial cancer cells

    SciTech Connect

    Choi, Hye Jin; Lee, Dong-Hyung; Park, Seong-Hwan; Kim, Juil; Do, Kee Hun; An, Tae Jin; Ahn, Young Sup; Park, Chung Berm; Moon, Yuseok

    2011-09-30

    Highlights: {yields} As a target of oncogene RhoA-linked signal, a prostaglandin metabolism is assessed. {yields} RhoA activation increases PGE{sub 2} levels and its metabolic enzyme mPGES-1. {yields} RhoA-activated NF-{kappa}B and EGR-1 are positively involved in mPGES-1 induction. -- Abstract: Oncogenic RhoA GTPase has been investigated as a mediator of pro-inflammatory responses and aggressive carcinogenesis. Among the various targets of RhoA-linked signals, pro-inflammatory prostaglandin E{sub 2} (PGE{sub 2}), a major prostaglandin metabolite, was assessed in epithelial cancer cells. RhoA activation increased PGE{sub 2} levels and gene expression of the rate-limiting PGE{sub 2} producing enzymes, cyclooxygenase-2 and microsomal prostaglandin E synthase 1 (mPGES-1). In particular, human mPGES-1 was induced by RhoA via transcriptional activation in control and interleukin (IL)-1{beta}-activated cancer cells. To address the involvement of potent signaling pathways in RhoA-activated mPGES-1 induction, various signaling inhibitors were screened for their effects on mPGES-1 promoter activity. RhoA activation enhanced basal and IL-1{beta}-mediated phosphorylated nuclear factor-{kappa}B and extracellular signal-regulated kinase1/2 proteins, all of which were positively involved in RhoA-induced gene expression of mPGES-1. As one potent down-stream transcription factor of ERK1/2 signals, early growth response gene 1 product also mediated RhoA-induced gene expression of mPGES-1 by enhancing transcriptional activity. Since oncogene-triggered PGE{sub 2} production is a critical modulator of epithelial tumor cells, RhoA-associated mPGES-1 represents a promising chemo-preventive or therapeutic target for epithelial inflammation and its associated cancers.

  8. Carnosol and carnosic acids from Salvia officinalis inhibit microsomal prostaglandin E2 synthase-1.

    PubMed

    Bauer, Julia; Kuehnl, Susanne; Rollinger, Judith M; Scherer, Olga; Northoff, Hinnak; Stuppner, Hermann; Werz, Oliver; Koeberle, Andreas

    2012-07-01

    Prostaglandin E(2) (PGE(2)), the most relevant eicosanoid promoting inflammation and tumorigenesis, is formed by cyclooxygenases (COXs) and PGE(2) synthases from free arachidonic acid. Preparations of the leaves of Salvia officinalis are commonly used in folk medicine as an effective antiseptic and anti-inflammatory remedy and possess anticancer activity. Here, we demonstrate that a standard ethyl acetate extract of S. officinalis efficiently suppresses the formation of PGE(2) in a cell-free assay by direct interference with microsomal PGE(2) synthase (mPGES)-1. Bioactivity-guided fractionation of the extract yielded closely related fractions that potently suppressed mPGES-1 with IC(50) values between 1.9 and 3.5 μg/ml. Component analysis of these fractions revealed the diterpenes carnosol and carnosic acid as potential bioactive principles inhibiting mPGES-1 activity with IC(50) values of 5.0 μM. Using a human whole-blood assay as a robust cell-based model, carnosic acid, but not carnosol, blocked PGE(2) generation upon stimulation with lipopolysaccharide (IC(50) = 9.3 μM). Carnosic acid neither inhibited the concomitant biosynthesis of other prostanoids [6-keto PGF(1α), 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid, and thromboxane B(2)] in human whole blood nor affected the activities of COX-1/2 in a cell-free assay. Together, S. officinalis extracts and its ingredients carnosol and carnosic acid inhibit PGE(2) formation by selectively targeting mPGES-1. We conclude that the inhibitory effect of carnosic acid on PGE(2) formation, observed in the physiologically relevant whole-blood model, may critically contribute to the anti-inflammatory and anticarcinogenic properties of S. officinalis.

  9. Carnosol and Carnosic Acids from Salvia officinalis Inhibit Microsomal Prostaglandin E2 Synthase-1

    PubMed Central

    Bauer, Julia; Kuehnl, Susanne; Rollinger, Judith M.; Scherer, Olga; Northoff, Hinnak; Stuppner, Hermann; Werz, Oliver; Koeberle, Andreas

    2012-01-01

    Prostaglandin E2 (PGE2), the most relevant eicosanoid promoting inflammation and tumorigenesis, is formed by cyclooxygenases (COXs) and PGE2 synthases from free arachidonic acid. Preparations of the leaves of Salvia officinalis are commonly used in folk medicine as an effective antiseptic and anti-inflammatory remedy and possess anticancer activity. Here, we demonstrate that a standard ethyl acetate extract of S. officinalis efficiently suppresses the formation of PGE2 in a cell-free assay by direct interference with microsomal PGE2 synthase (mPGES)-1. Bioactivity-guided fractionation of the extract yielded closely related fractions that potently suppressed mPGES-1 with IC50 values between 1.9 and 3.5 μg/ml. Component analysis of these fractions revealed the diterpenes carnosol and carnosic acid as potential bioactive principles inhibiting mPGES-1 activity with IC50 values of 5.0 μM. Using a human whole-blood assay as a robust cell-based model, carnosic acid, but not carnosol, blocked PGE2 generation upon stimulation with lipopolysaccharide (IC50 = 9.3 μM). Carnosic acid neither inhibited the concomitant biosynthesis of other prostanoids [6-keto PGF1α, 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid, and thromboxane B2] in human whole blood nor affected the activities of COX-1/2 in a cell-free assay. Together, S. officinalis extracts and its ingredients carnosol and carnosic acid inhibit PGE2 formation by selectively targeting mPGES-1. We conclude that the inhibitory effect of carnosic acid on PGE2 formation, observed in the physiologically relevant whole-blood model, may critically contribute to the anti-inflammatory and anticarcinogenic properties of S. officinalis. PMID:22511203

  10. Prognostic Value of Prostaglandin-endoperoxide Synthase 2 Polymorphisms in Prostate Cancer Recurrence after Radical Prostatectomy

    PubMed Central

    Lee, Cheng-Hsueh; Pao, Jiunn-Bey; Lu, Te-Ling; Lee, Hong-Zin; Lee, Yung-Chin; Liu, Chia-Chu; Huang, Chao-Yuan; Lin, Victor C.; Yu, Chia-Cheng; Yin, Hsin-Ling; Huang, Shu-Pin; Bao, Bo-Ying

    2016-01-01

    Backgroud: Increasing evidence suggests the involvement of chronic inflammation in the progression of prostate cancer, and prostaglandin-endoperoxide synthase 2 (PTGS2), also known as cyclooxygenase-2, catalyzes the rate-limiting steps of the pathway. We hypothesized that genetic variants of PTGS2 can influence the outcome of prostate cancer patients. Methods: We genotyped five haplotype-tagging single-nucleotide polymorphisms (SNPs) to detect common genetic variations across the PTGS2 region in 458 prostate cancer patients treated with radical prostatectomy. Results: One SNP, rs4648302, was associated with disease recurrence. Five-year recurrence-free survival rate increased according to the number of variant alleles inherited (55.6%, 70.7%, and 100.0% for patients with different genotypes; P = 0.037), and the effect was maintained in multivariable analysis. Public dataset analyses also suggested that PTGS2 expression was correlated with prostate cancer prognosis. Conclusion: Our results indicated that PTGS2 could be a potential prognostic marker to improve the prediction of disease recurrence in prostate cancer patients. PMID:27647999

  11. Prognostic Value of Prostaglandin-endoperoxide Synthase 2 Polymorphisms in Prostate Cancer Recurrence after Radical Prostatectomy

    PubMed Central

    Lee, Cheng-Hsueh; Pao, Jiunn-Bey; Lu, Te-Ling; Lee, Hong-Zin; Lee, Yung-Chin; Liu, Chia-Chu; Huang, Chao-Yuan; Lin, Victor C.; Yu, Chia-Cheng; Yin, Hsin-Ling; Huang, Shu-Pin; Bao, Bo-Ying

    2016-01-01

    Backgroud: Increasing evidence suggests the involvement of chronic inflammation in the progression of prostate cancer, and prostaglandin-endoperoxide synthase 2 (PTGS2), also known as cyclooxygenase-2, catalyzes the rate-limiting steps of the pathway. We hypothesized that genetic variants of PTGS2 can influence the outcome of prostate cancer patients. Methods: We genotyped five haplotype-tagging single-nucleotide polymorphisms (SNPs) to detect common genetic variations across the PTGS2 region in 458 prostate cancer patients treated with radical prostatectomy. Results: One SNP, rs4648302, was associated with disease recurrence. Five-year recurrence-free survival rate increased according to the number of variant alleles inherited (55.6%, 70.7%, and 100.0% for patients with different genotypes; P = 0.037), and the effect was maintained in multivariable analysis. Public dataset analyses also suggested that PTGS2 expression was correlated with prostate cancer prognosis. Conclusion: Our results indicated that PTGS2 could be a potential prognostic marker to improve the prediction of disease recurrence in prostate cancer patients.

  12. Prostaglandin D2 synthase/GPR44: a signaling axis in PNS myelination.

    PubMed

    Trimarco, Amelia; Forese, Maria Grazia; Alfieri, Valentina; Lucente, Alessandra; Brambilla, Paola; Dina, Giorgia; Pieragostino, Damiana; Sacchetta, Paolo; Urade, Yoshihiro; Boizet-Bonhoure, Brigitte; Martinelli Boneschi, Filippo; Quattrini, Angelo; Taveggia, Carla

    2014-12-01

    Neuregulin 1 type III is processed following regulated intramembrane proteolysis, which allows communication from the plasma membrane to the nucleus. We found that the intracellular domain of neuregulin 1 type III upregulated the prostaglandin D2 synthase (L-pgds, also known as Ptgds) gene, which, together with the G protein-coupled receptor Gpr44, forms a previously unknown pathway in PNS myelination. Neuronal L-PGDS is secreted and produces the PGD2 prostanoid, a ligand of Gpr44. We found that mice lacking L-PGDS were hypomyelinated. Consistent with this, specific inhibition of L-PGDS activity impaired in vitro myelination and caused myelin damage. Furthermore, in vivo ablation and in vitro knockdown of glial Gpr44 impaired myelination. Finally, we identified Nfatc4, a key transcription factor for myelination, as one of the downstream effectors of PGD2 activity in Schwann cells. Thus, L-PGDS and Gpr44 are previously unknown components of an axo-glial interaction that controls PNS myelination and possibly myelin maintenance.

  13. Effect of a selective thromboxane synthase inhibitor on arterial graft patency and platelet deposition in dogs

    SciTech Connect

    McDaniel, M.D.; Huntsman, W.T.; Miett, T.O.; Cronenwett, J.L.

    1987-08-01

    This study examined the effect of selective thromboxane synthase inhibition and nonselective cyclooxygenase inhibition on vascular graft patency and indium 111-labeled platelet deposition in 35 mongrel dogs undergoing carotid artery replacement with 4 mm X 4 cm polytetrafluoroethylene (PTFE) (one side) and Dacron (opposite side) end-to-end grafts. Aspirin-dipyridamole therapy improved one-week graft patency, from 46% in untreated dogs to 93% in treated dogs. Thromboxane synthase inhibition (U-63557A) improved graft patency in these dogs to 81%. Both drug treatments reduced platelet deposition on Dacron and PTFE grafts by 48% to 68% compared with control dogs. Dacron grafts accumulated significantly more platelets than PTFE grafts but had comparable patency rates. Low-dose aspirin therapy had no significant effect on either graft patency or platelet deposition. All treatment groups showed a 60% to 76% reduction in serum thromboxane B2, but only thromboxane synthase inhibitor treatment increased plasma 6-keto-prostaglandin F1 alpha by 100%. Selective thromboxane synthase inhibition improved small-caliber prosthetic graft patency to the same extent as did conventional cyclooxygenase inhibition in this preliminary study.

  14. Inhibition of hematopoietic prostaglandin D2 Synthase (H-PGDS) by an alkaloid extract from Combretum molle

    PubMed Central

    2014-01-01

    Background Hematopoietic prostaglandin D2 synthase (H-PGDS, GST Sigma) is a member of the glutathione S-transferase super family of enzymes that catalyses the conjugation of electrophilic substances with reduced glutathione. The enzyme catalyses the conversion of PGH2 to PGD2 which mediates inflammatory responses. The inhibition of H-PGDS is of importance in alleviating damage to tissues due to unwarranted synthesis of PGD2. Combretum molle has been used in African ethno medicinal practices and has been shown to reduce fever and pain. The effect of C. molle alkaloid extract on H-PGDS was thus, investigated. Methods H-PGDS was expressed in Escherichia coli XL1-Blue cells and purified using nickel immobilized metal affinity chromatography. The effect of C. molle alkaloid extract on H-PGDS activity was determined with 1-chloro-2, 4-dinitrobenzene (CDNB) as substrate. The effect of C. molle alkaloid extract with time on H-PGDS was determined. The mechanism of inhibition was then investigated using CDNB and glutathione (GSH) as substrates. Results A specific activity of 24 μmol/mg/min was obtained after H-PGDS had been purified. The alkaloid extract exhibited a 70% inhibition on H-PGDS with an IC50 of 13.7 μg/ml. C. molle alkaloid extract showed an uncompetitive inhibition of H-PGDS with Ki = 41 μg/ml towards GSH, and non-competitive inhibition towards CDNB with Ki = 7.7 μg/ml and Ki′ = 9.2 μg/ml. Conclusion The data shows that C. molle alkaloid extract is a potent inhibitor of H-PGDS. This study thus supports the traditional use of the plant for inflammation. PMID:24996417

  15. Prostaglandin synthesis inhibitors reduce Cannabis and restraint stress induced increase in rat brain serotonin concentrations.

    PubMed

    Bhattacharya, S K; Bhattacharya, D

    1983-01-01

    Cannabis resin (CI) produced a dose-related increase in rat brain serotonin concentrations, whereas restraint stress produced maximal rise of the neurotransmitter concentrations at 1 h, followed by a tendency to normalise by 4 h. The prostaglandin (PG) synthesis inhibitors, diclofenac and paracetamol, antagonized CI and restraint stress induced rise in serotonin concentrations. The findings lend credence to earlier reports that PG synthesis inhibitors antagonize serotonin-mediated neuropharmacological actions of CI and restraint stress in rats.

  16. Prostaglandin-E2 is a potent inhibitor of human interleukin 12 production

    PubMed Central

    1995-01-01

    During human immunodeficiency virus infection and allergic diseases, characterized by a dominant T helper (Th) 2 response, overproduction of prostaglandin E2 (PGE2) is observed. In this paper we studied the effect of PGE2 on interleukin (IL)-12 synthesis, because this cytokine has been described to be essential in induction of Th1 responses. IL-12 synthesis was induced in monocytes that were stimulated with Neisseria meningitidis-derived lipopolysaccharide in whole blood cultures. PGE2 almost completely inhibited lipopolysaccharide induced IL-12 production, whereas IL-6 production was only partially inhibited by PGE2. In contrast, the production of IL-10 was approximately twofold enhanced at these conditions. The effects of PGE2 were due to its cAMP- inducing capacity, since they could be mimicked by other cAMP inducers. Recombinant human IL-10 also inhibited IL-12 and IL-6 production. However, the inhibitory effect of PGE2 on IL-12 production was independent of IL-10 since neutralizing anti-IL-10 antibodies were unable to reverse this inhibition. These results suggest that the capacity of an antigen to induce PGE2 synthesis may play a crucial role in the development of either a Th1 or Th2 response. PMID:7836930

  17. Electron spin resonance investigation of tyrosyl radicals of prostaglandin H synthase. Relation to enzyme catalysis.

    PubMed

    Lassmann, G; Odenwaller, R; Curtis, J F; DeGray, J A; Mason, R P; Marnett, L J; Eling, T E

    1991-10-25

    We have examined, by low temperature ESR, the protein-derived radicals formed by reaction of purified ram seminal vesicle prostaglandin H synthase (PHS). Upon addition of arachidonic acid or 5-phenyl-4-pentenyl-1-hydroperoxide (PPHP) to PHS reconstituted with Fe(III)-protoporphyrin IX (Fe-PHS) at -12 degrees C, an ESR spectrum was observed at -196 degrees C containing a doublet that rapidly converted into a singlet. These protein-derived radicals were identified as tyrosyl radicals. The addition of a peroxidase substrate, phenol, completely abolished the appearance of the doublet and suppressed the formation of the singlet but did not inhibit eicosanoid formation. Incubation of arachidonic acid with PHS reconstituted with Mn(III)-protoporphyrin IX (Mn-PHS) produced only a broad singlet that exhibited different power saturation behavior than the tyrosyl radicals and decayed more rapidly. This broad singlet does not appear to be a tyrosyl radical. No ESR signals were observed on incubation of PPHP with Mn-PHS, which has cyclooxygenase but not peroxidase activity. Eicosanoid synthesis occurred very rapidly after addition of arachidonic acid and was complete within 1 min. In contrast, the protein-derived radicals appeared at a slower rate and after the addition of the substrate reached maximal levels between 1 and 2 min for Fe-PHS and 4-6 min for Mn-PHS. These results suggest that the observed protein-derived radicals are not catalytically competent intermediates in cyclooxygenase catalysis by either Fe-PHS or Mn-PHS. The peroxidase activity appears to play a major role in the formation of the tyrosyl radicals with Fe-PHS.

  18. Prostaglandin H synthase catalyzes regiospecific release of tritium from labeled estradiol

    SciTech Connect

    Degen, G.H.; Jellinck, P.H.; Hershcopf, R.J.

    1987-06-01

    Prostaglandin H synthase (PHS) from ram seminal vesicle microsomes was found to catalyze the release of tritium (3H) from estradiol (E2) regiospecifically labeled in position C-2 or C-4 of ring A but not from positions C-17 alpha, C-16 alpha, or C-6,7. Formation of 3H2O from ring A of E2 is dependent upon native enzyme supplemented with either arachidonic acid, eicosapentaenoic acid, or hydrogen peroxide and proceeds very rapidly as do other cooxidation reactions catalyzed by PHS-peroxidase. The 3H-loss from ring A of E2 reflecting oxidative displacement of this isotope by PHS increases linearly up to 100 microM under our conditions (8-45 nmol/mg x 5 min). Loss of tritium in various blanks is negligible by comparison. Indomethacin (0.07 and 0.2 mM) inhibited the PHS-dependent release of 3H2O from estradiol but less efficiently than it inhibited DES-cooxidation measured in parallel incubations under similar conditions. Addition of EDTA (0.5 mM) had no effect on the regiospecific transfer of 3H from E2 or on DES-oxidation; ascorbic acid (0.5 mM) or NADH (0.33 mM) clearly inhibited both reactions and to a similar extent. These data suggest that estradiol-2/4-hydroxylation can be catalyzed by PHS in vitro probably via its peroxidase activity and point to PHS as an enzyme that could contribute to catechol estrogen formation in vitro by tissue preparations in the presence of unsaturated fatty acids or peroxides.

  19. Lipocalin-type prostaglandin D synthase protects against oxidative stress-induced neuronal cell death.

    PubMed

    Fukuhara, Ayano; Yamada, Mao; Fujimori, Ko; Miyamoto, Yuya; Kusumoto, Toshihide; Nakajima, Hidemitsu; Inui, Takashi

    2012-04-01

    L-PGDS [lipocalin-type PGD (prostaglandin D) synthase] is a dual-functional protein, acting as a PGD2-producing enzyme and a lipid transporter. L-PGDS is a member of the lipocalin superfamily and can bind a wide variety of lipophilic molecules. In the present study we demonstrate the protective effect of L-PGDS on H2O2-induced apoptosis in neuroblastoma cell line SH-SY5Y. L-PGDS expression was increased in H2O2-treated neuronal cells, and the L-PGDS level was highly associated with H2O2-induced apoptosis, indicating that L-PGDS protected the neuronal cells against H2O2-mediated cell death. A cell viability assay revealed that L-PGDS protected against H2O2-induced cell death in a concentration-dependent manner. Furthermore, the titration of free thiols in H2O2-treated L-PGDS revealed that H2O2 reacted with the thiol of Cys65 of L-PGDS. The MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight)-MS spectrum of H2O2-treated L-PGDS showed a 32 Da increase in the mass relative to that of the untreated protein, showing that the thiol was oxidized to sulfinic acid. The binding affinities of oxidized L-PGDS for lipophilic molecules were comparable with those of untreated L-PGDS. Taken together, these results demonstrate that L-PGDS protected against neuronal cell death by scavenging reactive oxygen species without losing its ligand-binding function. The novel function of L-PGDS could be useful for the suppression of oxidative stress-mediated neurodegenerative diseases. PMID:22248185

  20. Prostaglandin inhibitor and radiotherapy in advanced head and neck cancers

    SciTech Connect

    Pillsbury, H.C. III; Webster, W.P.; Rosenman, J.

    1986-05-01

    Radiotherapy is the usual mode of treatment for unresectable head and neck cancer. To improve cure rates, extend survival, and reduce morbidity, we use accelerated hyperfractionation radiotherapy and an adjuvant drug to inhibit prostaglandin synthesis. In this study, 19 patients received 300 rad/day of radiotherapy in two equally divided doses to a total dose averaging 6,200 rad. Either indomethacin, 25 mg, or placebo was given four times a day in a double-blind fashion during therapy. Radiation mucositis was graded as 0 to 4+; pain, nutritional status, and tumor status were monitored daily and recorded biweekly. Evaluation of the data showed delayed mucositis in the experimental group for grades 1 to 3, with a significant difference at grade 3 compared with controls. The significance of a long-term comparison of cure rates would be doubtful considering the heterogeneity of the primary sites and regional disease in this group coupled with the small size of our study.

  1. TGF-beta 1 inhibits both endotoxin-induced prostaglandin synthesis and expression of the TIS10/prostaglandin synthase 2 gene in murine macrophages.

    PubMed

    Reddy, S T; Gilbert, R S; Xie, W; Luner, S; Herschman, H R

    1994-02-01

    Activated macrophages produce substantial quantities of paracrine mediators, including cytokines, nitric oxide, and prostaglandins. Transforming growth factor beta 1 (TGF-beta) is a potent modulator of immune function. TGF-beta inhibits the cytotoxic activity of endotoxin/lipopolysaccharide (LPS)-activated macrophage cell lines and primary macrophage cultures, reducing their expression of cytokines and nitric oxide. In this report we demonstrate that TGF-beta also attenuates the LPS-induced synthesis and secretion of prostaglandin E2 in murine RAW 264.7 macrophage cells. Macrophage activation also induces accumulation of the recently described ligand-responsive prostaglandin synthase (PGS) TIS10/PGS-2. While TGF-beta alone has no effect on expression from the TIS10/PGS-2 gene, this cytokine inhibits LPS-induced TIS10/PGS-2 protein accumulation and synthesis, as well as LPS-induced TIS10/PGS-2 message accumulation in RAW 264.7 cells. TGF-beta concentrations in the range of 0.1-1.0 ng/ml (4-40 pM) maximally inhibit LPS-induced TIS10/PGS-2 message accumulation. In contrast, neither LPS nor TGF-beta has any effect on the level of PGS-1 (EC 1.14.99.1) message. TGF-beta also attenuates LPS-induced accumulation of unspliced TIS10/PGS-2 transcripts in RAW 264.7 cells, suggesting that this cytokine exerts its effects on TIS10/PGS-2 expression at the transcriptional level. TGF-beta inhibits the LPS-induced accumulation of TIS10/PGS-2 protein and message in cultured murine peritoneal macrophages, as well as in macrophage cell lines.

  2. Arg126 and Asp49 Are Essential for the Catalytic Function of Microsomal Prostaglandin E2 Synthase 1 and Ser127 Is Not

    PubMed Central

    Rafique, Nazmi; Goodman, Michael Christopher; Idborg, Helena; Bergqvist, Filip; Jakobsson, Per-Johan

    2016-01-01

    Introduction Prostaglandins are signaling molecules that regulate different physiological processes, involving allergic and inflammatory responses and cardiovascular control. They are involved in several pathophysiological processes, including inflammation and cancer. The inducible terminal enzyme, microsomal prostaglandin E synthase 1 (MPGES1), catalyses prostaglandin E2 production during inflammation. MPGES1 has therefore been intensively studied as a pharmaceutical target and many competitive inhibitors targeting its active site have been developed. However, little is known about its catalytic mechanism. Aim The objective of this study was to investigate which amino acids play a key role in the catalytic mechanism of MPGES1. Materials and Methods Based on results and predictions from previous structural studies, the amino acid residues Asp49, Arg73, Arg126, and Ser127 were chosen and altered by site-directed mutagenesis. The mutated enzyme variants were cloned and expressed in both the E. coli and the Baculovirus expression systems. Their catalytic significance was evaluated by activity measurements with prostanoid profiling. Results and Conclusions Our study shows that Arg126 and Asp49 are absolutely required for the catalytic activity of MPGES1, as when exchanged, the enzyme variants loose activity. Ser127 and Arg73 on the other hand, don't seem to be central to the catalytic mechanism because when exchanged, their variants retain considerable activity. Our finding that the Ser127Ala variant retains activity was surprising since high-resolution structural data supported a role in glutathione activation. The close proximity of Ser127 to the active site is, however, supported since the Ser127Cys variant displays 80% lowered activity. PMID:27684486

  3. Protection of rabbit lungs from endotoxin injury by in vivo hyperexpression of the prostaglandin G/H synthase gene.

    PubMed Central

    Conary, J T; Parker, R E; Christman, B W; Faulks, R D; King, G A; Meyrick, B O; Brigham, K L

    1994-01-01

    A recombinant prostaglandin G/H (PGH) synthase gene has been expressed in vitro in bovine pulmonary artery endothelial cells and in vivo in rabbits by transfection with a plasmid using cationic liposomes. Transfection of bovine pulmonary artery endothelial cells with the PGH synthase cDNA resulted in increased intracellular PGH synthase protein (determined by Western blot analysis) and increased release of prostacyclin. Rabbits intravenously transfected with the PGH synthase gene had increased plasma levels of prostacyclin and PGE2, and their lungs produced increased amounts of the same eicosanoids. In an in situ, perfused preparation of PGH synthase transfected rabbit lungs, the pressor response to endotoxin was markedly attenuated. In addition, pulmonary edema and release of thromboxane B2 into the perfusate after endotoxin infusion were markedly decreased in transfected lungs compared to controls (animals transfected with a pCMV4 construct that did not contain a cDNA insert). The data suggest that augmented endogenous production of prostacyclin and PGE2, achieved by liposome-mediated gene transfer, protects the lungs from endotoxin. This may be caused in part by suppression of endotoxin-stimulated thromboxane B2 production. Modification of lipid mediator responses by in vivo transfection is a potential approach to the therapy of acute lung injury. Images PMID:8163682

  4. Structural similarity of bovine lung prostaglandin F synthase to lens epsilon-crystallin of the European common frog.

    PubMed Central

    Watanabe, K; Fujii, Y; Nakayama, K; Ohkubo, H; Kuramitsu, S; Kagamiyama, H; Nakanishi, S; Hayaishi, O

    1988-01-01

    Cloned cDNA sequences specific for prostaglandin F (PGF) synthase have been isolated from a cDNA library of bovine lung mRNA sequences. Nucleotide-sequence analyses of cloned cDNA inserts have revealed that PGF synthase consists of a 969-base pair open reading frame coding for a 323-amino acid polypeptide with a Mr of 36,666. The sequence analysis indicates that bovine lung PGF synthase shows 62% identical plus conservative substitutions compared with human liver aldehyde reductase [Wermuth, B., Omar, A., Forster, A., Francesco, C., Wolf, M., Wartburg, J.P., Bullock, B. & Gabbay, K.H. (1987) in Enzymology and Molecular Biology of Carbonyl Metabolism: Aldehyde Dehydrogenase, Aldo-Keto Reductase, and Alcohol Dehydrogenase, eds. Weiner, H. & Flynn, T.G. (Liss, New York), pp. 297-307], which is similar to PGF synthase in molecular weight and substrate specificity. However, comparison of the amino acid sequence of PGF synthase with the National Biomedical Research Foundation protein data base reveals that the sequences of 225 amino acids from C termini of epsilon-crystallin of the European common frog (Rana temporaria) [Tomarev, S.I., Zinovieva, R.D., Dolgilevich, S.M., Luchin, S.V., Krayev, A.S., Skryabin, K.G. & Gause, G.G. (1984) FEBS Lett. 171, 297-302] and of PGF synthase show 77% identical and conservative substitutions without deletions/additions. The result suggests that European common frog lens epsilon-crystallin is identical to bovine lung PGF synthase. Images PMID:2829166

  5. 2.0 Angstrom Structure of Prostaglandin H2 Synthase-1 Reconstituted with a Manganese Porphyrin Cofactor

    SciTech Connect

    Gupta,K.; Selinsky, B.; Loll, P.

    2006-01-01

    Prostaglandin H{sub 2} synthase (EC 1.14.99.1) is a clinically important drug target that catalyzes two key steps in the biosynthesis of the eicosanoid hormones. The enzyme contains spatially distinct cyclooxygenase and peroxidase active sites, both of which require a heme cofactor. Substitution of ferric heme by Mn{sup III} protoporphyrin IX greatly diminishes the peroxidase activity, but has little effect on the cyclooxygenase activity. Here, the 2.0 Angstrom resolution crystal structure of the Mn{sup III} form of ovine prostaglandin H{sub 2} synthase-1 is described (R = 21.8%, R{sub free} = 23.7%). Substitution of Mn{sup III} for Fe{sup III} causes no structural perturbations in the protein. However, the out-of-plane displacement of the manganese ion with respect to the porphyrin is greater than that of the iron by approximately 0.2 Angstroms. This perturbation may help to explain the altered catalytic properties of the manganese enzyme.

  6. New insights into the catalytic mechanism of Bombyx mori prostaglandin E synthase gained from structure–function analysis

    SciTech Connect

    Yamamoto, Kohji; Suzuki, Mamoru; Higashiura, Akifumi; Aritake, Kosuke; Urade, Yoshihiro; Uodome, Nobuko; Hossain, MD. Tofazzal; Nakagawa, Atsushi

    2013-11-01

    Highlights: •Structure of Bombyx mori prostaglandin E synthase is determined. •Bound glutathione sulfonic acid is located at the glutathione-binding site. •Electron-sharing network is present in this protein. •This network includes Asn95, Asp96, and Arg98. •Site-directed mutagenesis reveals that the residues contribute to the catalytic activity. -- Abstract: Prostaglandin E synthase (PGES) catalyzes the isomerization of PGH{sub 2} to PGE{sub 2}. We previously reported the identification and structural characterization of Bombyx mori PGES (bmPGES), which belongs to Sigma-class glutathione transferase. Here, we extend these studies by determining the structure of bmPGES in complex with glutathione sulfonic acid (GTS) at a resolution of 1.37 Å using X-ray crystallography. GTS localized to the glutathione-binding site. We found that electron-sharing network of bmPGES includes Asn95, Asp96, and Arg98. Site-directed mutagenesis of these residues to create mutant forms of bmPGES mutants indicate that they contribute to catalytic activity. These results are, to our knowledge, the first to reveal the presence of an electron-sharing network in bmPGES.

  7. Increased lethality and defective pulmonary clearance of Streptococcus pneumoniae in microsomal prostaglandin E synthase-1-knockout mice.

    PubMed

    Dolan, Jennifer M; Weinberg, Jason B; O'Brien, Edmund; Abashian, Anya; Procario, Megan C; Aronoff, David M; Crofford, Leslie J; Peters-Golden, Marc; Ward, Lindsay; Mancuso, Peter

    2016-06-01

    The production of prostaglandin E2 (PGE2) increases dramatically during pneumococcal pneumonia, and this lipid mediator impairs alveolar macrophage (AM)-mediated innate immune responses. Microsomal prostaglandin E synthase-1 (mPGES-1) is a key enzyme involved in the synthesis of PGE2, and its expression is enhanced during bacterial infections. Genetic deletion of mPGES-1 in mice results in diminished PGE2 production and elevated levels of other prostaglandins after infection. Since PGE2 plays an important immunoregulatory role during bacterial pneumonia we assessed the impact of mPGES-1 deletion in the host defense against pneumococcal pneumonia in vivo and in AMs in vitro. Wild-type (WT) and mPGES-1 knockout (KO) mice were challenged with Streptococcus pneumoniae via the intratracheal route. Compared with WT animals, we observed reduced survival and increased lung and spleen bacterial burdens in mPGES-1 KO mice 24 and 48 h after S. pneumoniae infection. While we found modest differences between WT and mPGES-1 KO mice in pulmonary cytokines, AMs from mPGES-1 KO mice exhibited defective killing of ingested bacteria in vitro that was associated with diminished inducible nitric oxide synthase expression and reduced nitric oxide (NO) synthesis. Treatment of AMs from mPGES-1 KO mice with an NO donor restored bacterial killing in vitro. These results suggest that mPGES-1 plays a critical role in bacterial pneumonia and that genetic ablation of this enzyme results in diminished pulmonary host defense in vivo and in vitro. These results suggest that specific inhibition of PGE2 synthesis by targeting mPGES-1 may weaken host defense against bacterial infections. PMID:27059285

  8. Inhibitors of glycogen synthase 3 kinase

    DOEpatents

    Schultz, Peter; Ring, David B.; Harrison, Stephen D.; Bray, Andrew M.

    2006-05-30

    Compounds of formula 1: ##STR00001## wherein R.sub.1 is alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl, substituted with 0 3 substituents selected from lower alkyl, halo, hydroxy, lower alkoxy, amino, lower alkyl-amino, and nitro; R.sub.2 is hydroxy, amino, or lower alkoxy; R.sub.3 is H, lower alkyl, lower acyl, lower alkoxy-acyl, or amino-acyl; R.sub.4 is H or lower alkyl; and pharmaceutically acceptable salts and esters thereof; are effective inhibitors of GSK3.

  9. Inhibitors of glycogen synthase 3 kinase

    DOEpatents

    Schultz, Peter; Ring, David B.; Harrison, Stephen D.; Bray, Andrew M.

    2000-01-01

    Compounds of formula 1: ##STR1## wherein R.sub.1 is alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl, substituted with 0-3 substituents selected from lower alkyl, halo, hydroxy, lower alkoxy, amino, lower alkyl-amino, and nitro; R.sub.2 is hydroxy, amino, or lower alkoxy; R.sub.3 is H, lower alkyl, lower acyl, lower alkoxy-acyl, or amnino-acyl; R.sub.4 is H or lower alkyl; and pharmaceutically acceptable salts and esters thereof; are effective inhibitors of GSK3.

  10. Seminal plasma induces prostaglandin-endoperoxide synthase (PTGS) 2 expression in immortalized human vaginal cells: involvement of semen prostaglandin E2 in PTGS2 upregulation.

    PubMed

    Joseph, Theresa; Zalenskaya, Irina A; Sawyer, Lyn C; Chandra, Neelima; Doncel, Gustavo F

    2013-01-01

    Inflammation of the cervicovaginal mucosa is considered a risk factor for HIV infection in heterosexual transmission. In this context, seminal plasma (SP) may play an important role that is not limited to being the main carrier for the virions. It is known that SP induces an inflammatory reaction in the cervix called postcoital leukocytic reaction, which has been associated with promotion of fertility. The mechanisms by which SP triggers this reaction, however, have not been clearly established. Previously we reported the expression of prostaglandin-endoperoxide synthase 2 (PTGS2), also known as cyclooxygenase 2 (COX-2), in human vaginal cells in response to toll-like receptor (TLR) ligands and other proinflammatory stimuli. In this study, we demonstrate that SP induces transcriptional and translational increase of COX-2 expression in human vaginal cells and cervicovaginal tissue explants. Furthermore, SP potentiates vaginal PTGS2 expression induced by other proinflammatory stimulants, such as TLR ligands and a vaginal mucosal irritant (nonoxynol-9) in a synergistic manner. SP-induced PTGS2 expression is mediated by intracellular signaling pathways involving MAPKs and NF-κB. Using fractionation and functional analysis, seminal prostaglandin (PG)-E(2) was identified as a one of the major factors in PTGS2 induction. Given the critical role of this PG-producing enzyme in mucosal inflammatory processes, the finding that SP induces and potentiates the expression of PTGS2 in cervicovaginal cells and tissues has mechanistic implications for the role of SP in fertility-associated mucosal leukocytic reaction and its potential HIV infection-enhancing effect. PMID:23153564

  11. The Prostaglandin F Synthase Activity of the Human Aldose Reductase AKR1B1 Brings New Lenses to Look at Pathologic Conditions

    PubMed Central

    Bresson, Eva; Lacroix-Pépin, Nicolas; Boucher-Kovalik, Sofia; Chapdelaine, Pierre; Fortier, Michel A.

    2012-01-01

    Prostaglandins are important regulators of female reproductive functions to which aldose reductases exhibiting hydroxysteroid dehydrogenase activity also contribute. Our work on the regulation of reproductive function by prostaglandins (PGs), lead us to the discovery that AKR1B5 and later AKR1B1were highly efficient and physiologically relevant PGF synthases. PGE2 and PGF2α are the main prostanoids produced in the human endometrium and proper balance in their relative production is important for normal menstruation and optimal fertility. Recent evidence suggests that PGE2/EP2 and PGF2α/FP may constitute a functional dyad with physiological relevance comparable to the prostacyclin-thromboxane dyad in the vascular system. We have recently reported that AKR1B1 was expressed and modulated in association with PGF2α production in response to IL-1β in the human endometrium. In the present study, we show that the human AKR1B1 (gene ID: 231) also known as ALDR1 or ALR2 is a functional PGF2α synthase in different models of living cells and tissues. Using human endometrial cells, prostate, and vascular smooth muscle cells, cardiomyocytes and endothelial cells we demonstrate that IL-1β is able to up regulate COX-2 and AKR1B1 proteins as well as PGF2α production under normal glucose concentrations. We show that the promoter activity of AKR1B1 gene is increased by IL-1β particularly around the multiple stress response region containing two putative antioxidant response elements adjacent to TonE and AP1. We also show that AKR1B1 is able to regulate PGE2 production through PGF2α acting on its FP receptor and that aldose reductase inhibitors like alrestatin, Statil (ponalrestat), and EBPC exhibit distinct and characteristic inhibition of PGF2α production in different cell models. The PGF synthase activity of AKR1B1 represents a new and important target to regulate ischemic and inflammatory responses associated with several human pathologies. PMID:22654757

  12. Bioactivation of aflatoxin B1 by lipoxygenases, prostaglandin H synthase and cytochrome P450 monooxygenase in guinea-pig tissues.

    PubMed

    Liu, L; Massey, T E

    1992-04-01

    In the present investigation, we have examined the role of lipoxygenases in the bioactivation of aflatoxin B1 (AFB1) in hepatic and extrahepatic tissues. The enzyme activities were evaluated by determining [3H]AFB1-DNA adduct formation. The results demonstrated that both purified soybean lipoxygenase and guinea-pig tissue cytosolic lipoxygenases were able to activate AFB1 to form [3H]AFB1-DNA adduct(s). The reaction was completely inhibited by nordihydroguaiaretic acid (NDGA, 0.1 mM), a lipoxygenase inhibitor and an antioxidant, but not by indomethacin (0.1 mM), an inhibitor of prostaglandin H synthase (PHS), indicating that this reaction is associated with lipoxygenase activity, and/or is involved in a peroxyl radical process. While purified lipoxygenase showed arachidonic acid (AA)-dependent properties, the omission of AA did not diminish guinea-pig tissue cytosolic [3H]AFB1-DNA adduct formation, possibly because AA was released from lipid particles by AFB1. Within the range of hemoglobin (Hb) concentrations found in lung, kidney and liver cytosols (1.4-11.1 microM) and microsomes (0-0.5 microM), neither pure Hb, nor Hb of cytosols or microsomes from whole blood caused detectable AA-dependent AFB1-DNA binding. This indicates that Hb, as a contaminant with quasi-lipoxygenase activity, did not contribute to AFB1 activation attributed to guinea-pig tissue lipoxygenases. [3H]AFB1 concentrations at half-maximal DNA binding rate of pulmonary cytochrome P450 monooxygenases (P450) and lipoxygenases were similar, though P450 had a much higher maximum DNA binding rate. Pulmonary microsomal PHS activity for AFB1 activation was too low for its half-maximal binding concentrations of [3H]AFB1 and maximum rate to be accurately determined. In kidney, maximum rates for lipoxygenase, PHS and P450 were similar, whereas half-maximal binding concentrations for reactions by lipoxygenase and P450 were lower compared to that of PHS. The half-maximal binding concentration of hepatic

  13. Farnesyl Diphosphate Synthase Inhibitors With Unique Ligand-Binding Geometries

    PubMed Central

    2015-01-01

    Farnesyl diphosphate synthase (FPPS) is an important drug target for bone resorption, cancer, and some infectious diseases. Here, we report five new structures including two having unique bound ligand geometries. The diamidine inhibitor 7 binds to human FPPS close to the homoallylic (S2) and allosteric (S3) sites and extends into a new site, here called S4. With the bisphosphonate inhibitor 8, two molecules bind to Trypanosoma brucei FPPS, one molecule in the allylic site (S1) and the other close to S2, the first observation of two bisphosphonate molecules bound to FPPS. We also report the structures of apo-FPPS from T. brucei, together with two more bisphosphonate-bound structures (2,9), for purposes of comparison. The diamidine structure is of particular interest because 7 could represent a new lead for lipophilic FPPS inhibitors, while 8 has low micromolar activity against T. brucei, the causative agent of human African trypanosomiasis. PMID:25815158

  14. Fine-tuned broad binding capability of human lipocalin-type prostaglandin D synthase for various small lipophilic ligands.

    PubMed

    Kume, Satoshi; Lee, Young-Ho; Nakatsuji, Masatoshi; Teraoka, Yoshiaki; Yamaguchi, Keisuke; Goto, Yuji; Inui, Takashi

    2014-03-18

    The hydrophobic cavity of lipocalin-type prostaglandin D synthase (L-PGDS) has been suggested to accommodate various lipophilic ligands through hydrophobic effects, but its energetic origin remains unknown. We characterized 18 buffer-independent binding systems between human L-PGDS and lipophilic ligands using isothermal titration calorimetry. Although the classical hydrophobic effect was mostly detected, all complex formations were driven by favorable enthalpic gains. Gibbs energy changes strongly correlated with the number of hydrogen bond acceptors of ligand. Thus, the broad binding capability of L-PGDS for ligands should be viewed as hydrophilic interactions delicately tuned by enthalpy-entropy compensation using combined effects of hydrophilic and hydrophobic interactions. PMID:24530534

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

    PubMed

    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. PMID:19929706

  16. Aldosterone synthase inhibitors in hypertension: current status and future possibilities

    PubMed Central

    Hargovan, Milan

    2014-01-01

    The renin-angiotensin aldosterone system is a critical mechanism for controlling blood pressure, and exerts most of its physiological effects through the action of angiotensin II. In addition to increasing blood pressure by increasing vascular resistance, angiotensin II also stimulates aldosterone secretion from the adrenal gland. Aldosterone acts to cause an increase in sodium and water reabsorption, thus elevating blood pressure. Although treatment with angiotensin converting enzyme inhibitors initially lowers circulating aldosterone, with chronic treatment aldosterone levels increase back to baseline, a phenomenon termed aldosterone escape; aldosterone blockade may therefore give added value in the treatment of hypertension. The first mineralocorticoid receptor antagonist developed was spironolactone, but its use has been severely hampered by adverse (notably oestrogenic) effects. The more recently developed mineralocorticoid receptor antagonist eplerenone exhibits a better adverse effect profile, although it is not devoid of effects similar to spironolactone. In addition, aldosterone activates non-genomic receptors that are not inhibited by either eplerenone or spironolactone. It is believed that deleterious organ remodelling is mediated by aldosterone via such non-genomic pathways. A new class of drugs, the aldosterone synthase inhibitors, is currently under development. These may offer a novel therapeutic approach for both lowering blood pressure and preventing the non-genomic effects of aldosterone. Here, we will review the cardiovascular effects of aldosterone and review the drugs available that target this hormone, with a particular focus on the aldosterone synthase inhibitors. PMID:24570839

  17. Pheophytin a Inhibits Inflammation via Suppression of LPS-Induced Nitric Oxide Synthase-2, Prostaglandin E2, and Interleukin-1β of Macrophages

    PubMed Central

    Lin, Chun-Yu; Lee, Chien-Hsing; Chang, Yu-Wei; Wang, Hui-Min; Chen, Chung-Yi; Chen, Yen-Hsu

    2014-01-01

    Inflammation is a serious health issue worldwide that induces many diseases such as sepsis. There has been a vast search for potentially effective drugs to decrease mortality from sepsis. Pheophytin a is a chlorophyll-related compound derived from green tea. We found that pre-treatment with pheophytin a suppressed lipopolysaccharide (LPS)-induced nitric oxide (NO), prostaglandin E2 (PGE2), and interleukin-1β in RAW 264.7 macrophages. NO synthase-2 (NOS2) and cyclooxygenase-2 (COX-2) expression levels were repressed by pre-treatment with pheophytin a at both the transcriptional and translational levels. Pheophytin a inhibited NOS2 promoter activity, but not its mRNA stability, through extracellular signal-regulated kinase (ERK1/2). This suppression was reversed by ERK1/2 inhibitor (U0126). Pheophytin a reduced signal transducers and activators of transcription 1 (STAT-1) activation, without an obvious influence on activator protein-1 (AP-1) and nuclear factor κB (NF-κB). These results suggest that pheophytin a functions by down-regulating the transcriptional levels of inflammatory mediators and blocking the ERK and STAT-1 pathways. PMID:25501336

  18. Embryopathic effects of thalidomide and its hydrolysis products in rabbit embryo culture: evidence for a prostaglandin H synthase (PHS)-dependent, reactive oxygen species (ROS)-mediated mechanism.

    PubMed

    Lee, Crystal J J; Gonçalves, Luisa L; Wells, Peter G

    2011-07-01

    Thalidomide (TD) causes birth defects in humans and rabbits via several potential mechanisms, including bioactivation by embryonic prostaglandin H synthase (PHS) enzymes to a reactive intermediate that enhances reactive oxygen species (ROS) formation. We show herein that TD in rabbit embryo culture produces relevant embryopathies, including decreases in head/brain development by 28% and limb bud growth by 71% (P<0.05). Two TD hydrolysis products, 2-phthalimidoglutaramic acid (PGMA) and 2-phthalimidoglutaric acid (PGA), were similarly embryopathic, attenuating otic vesicle (ear) and limb bud formation by up to 36 and 77%, respectively (P<0.05). TD, PGMA, and PGA all increased embryonic DNA oxidation measured as 8-oxoguanine (8-oxoG) by up to 2-fold (P<0.05). Co- or pretreatment with the PHS inhibitors eicosatetraynoic acid (ETYA) or acetylsalicylic acid (ASA), or the free-radical spin trap phenylbutylnitrone (PBN), completely blocked embryonic 8-oxoG formation and/or embryopathies initiated by TD, PGMA, and PGA. This is the first demonstration of limb bud embryopathies initiated by TD, as well as its hydrolysis products, in a mammalian embryo culture model of a species susceptible to TD in vivo, indicating that all likely contribute to TD teratogenicity in vivo, in part through PHS-dependent, ROS-mediated mechanisms.

  19. Catalytic mechanism of the primary human prostaglandin F2α synthase, aldo-keto reductase 1B1--prostaglandin D2 synthase activity in the absence of NADP(H).

    PubMed

    Nagata, Nanae; Kusakari, Yukiko; Fukunishi, Yoshifumi; Inoue, Tsuyoshi; Urade, Yoshihiro

    2011-04-01

    Aldo-keto reductase 1B1 and 1B3 (AKR1B1 and AKR1B3) are the primary human and mouse prostaglandin F(2α) (PGF(2α)) synthases, respectively, which catalyze the NADPH-dependent reduction of PGH(2), a common intermediate of various prostanoids, to form PGF(2α). In this study, we found that AKR1B1 and AKR1B3, but not AKR1B7 and AKR1C3, also catalyzed the isomerization of PGH(2) to PGD(2) in the absence of NADPH or NADP(+). Both PGD(2) and PGF(2α) synthase activities of AKR1B1 and AKR1B3 completely disappeared in the presence of NADP(+) or after heat treatment of these enzymes at 100 °C for 5 min. The K(m), V(max), pK and optimum pH values of the PGD(2) synthase activities of AKR1B1 and AKR1B3 were 23 and 18 μM, 151 and 57 nmol·min(-1)·(mg protein)(-1), 7.9 and 7.6, and pH 8.5 for both AKRs, respectively, and those of PGF(2α) synthase activity were 29 and 33 μM, 169 and 240 nmol·min(-1)·(mg protein)(-1), 6.2 and 5.4, and pH 5.5 and pH 5.0, respectively, in the presence of 0.5 mm NADPH. Site-directed mutagenesis of the catalytic tetrad of AKR1B1, composed of Tyr, Lys, His and Asp, revealed that the triad of Asp43, Lys77 and His110, but not Tyr48, acts as a proton donor in most AKR activities, and is crucial for PGD(2) and PGF(2α) synthase activities. These results, together with molecular docking simulation of PGH(2) to the crystallographic structure of AKR1B1, indicate that His110 acts as a base in concert with Asp43 and Lys77 and as an acid to generate PGD(2) and PGF(2α) in the absence of NADPH or NADP(+) and in the presence of NADPH, respectively.

  20. Structure-based design of bacterial nitric oxide synthase inhibitors.

    PubMed

    Holden, Jeffrey K; Kang, Soosung; Hollingsworth, Scott A; Li, Huiying; Lim, Nathan; Chen, Steven; Huang, He; Xue, Fengtian; Tang, Wei; Silverman, Richard B; Poulos, Thomas L

    2015-01-22

    Inhibition of bacterial nitric oxide synthase (bNOS) has the potential to improve the efficacy of antimicrobials used to treat infections by Gram-positive pathogens Staphylococcus aureus and Bacillus anthracis. However, inhibitor specificity toward bNOS over the mammalian NOS (mNOS) isoforms remains a challenge because of the near identical NOS active sites. One key structural difference between the NOS isoforms is the amino acid composition of the pterin cofactor binding site that is adjacent to the NOS active site. Previously, we demonstrated that a NOS inhibitor targeting both the active and pterin sites was potent and functioned as an antimicrobial ( Holden , , Proc. Natl. Acad. Sci. U.S.A. 2013 , 110 , 18127 ). Here we present additional crystal structures, binding analyses, and bacterial killing studies of inhibitors that target both the active and pterin sites of a bNOS and function as antimicrobials. Together, these data provide a framework for continued development of bNOS inhibitors, as each molecule represents an excellent chemical scaffold for the design of isoform selective bNOS inhibitors.

  1. Structure-Based Discovery of Inhibitors of Thymidylate Synthase

    NASA Astrophysics Data System (ADS)

    Shoichet, Brian K.; Stroud, Robert M.; Santi, Daniel V.; Kuntz, Irwin D.; Perry, Kathy M.

    1993-03-01

    A molecular docking computer program (DOCK) was used to screen the Fine Chemical Directory, a database of commercially available compounds, for molecules that are complementary to thymidylate synthase (TS), a chemotherapeutic target. Besides retrieving the substrate and several known inhibitors, DOCK proposed putative inhibitors previously unknown to bind to the enzyme. Three of these compounds inhibited Lactobacillus caser TS at submillimolar concentrations. One of these inhibitors, sulisobenzone, crystallized with TS in two configurations that differed from the DOCK-favored geometry: a counterion was bound in the substrate site, which resulted in a 6 to 9 angstrom displacement of the inhibitor. The structure of the complexes suggested another binding region in the active site that could be exploited. This region was probed with molecules sterically similar to sulisobenzone, which led to the identification of a family of phenolphthalein analogs that inhibit TS in the 1 to 30 micromolar range. These inhibitors do not resemble the substrates of the enzyme. A crystal structure of phenolphthalein with TS shows that it binds in the target site in a configuration that resembles the one suggested by DOCK.

  2. Structure-Based Design of Bacterial Nitric Oxide Synthase Inhibitors

    PubMed Central

    2015-01-01

    Inhibition of bacterial nitric oxide synthase (bNOS) has the potential to improve the efficacy of antimicrobials used to treat infections by Gram-positive pathogens Staphylococcus aureus and Bacillus anthracis. However, inhibitor specificity toward bNOS over the mammalian NOS (mNOS) isoforms remains a challenge because of the near identical NOS active sites. One key structural difference between the NOS isoforms is the amino acid composition of the pterin cofactor binding site that is adjacent to the NOS active site. Previously, we demonstrated that a NOS inhibitor targeting both the active and pterin sites was potent and functioned as an antimicrobial (Holden, , Proc. Natl. Acad. Sci. U.S.A.2013, 110, 1812724145412). Here we present additional crystal structures, binding analyses, and bacterial killing studies of inhibitors that target both the active and pterin sites of a bNOS and function as antimicrobials. Together, these data provide a framework for continued development of bNOS inhibitors, as each molecule represents an excellent chemical scaffold for the design of isoform selective bNOS inhibitors. PMID:25522110

  3. Structural Studies of Pterin-Based Inhibitors of Dihydropteroate Synthase

    SciTech Connect

    Hevener, Kirk E.; Yun, Mi-Kyung; Qi, Jianjun; Kerr, Iain D.; Babaoglu, Kerim; Hurdle, Julian G.; Balakrishna, Kanya; White, Stephan W.; Lee, Richard E.

    2010-01-12

    Dihydropteroate synthase (DHPS) is a key enzyme in bacterial folate synthesis and the target of the sulfonamide class of antibacterials. Resistance and toxicities associated with sulfonamides have led to a decrease in their clinical use. Compounds that bind to the pterin binding site of DHPS, as opposed to the p-amino benzoic acid (pABA) binding site targeted by the sulfonamide agents, are anticipated to bypass sulfonamide resistance. To identify such inhibitors and map the pterin binding pocket, we have performed virtual screening, synthetic, and structural studies using Bacillus anthracis DHPS. Several compounds with inhibitory activity have been identified, and crystal structures have been determined that show how the compounds engage the pterin site. The structural studies identify the key binding elements and have been used to generate a structure-activity based pharmacophore map that will facilitate the development of the next generation of DHPS inhibitors which specifically target the pterin site.

  4. Inhibitors to Polyhydroxyalkanoate (PHA) Synthases: Synthesis, Molecular Docking, and Implications

    PubMed Central

    Cao, Ruikai; Maurmann, Leila; Li, Ping

    2015-01-01

    Polyhydroxyalkanoate (PHA) synthases (PhaCs) catalyze the formation of biodegradable PHAs that are considered as an ideal alternative to nonbiodegradable synthetic plastics. However, study of PhaC has been challenging because the rate of PHA chain elongation is much faster than that of initiation. This difficulty along with lack of a structure has become the main hurdle to understand and engineer PhaCs for economical PHA production. Here we reported the synthesis of two carbadethia CoA analogs, sT-CH2-CoA 26a and sTet-CH2-CoA 26b as well as sT-aldehyde 29 as new PhaC inhibitors. Study of these analogs with PhaECAv revealed that 26a/b and 29 are competitive and mixed inhibitors, respectively. It was observed that CoA moiety and PHA chain extension can increase binding affinity, which is consistent with the docking study. Estimation from Kic of 26a/b predicts that a CoA analog attached with an octameric-HB chain may facilitate the formation of a kinetically well-behaved synthase. PMID:25394180

  5. Inhibitors of polyhydroxyalkanoate (PHA) synthases: synthesis, molecular docking, and implications.

    PubMed

    Zhang, Wei; Chen, Chao; Cao, Ruikai; Maurmann, Leila; Li, Ping

    2015-01-01

    Polyhydroxyalkanoate (PHA) synthases (PhaCs) catalyze the formation of biodegradable PHAs that are considered to be ideal alternatives to non-biodegradable synthetic plastics. However, study of PhaCs has been challenging because the rate of PHA chain elongation is much faster than that of initiation. This difficulty, along with lack of a crystal structure, has become the main hurdle to understanding and engineering PhaCs for economical PHA production. Here we report the synthesis of two carbadethia CoA analogues--sT-CH2-CoA (26 a) and sTet-CH2-CoA (26 b)--as well as sT-aldehyde (saturated trimer aldehyde, 29), as new PhaC inhibitors. Study of these analogues with PhaECAv revealed that 26 a/b and 29 are competitive and mixed inhibitors, respectively. Both the CoA moiety and extension of PHA chain will increase binding affinity; this is consistent with our docking study. Estimation of the Kic values of 26 a and 26 b predicts that a CoA analogue incorporating an octameric hydroxybutanoate (HB) chain might facilitate the formation of a kinetically well-behaved synthase.

  6. Glycogen synthase kinase-3 inhibitors: Rescuers of cognitive impairments

    PubMed Central

    King, Margaret K.; Pardo, Marta; Cheng, Yuyan; Downey, Kimberlee; Jope, Richard S.; Beurel, Eléonore

    2013-01-01

    Impairment of cognitive processes is a devastating outcome of many diseases, injuries, and drugs affecting the central nervous system (CNS). Most often, very little can be done by available therapeutic interventions to improve cognitive functions. Here we review evidence that inhibition of glycogen synthase kinase-3 (GSK3) ameliorates cognitive deficits in a wide variety of animal models of CNS diseases, including Alzheimer's disease, Fragile X syndrome, Down syndrome, Parkinson's disease, spinocerebellar ataxia type 1, traumatic brain injury, and others. GSK3 inhibitors also improve cognition following impairments caused by therapeutic interventions, such as cranial irradiation for brain tumors. These findings demonstrate that GSK3 inhibitors are able to ameliorate cognitive impairments caused by a diverse array of diseases, injury, and treatments. The improvements in impaired cognition instilled by administration of GSK3 inhibitors appear to involve a variety of different mechanisms, such as supporting long-term potentiation and diminishing long-term depression, promotion of neurogenesis, reduction of inflammation, and increasing a number of neuroprotective mechanisms. The potential for GSK3 inhibitors to repair cognitive deficits associated with many conditions warrants further investigation of their potential for therapeutic interventions, particularly considering the current dearth of treatments available to reduce loss of cognitive functions. PMID:23916593

  7. Low survival of mice following lethal gamma-irradiation after administration of inhibitors of prostaglandin synthesis.

    PubMed

    Hofer, M; Pospísil, M; Tkadlecek, L; Viklická, S; Pipalová, I; Holá, J

    1992-01-01

    An impairment of the survival of mice subjected to whole-body gamma-irradiation with a lethal dose of 10 Gy and treated with a repeated postirradiation administration of prostaglandin synthesis inhibitors (PGSIs), indomethacin or diclofenac, was observed. Morphological examination of the gastrointestinal tract and the estimation of blood loss into its lumen in animals treated with diclofenac did not show serious damage such as haemorrhages or perforation, but revealed structural injury to the intestinal mucosa indicating inflammatory processes. The lesions found are supposed to be connected with increased intestinal permeability which leads to endotoxin escape from the gut and a subsequent increased mortality rate of irradiated animals. It may be concluded that PGSIs are not suitable for the management of radiation sickness after an exposure to lethal doses of ionizing radiation.

  8. Canine placental prostaglandin E2 synthase: expression, localization, and biological functions in providing substrates for prepartum PGF2alpha synthesis.

    PubMed

    Gram, Aykut; Fox, Barbara; Büchler, Urs; Boos, Alois; Hoffmann, Bernd; Kowalewski, Mariusz P

    2014-12-01

    The prepartum output of PGF2alpha in the bitch is associated with increased placental PGE2-synthase (PTGES) mRNA levels. Contrasting with this is a decreased expression of PGF2alpha-synthase (PGFS/AKR1C3) in uteroplacental compartments during prepartum luteolysis, suggesting an involvement of alternative synthetic pathways in PGF2alpha synthesis, for example, conversion of PGE2 to PGF2alpha. However, because the expression and possible functions of the respective PTGES proteins remained unknown, no further conclusion could be drawn. Therefore, a canine-specific PTGES antibody was generated and used to investigate the expression, cellular localization, and biochemical activities of canine uteroplacental PTGES throughout pregnancy and at prepartum luteolysis. Additionally, the biochemical activities of these tissues involved in the conversion of PGE2 to PGF2alpha were investigated. The endometrial PTGES was localized in the uterine surface epithelium at preimplantation and in superficial and deep uterine glands, endothelial cells, and myometrium throughout pregnancy and at parturition. Placental signals were mostly in the trophoblast. The biochemical properties of recombinant PTGES protein were confirmed. Additionally, expression of two PGE2-receptors, PTGER2/EP2 and PTGER4/EP4, revealed their decreasing expression during luteolysis. In contrast, the uteroplacental expression of prostaglandin transporter (PGT) was strongly elevated prior to parturition. These localization patterns resembled that of PTGES. The increased expression of PTGES and PGT at parturition, together with the accompanying decreased levels of PGE2-receptors and the capability of canine uterine and placental homogenates to take part in the conversion of PGE2 to PGF2alpha, as found in this study, suggest that PGE2 could be used locally as a substrate for prepartum PGF2alpha synthesis in the dog.

  9. Modulation of lipocalin-type prostaglandin D2 synthase expression in catfish seminal vesicles by thyroid disrupting agents and hormones.

    PubMed

    Sreenivasulu, Gunti; Pavani, Ayinampudi; Sudhakumari, Cheni-Chery; Dutta-Gupta, Aparna; Senthilkumaran, Balasubramanian

    2013-11-01

    Thyroid hormones play crucial role in several biological processes including reproduction. Disruption of normal thyroid status by environmental contaminants can cause severe impairment in reproductive functions. In our previous study, we reported down-regulation of a protein in seminal vesicular fluid of air-breathing catfish, Clarias gariepinus during experimentally induced hyperthyroidism. N-terminal amino acid sequence analysis followed by search in sequence database denoted it to be lipocalin-type prostaglandin D2 synthase (ptgds-b). In the present study, we cloned full-length cDNA of ptgds-b based on the N-terminal amino acid sequence. Surprisingly, Northern blot as well as RT-PCR analysis demonstrated the presence of ptgds-b transcript predominantly in seminal vesicles and developing testis. Further, ptgds-b mRNA significantly decreased in seminal vesicles following L-thyroxine overdose while there was an increased expression of ptgds-b after depletion of thyroid hormone by thiourea and withdrawal of the treatments reverted this effect. Treatment of catfish with human chorionic gonadotropin and estradiol significantly reduced ptgds-b expression. Taken together, we report ptgds-b as a thyroid hormone regulated protein in the seminal vesicles in addition to gonadotropin and estradiol. Further studies might explain the exclusive presence of ptgds-b in seminal vesicles and developing testis yet present data evaluated it as a putative biomarker for thyroid hormone disruption.

  10. L-NAME, a nitric oxide synthase inhibitor, as a potential countermeasure to post-suspension hypotension in rats

    NASA Technical Reports Server (NTRS)

    Bayorh, M. A.; Socci, R. R.; Watts, S.; Wang, M.; Eatman, D.; Emmett, N.; Thierry-Palmer, M.

    2001-01-01

    A large number of astronauts returning from spaceflight experience orthostatic hypotension. This hypotension may be due to overproduction of vasodilatory mediators, such as nitric oxide (NO) and prostaglandins. To evaluate the role of the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) as a countermeasure against the post-suspension reduction in mean arterial pressure (MAP), we assessed the cardiovascular responses and vascular reactivity to 7-day 30 degrees tail-suspension and a subsequent 6 hr post-suspension period in conscious rats. After a pre-suspension reading, direct MAP and heart rate (HR) were measured daily and every 2 hrs post-suspension. The NO synthase inhibitor L-NAME (20 mg/kg, i.v.), or saline, were administered after the 7th day reading prior to release from suspension and at 2 and 4 hrs post-suspension. At 6 hrs post-suspension, vascular reactivity was assessed. While MAP did not change during the suspension period, it was reduced post-suspension. Heart rate was not significantly altered. L-NAME administration reversed the post-suspension reduction in MAP. In addition, the baroreflex sensitivity for heart rate was modified by L-NAME. Thus, the post-suspension reduction in MAP may be due to overproduction of NO and altered baroreflex activity.

  11. Stable expression of lipocalin-type prostaglandin D synthase in cultured preadipocytes impairs adipogenesis program independently of endogenous prostanoids

    SciTech Connect

    Hossain, Mohammad Salim; Chowdhury, Abu Asad; Rahman, Mohammad Sharifur; Nishimura, Kohji; Jisaka, Mitsuo; Nagaya, Tsutomu; Shono, Fumiaki; Yokota, Kazushige

    2012-02-15

    Lipocalin-type prostaglandin D synthase (L-PGDS) expressed preferentially in adipocytes is responsible for the synthesis of PGD{sub 2} and its non-enzymatic dehydration products, PGJ{sub 2} series, serving as pro-adipogenic factors. However, the role of L-PGDS in the regulation of adipogenesis is complex because of the occurrence of several derivatives from PGD{sub 2} and their distinct receptor subtypes as well as other functions such as a transporter of lipophilic molecules. To manipulate the expression levels of L-PGDS in cultured adipocytes, cultured preadipogenic 3T3-L1 cells were transfected stably with a mammalian expression vector having cDNA encoding murine L-PGDS oriented in the sense direction. The isolated cloned stable transfectants with L-PGDS expressed higher levels of the transcript and protein levels of L-PGDS, and synthesized PGD{sub 2} from exogenous arachidonic acid at significantly higher levels. By contrast, the synthesis of PGE{sub 2} remained unchanged, indicating no influence on the reactions of cyclooxygenase (COX) and PGE synthase. Furthermore, the ability of those transfectants to synthesize {Delta}{sup 12}-PGJ{sub 2} increased more greatly during the maturation phase. The sustained expression of L-PGDS in cultured stable transfectants hampered the storage of fats during the maturation phase of adipocytes, which was accompanied by the reduced gene expression of adipocyte-specific markers reflecting the down-regulation of the adipogenesis program. The suppressed adipogenesis was not rescued by either exogenous aspirin or peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) agonists including troglitazone and {Delta}{sup 12}-PGJ{sub 2}. Taken together, the results indicate the negative regulation of the adipogenesis program by the enhanced expression of L-PGDS through a cellular mechanism involving the interference of the PPAR{gamma} signaling pathway without the contribution of endogenous pro-adipogenic prostanoids

  12. A nitric oxide synthase inhibitor impairs memory storage in mice.

    PubMed

    Baratti, C M; Kopf, S R

    1996-05-01

    Posttraining administration of the L-enantiomer of the competitive inhibitor of nitric oxide synthase, NG-nitro-L-arginine methyl ester (L-NAME, 3-100 mg/kg, ip), impaired 48-h retention of a one-trial step-through inhibitory shock-avoidance task in male Swiss mice. The effects were dose-dependent and were not observed when the D-enantiomer (D-NAME, 3-100 mg/kg, ip) was injected instead of L-NAME. Retention latencies of mice that had not received a footshock during training were not affected by L-NAME. The memory impairment produced by L-NAME was time-dependent, suggesting an action on memory storage. The effects of L-NAME on memory were overcome by the injection of L-(but not D-)arginine (300 mg/kg, ip) along with the inhibitor. Considered together, these findings suggest that the L-arginine/nitric oxide pathway may be involved in memory storage of an inhibitory avoidance response in mice. PMID:8616582

  13. A nitric oxide synthase inhibitor impairs memory storage in mice.

    PubMed

    Baratti, C M; Kopf, S R

    1996-05-01

    Posttraining administration of the L-enantiomer of the competitive inhibitor of nitric oxide synthase, NG-nitro-L-arginine methyl ester (L-NAME, 3-100 mg/kg, ip), impaired 48-h retention of a one-trial step-through inhibitory shock-avoidance task in male Swiss mice. The effects were dose-dependent and were not observed when the D-enantiomer (D-NAME, 3-100 mg/kg, ip) was injected instead of L-NAME. Retention latencies of mice that had not received a footshock during training were not affected by L-NAME. The memory impairment produced by L-NAME was time-dependent, suggesting an action on memory storage. The effects of L-NAME on memory were overcome by the injection of L-(but not D-)arginine (300 mg/kg, ip) along with the inhibitor. Considered together, these findings suggest that the L-arginine/nitric oxide pathway may be involved in memory storage of an inhibitory avoidance response in mice.

  14. Transforming growth factor beta 1 augments mitogen-induced prostaglandin synthesis and expression of the TIS10/prostaglandin synthase 2 gene both in Swiss 3T3 cells and in murine embryo fibroblasts.

    PubMed

    Gilbert, R S; Reddy, S T; Kujubu, D A; Xie, W; Luner, S; Herschman, H R

    1994-04-01

    Transforming growth factor-beta (TGF-beta), a potent cytokine, modulates a wide variety of biological responses. Among its actions, TGF-beta can augment prostaglandin synthesis in several cell types. Although TGF-beta alone has no effect on prostaglandin production in Swiss 3T3 cells, we find that TGF-beta augments the ability of tetradecanoyl phorbol acetate (TPA) or serum to stimulate PGE2 production. The TIS10 gene is a primary response gene encoding a second form of prostaglandin synthase (PGS), the rate-limiting enzyme in the biosynthesis of prostaglandins, thromboxanes, and prostacyclins from arachidonic acid. TIS10/PGS-2 expression is induced by mitogens in Swiss 3T3 cells. TGF-beta also augments mitogen-induced synthesis and accumulation of TIS10/PGS-2 protein and induction of TIS10/PGS-2 message in Swiss 3T3 cells. In contrast, TGF-beta has little or no effect on the level of PGS-1 (EC1.14.99.1) message, either alone or in concert with TPA or serum. TGF-beta concentrations in the range of 0.01-0.10 ng/ml (0.4-4.0 pM) maximally enhance mitogen induction of TIS10/PGS-2 message. TPA-induced accumulation of unspliced TIS10/PGS-2 transcript is augmented by TGF-beta, suggesting that this cytokine exerts its effect on expression of the TIS10/PGS-2 gene by transcriptional regulation. TGF-beta also augments TPA-induced prostaglandin production, TIS10/PGS-2 antigen accumulation, and TIS10/PGS-2 message induction in primary cultures of mouse embryo fibroblasts. Dexamethasone attenuates TGF-beta enhancement of all these mitogen-induced responses: PGE2 accumulation, appearance of TIS10/PGS-2 protein and message, and accumulation of TIS10/PGS-2 unprocessed transcript.

  15. Prostaglandin synthase-mediated metabolism of carcinogens and a potential role for peroxyl radicals as reactive intermediates

    SciTech Connect

    Marnett, L.J. )

    1990-08-01

    Prostaglandin-H synthase is unique among enzymes of the plant and animal kingdom in its ability to biosynthesize and metabolize hydroperoxides. Higher oxidation states of the peroxidase oxidize reducing substrates to electron-deficient derivatives that react with macromolecular nucleophiles. In the case of aromatic amines, the electron-deficient derivatives are mutagenic to bacterial and mammalian cells. {beta}-Dicarbonyl compounds and retinoic acid are oxidized to carbon-centered radicals that react with O{sub 2} to form peroxyl free radicals. Peroxyl radicals are the most stable oxy radicals and are able to diffuse some distance from the site of their generation. Peroxyl radicals are also formed during lipid peroxidation and in the reaction of polyunsaturated fatty acid hydroperoxides with metal complexes and metalloproteins. Peroxyl radicals epoxidize isolated doubled bonds of compounds such as 7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene (BP-7,8-diol); 3,4-dihydroxy-3,4-dihydrobenzo(a)anthracene; and aflatoxin B{sub 1}. The epoxide products represent the ultimate carcinogenic forms of the respective compounds. Techniques for quantitating the extent of peroxidase dependent or peroxyl radical-dependent metabolism in vivo make use of differences in the structure or stereochemistry of reactive intermediates formed by peroxidases relative to cytochromes P-450. Differences in the relative amounts of hydrolysis products and DNA adducts derived from anti- and syn-dihydrodiolepoxides following application of BP-7,8-diol to mouse skin in vivo indicate peroxyl radicals play a significant role in metabolism of BP-7,8-diol in uninduced animals.

  16. Prostaglandin synthase-mediated metabolism of carcinogens and a potential role for peroxyl radicals as reactive intermediates.

    PubMed Central

    Marnett, L J

    1990-01-01

    Prostaglandin-H synthase is unique among enzymes of the plant and animal kingdom in its ability to biosynthesize and metabolize hydroperoxides. Its cyclooxygenase activity oxygenates polyunsaturated fatty acids to hydroperoxy endoperoxides, and its peroxidase activity reduces the hydroperoxy group to hydroxy groups. Higher oxidation states of the peroxidase oxidize reducing substrates to electron-deficient derivatives that react with macromolecular nucleophiles. In the case of aromatic amines, the electron-deficient derivatives are mutagenic to bacterial and mammalian cells. beta-Dicarbonyl compounds and retinoic acid are oxidized to carbon-centered radicals that react with O2 to form peroxyl free radicals. Peroxyl radicals are the most stable oxy radicals and are able to diffuse some distance from the site of their generation. Peroxyl radicals are also formed during lipid peroxidation and in the reaction of polyunsaturated fatty acid hydroperoxides with metal complexes and metalloproteins. Peroxyl radicals epoxidize isolated doubled bonds of compounds such as 7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene (BP-7,8-diol); 3,4-dihydroxy-3,4-dihydrobenzo(a)anthracene; and aflatoxin B1. The epoxide products represent the ultimate carcinogenic forms of the respective compounds. Techniques for quantitating the extent of peroxidase dependent or peroxyl radical-dependent metabolism in vivo make use of differences in the structure or stereochemistry of reactive intermediates formed by peroxidases relative to cytochromes P-450. Differences in the relative amounts of hydrolysis products and DNA adducts derived from anti- and syn-dihydrodiolepoxides following application of BP-7,8-diol to mouse skin in vivo indicate peroxyl radicals play a significant role in metabolism of BP-7,8-diol in uninduced animals. PMID:2125560

  17. The impact of lipocalin-type-prostaglandin-D-synthase as a predictor of kidney disease in patients with type 2 diabetes.

    PubMed

    Bacci, Marcelo Rodrigues; Cavallari, Marina Romera; de Rozier-Alves, Ross Martin; Alves, Beatriz da Costa Aguiar; Fonseca, Fernando Luiz Affonso

    2015-01-01

    Hypertension and diabetes are clinical conditions which contribute to the development of chronic kidney disease as well as risk factors for cardiovascular events. In recent years, lipocalin-type-prostaglandin-D-synthase (beta trace protein; BTP) has increasingly been studied as an alternative to creatinine for the evaluation of renal function as well as for being a possible biomarker for cardiovascular disease. It is expected that the levels of BTP in patients with cardiovascular disease are elevated, as is the case with patients with renal dysfunction. The objective of this study is to realize a systematic review of the pertinent literature in respect to BTP as a biomarker of renal dysfunction in diabetic patients. Using the database MEDLINE, a search up to year 2014 was conducted using the follow descriptors: "lipocalin type prostaglandin d synthase" AND "diabetes"; "lipocalin type prostaglandin d synthase" and "diabetic nephropathy"; "beta trace protein" AND "diabetes"; "beta trace protein" AND "diabetic nephropathy". The criteria used for inclusion were the presence of the referring to terms in title or abstract and study conducted in humans. About 17 articles were selected, of which six articles were duplicates, and of which six articles did not investigate any possible relationship between the protein (BTP) and either diabetes or nephropathy. The final result yielded five articles to be analyzed. This review found BTP is not influenced by race, by body mass index nor by patient's sex. BTP can be considered as a reliable early biomarker of renal dysfunction in diabetics. BTP is associated with metabolic syndrome and is also associated with greater cardiovascular risk. Prospective data establishing a correlation between BTP and mortality would have been of great interest, but such articles were not found in this review. PMID:26124640

  18. Positive correlation between patency and mRNA levels for cyclooxygenase-2 and prostaglandin E synthase in the uterine cervix of bitches with pyometra

    PubMed Central

    TAMADA, Hiromichi; ADACHI, Nahoko; KAWATE, Noritoshi; INABA, Toshio; HATOYA, Shingo; SAWADA, Tsutomu

    2015-01-01

    Factors involved in patency of uterine cervices in the bitch with pyometra remain to be clarified. This study examined relationship between patency and mRNA levels for inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-1, COX-2 and prostaglandin E synthase (PGES) in the uterine cervix of bitches with pyometra. Cervical patency was measured by inserting the stainless steel rods with different diameter into cervical canals. Levels of mRNA expression were determined by semi-quantitative reverse transcription-polymerase chain reaction. The cervical patency was positively correlated with mRNA levels for COX-2 and PGES, but not those for iNOS and COX-1. The results suggest that gene expression of COX-2 and PGES may be involved in the regulation of patency in the uterine cervix of bitches with pyometra. PMID:26596635

  19. Prostaglandin endoperoxide synthase kinetics in human amnion before and after labor at term and following preterm labor.

    PubMed

    Smieja, Z; Zakar, T; Walton, J C; Olson, D M

    1993-01-01

    To determine whether the kinetics of prostaglandin endoperoxide synthase (PGHS, commonly known as cyclooxygenase) in human amnion change with labor onset or between preterm and term labor, a specific enzyme assay was developed and characterized. The assay was linear for time (0-8 min) and protein concentration (5-30 micrograms/250 microliters incubation volume). The optimum pH was 8.0-8.5, and the enzyme reaction reached saturation at 10-20 microM arachidonic acid. Flufenamic acid was more efficacious than ibuprofen in the presence of 1 mM tryptophan in inhibiting enzyme activity. The Km and Vmax of PGHS were determined in 10 amnions obtained at elective caesarean section before labor onset (CS) at 39.3 +/- 0.8 wk gestational age (mean +/- SD, range = 38.5-41 wk) and 9 amnions obtained following spontaneous labor and vaginal delivery (SL) at 39.6 +/- 0.8 wk (range = 38.5-41 wk). The Km values were 1.4 +/- 1.2 mumol/l (CS) and 2.2 +/- 1.5 mumol/l (SL) (not different). However, the Vmax increased significantly (p < 0.05) from 11 +/- 8 (CS) to 19 +/- 4 (SL) pg PGE2/micrograms protein/min. In eight preterm amnions obtained following spontaneous labor and delivery at 32.9 +/- 2.1 wk (range = 29-36 wk), the Km and Vmax were 2.0 +/- 1.2 mumol/l and 17 +/- 9 pg PGE2/micrograms protein/min, respectively. Neither of these values was different from those of CS or SL amnions. None of the preterm pregnancies displayed histological evidence of infection. These results suggest that an increase in the mean amnion PGHS maximum velocity occurs in association with the onset of labor at term. The mean Vmax of PGHS in amnions obtained from idiopathic preterm spontaneous deliveries is between the CS and SL term values, reflecting, perhaps, multiple etiologies for preterm delivery.

  20. Microsomal prostaglandin E2 synthase-1 is induced by conditional expression of RET/PTC in thyroid PCCL3 cells through the activation of the MEK-ERK pathway.

    PubMed

    Puxeddu, Efisio; Mitsutake, Norisato; Knauf, Jeffrey A; Moretti, Sonia; Kim, Hei W; Seta, Karen A; Brockman, Diane; Myatt, Leslie; Millhorn, David E; Fagin, James A

    2003-12-26

    RET/PTC rearrangements are believed to be tumor-initiating events in papillary thyroid carcinomas. We identified microsomal prostaglandin E2 synthase-1 (mPGES-1) as a RET/PTC-inducible gene through subtraction hybridization cloning and expression profiling with custom microarrays. The inducible prostaglandin E2 (PGE2) biosynthetic enzymes cyclooxygenase-2 (COX-2) and mPGES-1 are up-regulated in many cancers. COX-2 is overexpressed in thyroid malignancies compared with benign nodules and normal thyroid tissues. Eicosanoids may promote tumorigenesis through effects on tumor cell growth, immune surveillance, and angiogenesis. Conditional RET/PTC1 or RET/PTC3 expression in PCCL3 thyroid cells markedly induced mPGES-1 and COX-2. PGE2 was the principal prostanoid and up-regulated (by approximately 60-fold), whereas hydroxyeicosatetraenoic acid metabolites were decreased, consistent with shunting of prostanoid biosynthesis toward PGE2 by coactivation of the two enzymes. RET/PTC activated mPGES-1 gene transcription. Based on experiments with kinase inhibitors, with PCCL3 cell lines with doxycycline-inducible expression of RET/PTC mutants with substitutions of critical tyrosine residues in the kinase domain, and lines with inducible expression of activated mutants of H-RAS and MEK1, RET/PTC was found to regulate mPGES-1 through Shc-RAS-MEK-ERK. These data show a direct relationship between activation of a tyrosine kinase receptor oncogene and regulation of PGE2 biosynthesis. As enzymes involved in prostanoid biosynthesis can be targeted with pharmacological inhibitors, these findings may have therapeutic implications. PMID:14555660

  1. Studies of inositol 1-phosphate analogues as inhibitors of the phosphatidylinositol phosphate synthase in mycobacteria.

    PubMed

    Morii, Hiroyuki; Okauchi, Tatsuo; Nomiya, Hiroki; Ogawa, Midori; Fukuda, Kazumasa; Taniguchi, Hatsumi

    2013-03-01

    We previously reported a novel pathway for the biosynthesis of phosphatidylinositol in mycobacteria via phosphatidylinositol phosphate (PIP) [Morii H., Ogawa, M., Fukuda, K., Taniguchi, H., and Koga, Y (2010) J. Biochem. 148, 593-602]. PIP synthase in the pathway is a promising target for the development of new anti-mycobacterium drugs. In the present study, we evaluated the characteristics of the PIP synthase of Mycobacterium tuberculosis. Four types of compounds were chemically synthesized based on the assumption that structural homologues of inositol 1-phosphate, a PIP synthase substrate, would act as PIP synthase inhibitors, and the results confirmed that all synthesized compounds inhibited PIP synthase activity. The phosphonate analogue of inositol 1-phosphate (Ino-C-P) had the greatest inhibitory effect among the synthesized compounds examined. Kinetic analysis indicated that Ino-C-P acted as a competitive inhibitor of inositol 1-phosphate. The IC(50) value for Ino-C-P inhibition of the PIP synthase activity was estimated to be 2.0 mM. Interestingly, Ino-C-P was utilized in the same manner as the normal PIP synthase substrate, leading to the synthesis of a phosphonate analogue of PIP (PI-C-P), which had a structure similar to that of the natural product, PIP. In addition, PI-C-P had high inhibitory activity against PIP synthase.

  2. Hypercalcemia stimulates expression of intrarenal phospholipase A2 and prostaglandin H synthase-2 in rats. Role of angiotensin II AT1 receptors.

    PubMed Central

    Mangat, H; Peterson, L N; Burns, K D

    1997-01-01

    In chronic hypercalcemia, inhibition of thick ascending limb sodium chloride reabsorption is mediated by elevated intrarenal PGE2. The mechanisms and source of elevated PGE2 in hypercalcemia are not known. We determined the effect of hypercalcemia on intrarenal expression of cytosolic phospholipase A2 (cPLA2), prostaglandin H synthase-1 (PGHS-1), and prostaglandin H synthase-2 (PGHS-2), enzymes important in prostaglandin production. In rats fed dihydrotachysterol to induce hypercalcemia, Western blot analysis revealed significant upregulation of both cPLA2 and PGHS-2 in the kidney cortex and the inner and outer medulla. Immunofluorescence localized intrarenal cPLA2 and PGHS-2 to interstitial cells of the inner and outer medulla, and to macula densa and cortical thick ascending limbs in both control and hypercalcemic rats. Hypercalcemia had no effect on intrarenal expression of PGHS-1. To determine if AT1 angiotensin II receptor activation was involved in the stimulation of cPLA2 and PGHS-2 in hypercalcemia, we treated rats with the AT1 receptor antagonist, losartan. Losartan abolished the polydipsia associated with hypercalcemia, prevented the increase in cPLA2 protein in all regions of the kidney, and diminished PGHS-2 expression in the inner medulla. In addition, losartan completely prevented the increase in urinary PGE2 excretion in hypercalcemic rats. Intrarenal levels of angiotensin II were unchanged in hypercalcemia. These data indicate that hypercalcemia stimulates intrarenal cPLA2 and PGHS-2 protein expression. Our results further support a role for angiotensin II, acting on AT1 receptors, in mediating this stimulation. PMID:9329957

  3. Investigation of potential glycogen synthase kinase 3 inhibitors using pharmacophore mapping and virtual screening.

    PubMed

    Dessalew, Nigus; Bharatam, Prasad V

    2006-09-01

    Glycogen synthase kinase-3 is a serine/threonine kinase that has attracted significant drug discovery attention in recent years. To investigate the identification of new potential glycogen synthase kinase-3 inhibitors, a pharmacophore mapping study was carried out using a set of 21 structurally diverse glycogen synthase kinase-3 inhibitors. A hypothesis containing four features: two hydrophobic, one hydrogen bond donor and another hydrogen bond acceptor was found to be the best from the 10 common feature hypotheses produced by HipHop module of Catalyst. The best hypothesis has a high cost of 156.592 and higher best fit values were obtained for the 21 inhibitors using this best hypothesis than the other HipHop hypotheses. The best hypothesis was then used to screen electronically the NCI2000 database. The hits obtained were docked into glycogen synthase kinase-3beta active site. A total of five novel potential leads were proposed after: (i) visual examination of how well they dock into the glycogen synthase kinase-3beta-binding site, (ii) comparative analysis of their FlexX, G-Score, PMF-Score, ChemScore and D-Scores values, (iii) comparison of their best fit value with the known inhibitors and (iv) examination of the how the hits retain interactions with the important amino acid residues of glycogen synthase kinase-3beta-binding site. PMID:17062013

  4. Investigation of potential glycogen synthase kinase 3 inhibitors using pharmacophore mapping and virtual screening.

    PubMed

    Dessalew, Nigus; Bharatam, Prasad V

    2006-09-01

    Glycogen synthase kinase-3 is a serine/threonine kinase that has attracted significant drug discovery attention in recent years. To investigate the identification of new potential glycogen synthase kinase-3 inhibitors, a pharmacophore mapping study was carried out using a set of 21 structurally diverse glycogen synthase kinase-3 inhibitors. A hypothesis containing four features: two hydrophobic, one hydrogen bond donor and another hydrogen bond acceptor was found to be the best from the 10 common feature hypotheses produced by HipHop module of Catalyst. The best hypothesis has a high cost of 156.592 and higher best fit values were obtained for the 21 inhibitors using this best hypothesis than the other HipHop hypotheses. The best hypothesis was then used to screen electronically the NCI2000 database. The hits obtained were docked into glycogen synthase kinase-3beta active site. A total of five novel potential leads were proposed after: (i) visual examination of how well they dock into the glycogen synthase kinase-3beta-binding site, (ii) comparative analysis of their FlexX, G-Score, PMF-Score, ChemScore and D-Scores values, (iii) comparison of their best fit value with the known inhibitors and (iv) examination of the how the hits retain interactions with the important amino acid residues of glycogen synthase kinase-3beta-binding site.

  5. The Prostaglandin Transporter: Eicosanoid Reuptake, Control of Signaling, and Development of High-Affinity Inhibitors as Drug Candidates

    PubMed Central

    Schuster, Victor L.; Chi, Yuling; Lu, Run

    2015-01-01

    We discovered the prostaglandin transporter (PGT) and cloned the human cDNA and gene. PGT transports extracellular prostaglandins (PGs) into the cytoplasm for enzymatic inactivation. PGT knockout mice have elevated prostaglandin E2 (PGE2) and neonatal patent ductus arteriosus, which reflects PGT's control over PGE2 signaling at EP1/EP4 cell-surface receptors. Interestingly, rescued PGT knockout pups have a nearly normal phenotype, as do human PGT nulls. Given the benign phenotype of PGT genetic nulls, and because PGs are useful medicines, we have approached PGT as a drug target. Triazine library screening yielded a lead compound of inhibitory constant 50% (IC50) = 3.7 μM, which we developed into a better inhibitor of IC50 378 nM. Further structural improvements have yielded 26 rationally designed derivatives with IC50 < 100 nM. The therapeutic approach of increasing endogenous PGs by inhibiting PGT offers promise in diseases such as pulmonary hypertension and obesity. PMID:26330684

  6. Reviewing Ligand-Based Rational Drug Design: The Search for an ATP Synthase Inhibitor

    PubMed Central

    Lee, Chia-Hsien; Huang, Hsuan-Cheng; Juan, Hsueh-Fen

    2011-01-01

    Following major advances in the field of medicinal chemistry, novel drugs can now be designed systematically, instead of relying on old trial and error approaches. Current drug design strategies can be classified as being either ligand- or structure-based depending on the design process. In this paper, by describing the search for an ATP synthase inhibitor, we review two frequently used approaches in ligand-based drug design: The pharmacophore model and the quantitative structure-activity relationship (QSAR) method. Moreover, since ATP synthase ligands are potentially useful drugs in cancer therapy, pharmacophore models were constructed to pave the way for novel inhibitor designs. PMID:21954360

  7. Comprehensive analysis of prostaglandin metabolic enzyme expression during pregnancy and the characterization of AKR1B1 as a prostaglandin F synthase at the maternal-conceptus interface in pigs.

    PubMed

    Seo, Heewon; Choi, Yohan; Shim, Jangsoo; Yoo, Inkyu; Ka, Hakhyun

    2014-05-01

    Prostaglandins (PGs) are important lipid mediators regulating various reproductive processes in many species. In pigs, the expression pattern of PGE2 and PGF2α metabolic enzymes and the regulatory mechanism controlling PGE2 and PGF2α levels in the uterus during pregnancy are not completely understood. This study determined endometrial expression of the genes (PLA2G4A, PTGS1, PTGS2, PTGES, PTGES2, PTGES3, AKR1B1, CBR1, and HPGD) involved in PGE2 and PGF2α metabolism during the estrous cycle and pregnancy and measured levels of PGE2 and PGF2α in uterine endometrial tissues and uterine flushings at the time of conceptus implantation in pigs. Except PTGES3, expression of the genes studied changed in a pregnancy-stage-specific manner, and localization of PTGES, AKR1B1, CBR1, and HPGD mRNAs were cell-type specific in the uterine endometrium. Levels of both PGE2 and PGF2α in uterine endometrial tissues and uterine lumen were higher on Day 12 of pregnancy than those of the estrous cycle and affected by different morphology of spherical and filamentous conceptuses. Furthermore, we determined that endometrial expression of AKR1B1, known to encode a PGF2α synthase in other species, was increased by estrogen and interleukin-1beta and that AKR1B1 exhibited PGF2α synthase activity in the porcine uterine endometrium. These results in pigs indicate that the PGE2 and PGF2α metabolic enzymes are expressed stage specifically in the endometrium during pregnancy and regulate the abundance of PGE2 and PGF2α in the uterus at the time of implantation and that AKR1B1 may act as a major PGF synthase in the endometrium during early pregnancy.

  8. Comprehensive analysis of prostaglandin metabolic enzyme expression during pregnancy and the characterization of AKR1B1 as a prostaglandin F synthase at the maternal-conceptus interface in pigs.

    PubMed

    Seo, Heewon; Choi, Yohan; Shim, Jangsoo; Yoo, Inkyu; Ka, Hakhyun

    2014-05-01

    Prostaglandins (PGs) are important lipid mediators regulating various reproductive processes in many species. In pigs, the expression pattern of PGE2 and PGF2α metabolic enzymes and the regulatory mechanism controlling PGE2 and PGF2α levels in the uterus during pregnancy are not completely understood. This study determined endometrial expression of the genes (PLA2G4A, PTGS1, PTGS2, PTGES, PTGES2, PTGES3, AKR1B1, CBR1, and HPGD) involved in PGE2 and PGF2α metabolism during the estrous cycle and pregnancy and measured levels of PGE2 and PGF2α in uterine endometrial tissues and uterine flushings at the time of conceptus implantation in pigs. Except PTGES3, expression of the genes studied changed in a pregnancy-stage-specific manner, and localization of PTGES, AKR1B1, CBR1, and HPGD mRNAs were cell-type specific in the uterine endometrium. Levels of both PGE2 and PGF2α in uterine endometrial tissues and uterine lumen were higher on Day 12 of pregnancy than those of the estrous cycle and affected by different morphology of spherical and filamentous conceptuses. Furthermore, we determined that endometrial expression of AKR1B1, known to encode a PGF2α synthase in other species, was increased by estrogen and interleukin-1beta and that AKR1B1 exhibited PGF2α synthase activity in the porcine uterine endometrium. These results in pigs indicate that the PGE2 and PGF2α metabolic enzymes are expressed stage specifically in the endometrium during pregnancy and regulate the abundance of PGE2 and PGF2α in the uterus at the time of implantation and that AKR1B1 may act as a major PGF synthase in the endometrium during early pregnancy. PMID:24695626

  9. Isolation of the peptide inhibitor of H+-ATP synthase from Crithidia fasciculata and Trypanosoma cruzi.

    PubMed

    Rilo, M C; Cataldi de Flombaum, M A; Stoppani, A O

    1989-02-01

    An inhibitor of Crithidia fasciculata and Trypanosoma cruzi H+ -ATP synthase (ATPase) was isolated from these organims mitochondrial particles, either by (a) ammonium sulfate-cholate extraction followed by heat treatment and ethanol precipitation, or (b) gel-filtration on Sephadex G-50, followed by a similar purification procedure. Inactivation by trypsin supported the inhibitor peptide structure. Removal of the peptide inhibitor increased about three-fold the specific activity of the protozoan ATPases. The isolated peptides and a highly purified bovine heart ATPase inhibitor inhibited C. fasciculata ATPase as a function of the peptide concentration.

  10. Treatment of obesity and pulmonary arterial hypertension with inhibitors of the prostaglandin transporter: evaluation of patent WO2014/204895A1.

    PubMed

    Dingemanse, Jasper; Bolli, Martin; Iglarz, Marc

    2015-01-01

    Prostaglandins display a wide array of pharmacological effects and prostaglandin analogs are already used in the treatment of pulmonary arterial hypertension (PAH). After synthesis and release from cells, prostaglandins undergo reuptake by the prostaglandin transporter (PGT). WO2014/204895 claims the use of a series of trisubstituted triazine derivatives for the treatment of obesity and PAH. Composition of matter of these triazines has been claimed in WO2011/037610 and the compounds are described as potent inhibitors of the PGT. One compound (nr 146) was shown to improve high fat diet-induced glucose tolerance in a mouse model. In addition, this compound has been explored in the rat monocrotaline model of PAH and reduced characteristic features of the pathology. This class of compounds presents a potential new treatment paradigm in the treatment of obesity-related disorders and PAH. PMID:26099857

  11. Screening of plants used by Southern African traditional healers in the treatment of dysmenorrhoea for prostaglandin-synthesis inhibitors and uterine relaxing activity.

    PubMed

    Lindsey, K; Jäger, A K; Raidoo, D M; van Staden, J

    1999-01-01

    Plants used by Southern African traditional healers for the treatment of menstrual pains were screened for prostaglandin-synthesis inhibitors and the ability to reduce isolated uterine muscle contraction using the cyclooxygenase and in vitro uterine bioassays respectively. Prostaglandins are synthesized from arachidonic acid and the enzyme that drives this reaction is cyclooxygenase. The excessive production of prostaglandins by the myometrium and endometrium induces uterine contractions. Inhibition of cyclooxygenase and hence of the prostaglandin biosynthetic pathway may lead to relief of menstrual pain. Ten plants used by traditional healers for menstrual pains were assayed for cyclooxygenase inhibitory activity. Several plant extracts exhibited high inhibitory activity in the assay. The highest activities were obtained with ethanolic extracts of Siphonochilus aethiopicus, Cenchrus ciliaris and Solanum mauritianum. Generally ethanolic extracts gave higher activity than the aqueous extracts. None of the ethanolic plant extracts were able to relax or reduce the contractions of the precontracted guinea pig uterus. PMID:10075117

  12. Discovery of two new inhibitors of Botrytis cinerea chitin synthase by a chemical library screening.

    PubMed

    Magellan, Hervé; Boccara, Martine; Drujon, Thierry; Soulié, Marie-Christine; Guillou, Catherine; Dubois, Joëlle; Becker, Hubert F

    2013-09-01

    Chitin synthases polymerize UDP-GlcNAC to form chitin polymer, a key component of fungal cell wall biosynthesis. Furthermore, chitin synthases are desirable targets for fungicides since chitin is absent in plants and mammals. Two potent Botrytis cinerea chitin synthase inhibitors, 2,3,5-tri-O-benzyl-d-ribose (compound 1) and a 2,5-functionalized imidazole (compound 2) were identified by screening a chemical library. We adapted the wheat germ agglutinin (WGA) test for chitin synthase activity detection to allow miniaturization and robotization of the screen. Both identified compounds inhibited chitin synthases in vitro with IC50 values of 1.8 and 10μM, respectively. Compounds 1 and 2 were evaluated for their antifungal activity and were found to be active against B. cinerea BD90 strain with MIC values of 190 and 100μM, respectively. Finally, we discovered that both compounds confer resistance to plant leaves against the attack of the fungus by reducing the propagation of lesions by 37% and 23%, respectively. Based on the inhibitory properties found in different assays, compounds 1 and 2 can be considered as antifungal hit inhibitors of chitin synthase, allowing further optimization of their pharmacological profile to improve their antifungal properties.

  13. Molecular docking studies on quinazoline antifolate derivatives as human thymidylate synthase inhibitors

    PubMed Central

    Srivastava, Vivek; Gupta, Satya Prakash; Siddiqi, Mohd. Imran; Mishra, Bhartendu Nath

    2010-01-01

    We have performed molecular docking on quinazoline antifolates complexed with human thymidylate synthase to gain insight into the structural preferences of these inhibitors. The study was conducted on a selected set of one hundred six compounds with variation in structure and activity. The structural analyses indicate that the coordinate bond interactions, the hydrogen bond interactions, the van der Waals interactions as well as the hydrophobic interactions between ligand and receptor are responsible simultaneously for the preference of inhibition and potency. In this study, fast flexible docking simulations were performed on quinazoline antifolates derivatives as human thymidylate synthase inhibitors. The results indicated that the quinazoline ring of the inhibitors forms hydrophobic contacts with Leu192, Leu221 and Tyr258 and stacking interaction is conserved in complex with the inhibitor and cofactor. PMID:20975900

  14. Selenoprotein-dependent Up-regulation of Hematopoietic Prostaglandin D2 Synthase in Macrophages Is Mediated through the Activation of Peroxisome Proliferator-activated Receptor (PPAR) γ*

    PubMed Central

    Gandhi, Ujjawal H.; Kaushal, Naveen; Ravindra, Kodihalli C.; Hegde, Shailaja; Nelson, Shakira M.; Narayan, Vivek; Vunta, Hema; Paulson, Robert F.; Prabhu, K. Sandeep

    2011-01-01

    The plasticity of macrophages is evident from their dual role in inflammation and resolution of inflammation that are accompanied by changes in the transcriptome and metabolome. Along these lines, we have previously demonstrated that the micronutrient selenium increases macrophage production of arachidonic acid (AA)-derived anti-inflammatory 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) and decreases the proinflammatory PGE2. Here, we hypothesized that selenium modulated the metabolism of AA by a differential regulation of various prostaglandin (PG) synthases favoring the production of PGD2 metabolites, Δ12-PGJ2 and 15d-PGJ2. A dose-dependent increase in the expression of hematopoietic-PGD2 synthase (H-PGDS) by selenium and a corresponding increase in Δ12-PGJ2 and 15d-PGJ2 in RAW264.7 macrophages and primary bone marrow-derived macrophages was observed. Studies with organic non-bioavailable forms of selenium and the genetic manipulation of cellular selenium incorporation machinery indicated that selenoproteins were necessary for H-PGDS expression and 15d-PGJ2 production. Treatment of selenium-deficient macrophages with rosiglitazone, a peroxisome proliferator-activated receptor γ ligand, up-regulated H-PGDS. Furthermore, electrophoretic mobility shift assays indicated the presence of an active peroxisome proliferator-activated receptor-response element in murine Hpgds promoter suggesting a positive feedback mechanism of H-PGDS expression. Alternatively, the expression of nuclear factor-κB-dependent thromboxane synthase and microsomal PGE2 synthase was down-regulated by selenium. Using a Friend virus infection model of murine leukemia, the onset of leukemia was observed only in selenium-deficient and indomethacin-treated selenium-supplemented mice but not in the selenium-supplemented group or those treated with 15d-PGJ2. These results suggest the importance of selenium in the shunting of AA metabolism toward the production of PGD2 metabolites, which may have

  15. Methylmercury Alters the Activities of Hsp90 Client Proteins, Prostaglandin E Synthase/p23 (PGES/23) and nNOS

    PubMed Central

    Caito, Samuel; Zeng, Heng; Aschner, Judy L.; Aschner, Michael

    2014-01-01

    Methylmercury (MeHg) is a persistent pollutant with known neurotoxic effects. We have previously shown that astrocytes accumulate MeHg and play a prominent role in mediating MeHg toxicity in the central nervous system (CNS) by altering glutamate signaling, generating oxidative stress, depleting glutathione (GSH) and initiating lipid peroxidation. Interestingly, all of these pathways can be regulated by the constitutively expressed, 90-kDa heat shock protein, Hsp90. As Hsp90 function is regulated by oxidative stress, we hypothesized that MeHg disrupts Hsp90-client protein functions. Astrocytes were treated with MeHg and expression of Hsp90, as well as the abundance of complexes of Hsp90-neuronal nitric oxide synthase (nNOS) and Hsp90-prostaglandin E synthase/p23 (PGES/p23) were assessed. MeHg exposure decreased Hsp90 protein expression following 12 h of treatment while shorter exposures had no effect on Hsp90 protein expression. Interestingly, following 1 or 6 h of MeHg exposure, Hsp90 binding to PGES/p23 or nNOS was significantly increased, resulting in increased prostaglandin E2 (PGE2) synthesis from MeHg-treated astrocytes. These effects were attenuated by the Hsp90 antagonist, geldanmycin. NOS activity was increased following MeHg treatment while cGMP formation was decreased. This was accompanied by an increase in •O2− and H2O2 levels, suggesting that MeHg uncouples NO formation from NO-dependent signaling and increases oxidative stress. Altogether, our data demonstrates that Hsp90 interactions with client proteins are increased following MeHg exposure, but over time Hsp90 levels decline, contributing to oxidative stress and MeHg-dependent excitotoxicity. PMID:24852575

  16. Nitric oxide synthase inhibitors in post-myocardial infarction cardiogenic shock--an update.

    PubMed

    Kaluski, Edo; Hendler, Alberto; Blatt, Alex; Uriel, Nir

    2006-11-01

    Cardiogenic shock (CS) in acute myocardial infarction, after successful coronary angioplasty, still carries a case fatality rate of 50%. These patients succumb to a systemic metabolic storm, superimposed on extensive myocardial necrosis and stunning. Nitric oxide (NO) overproduction contributes to the pathophysiology of this morbid state. Current data regarding the physiologic effects of NO and nitric oxide synthase (NOS) inhibitors on the cardiovascular system are reviewed. Clinical trials assessing the safety and efficacy of NOS inhibitors in CS are summarized.

  17. Nitric oxide synthase inhibitors containing the carboxamidine group or its isosteres

    NASA Astrophysics Data System (ADS)

    Proskuryakov, Sergei Ya; Konoplyannikov, Anatoly G.; Skvortzov, Valery G.; Mandrugin, Andrey A.; Fedoseev, Vladimir M.

    2005-09-01

    The review summarises structures, activities and selectivity of NO-synthase (NOS) inhibitors belonging to various classes of chemical compounds. Linear, cyclic and heterocyclic structures containing guanidine, amidine and/or isothiourea fragments are considered. The structure-activity relationships for these inhibitors were analysed in relation to their action on the inducible NOS isoform. This analysis can provide the basis for the synthesis of new more efficient compounds.

  18. Enantioselective synthesis of the novel chiral sulfoxide derivative as a glycogen synthase kinase 3beta inhibitor.

    PubMed

    Saitoh, Morihisa; Kunitomo, Jun; Kimura, Eiji; Yamano, Toru; Itoh, Fumio; Kori, Masakuni

    2010-09-01

    Glycogen synthase kinase 3beta (GSK-3beta) inhibitors are expected to be attractive therapeutic agents for the treatment of Alzheimer's disease (AD). Recently we discovered sulfoxides (S)-1 as a novel GSK-3beta inhibitor having in vivo efficacy. We investigated practical asymmetric preparation methods for the scale-up synthesis of (S)-1. The highly enantioselective synthesis of (S)-1 (94% ee) was achieved by titanium-mediated oxidation with D-(-)-diethyl tartrate on gram scale.

  19. Antagonism screen for inhibitors of bacterial cell wall biogenesis uncovers an inhibitor of undecaprenyl diphosphate synthase

    PubMed Central

    Farha, Maya A.; Czarny, Tomasz L.; Myers, Cullen L.; Worrall, Liam J.; French, Shawn; Conrady, Deborah G.; Wang, Yang; Oldfield, Eric; Strynadka, Natalie C. J.; Brown, Eric D.

    2015-01-01

    Drug combinations are valuable tools for studying biological systems. Although much attention has been given to synergistic interactions in revealing connections between cellular processes, antagonistic interactions can also have tremendous value in elucidating genetic networks and mechanisms of drug action. Here, we exploit the power of antagonism in a high-throughput screen for molecules that suppress the activity of targocil, an inhibitor of the wall teichoic acid (WTA) flippase in Staphylococcus aureus. Well-characterized antagonism within the WTA biosynthetic pathway indicated that early steps would be sensitive to this screen; however, broader interactions with cell wall biogenesis components suggested that it might capture additional targets. A chemical screening effort using this approach identified clomiphene, a widely used fertility drug, as one such compound. Mechanistic characterization revealed the target was the undecaprenyl diphosphate synthase, an enzyme that catalyzes the synthesis of a polyisoprenoid essential for both peptidoglycan and WTA synthesis. The work sheds light on mechanisms contributing to the observed suppressive interactions of clomiphene and in turn reveals aspects of the biology that underlie cell wall synthesis in S. aureus. Further, this effort highlights the utility of antagonistic interactions both in high-throughput screening and in compound mode of action studies. Importantly, clomiphene represents a lead for antibacterial drug discovery. PMID:26283394

  20. Antagonism screen for inhibitors of bacterial cell wall biogenesis uncovers an inhibitor of undecaprenyl diphosphate synthase.

    PubMed

    Farha, Maya A; Czarny, Tomasz L; Myers, Cullen L; Worrall, Liam J; French, Shawn; Conrady, Deborah G; Wang, Yang; Oldfield, Eric; Strynadka, Natalie C J; Brown, Eric D

    2015-09-01

    Drug combinations are valuable tools for studying biological systems. Although much attention has been given to synergistic interactions in revealing connections between cellular processes, antagonistic interactions can also have tremendous value in elucidating genetic networks and mechanisms of drug action. Here, we exploit the power of antagonism in a high-throughput screen for molecules that suppress the activity of targocil, an inhibitor of the wall teichoic acid (WTA) flippase in Staphylococcus aureus. Well-characterized antagonism within the WTA biosynthetic pathway indicated that early steps would be sensitive to this screen; however, broader interactions with cell wall biogenesis components suggested that it might capture additional targets. A chemical screening effort using this approach identified clomiphene, a widely used fertility drug, as one such compound. Mechanistic characterization revealed the target was the undecaprenyl diphosphate synthase, an enzyme that catalyzes the synthesis of a polyisoprenoid essential for both peptidoglycan and WTA synthesis. The work sheds light on mechanisms contributing to the observed suppressive interactions of clomiphene and in turn reveals aspects of the biology that underlie cell wall synthesis in S. aureus. Further, this effort highlights the utility of antagonistic interactions both in high-throughput screening and in compound mode of action studies. Importantly, clomiphene represents a lead for antibacterial drug discovery. PMID:26283394

  1. Biosynthesis and degradation of canine placental prostaglandins: prepartum changes in expression and function of prostaglandin F2α-synthase (PGFS, AKR1C3) and 15-hydroxyprostaglandin dehydrogenase (HPGD).

    PubMed

    Gram, Aykut; Büchler, Urs; Boos, Alois; Hoffmann, Bernd; Kowalewski, Mariusz P

    2013-07-01

    There is no distinct explanation of the mechanism for the prepartal prostaglandin F2alpha (PGF2alpha) increase in pregnant dogs. Although the PGF2alpha-synthase (PGFS [AKR1C3]) mRNA expression and localization profiles have been previously investigated in canine utero/placental compartments, the availability and biochemical activity of the PGFS (AKR1C3) protein remain unknown. In order to better understand the regulation of canine uterine PGF2alpha availability and eventual prepartum release in luteolytic amounts in dogs, canine-specific PGFS (AKR1C3) and 15-hydroxyprostaglandin dehydrogenase (HPGD) antibodies were generated and used to characterize the expression, cellular localization, and biochemical properties of PGFS (AKR1C3) and HPGD in the utero/placental compartments and corpus luteum throughout pregnancy and at prepartum luteolysis. PGFS (AKR1C3) expression was weak or absent in luteal samples. Uterine PGFS (AKR1C3) was up-regulated postimplantation and declined prepartum. The utero/placental expression of PGFS (AKR1C3) was identified in the superficial uterine glands throughout gestation and in the trophoblast cells within the feto-maternal contact zone during placentation, suggesting a possible role for PGFS (AKR1C3) in the trophoblast invasion. Utero-placental HPGD was up-regulated until postimplantation, lower at midgestation, and greatly suppressed at prepartum. Expression was routinely identified in the endometrial surface and glandular epithelia, and positive signals were also observed in the trophoblast cells at the feto-maternal contact zone. The biochemical activity of recombinant PGFS (AKR1C3) and HPGD was confirmed after its expression in a heterologous system. The colocalization of HPGD with PGFS (AKR1C3) expression suggests a modulatory role for HPGD as a gatekeeper of the supply of prostaglandin in the pregnant canine uterus.

  2. Biosynthesis and degradation of canine placental prostaglandins: prepartum changes in expression and function of prostaglandin F2α-synthase (PGFS, AKR1C3) and 15-hydroxyprostaglandin dehydrogenase (HPGD).

    PubMed

    Gram, Aykut; Büchler, Urs; Boos, Alois; Hoffmann, Bernd; Kowalewski, Mariusz P

    2013-07-01

    There is no distinct explanation of the mechanism for the prepartal prostaglandin F2alpha (PGF2alpha) increase in pregnant dogs. Although the PGF2alpha-synthase (PGFS [AKR1C3]) mRNA expression and localization profiles have been previously investigated in canine utero/placental compartments, the availability and biochemical activity of the PGFS (AKR1C3) protein remain unknown. In order to better understand the regulation of canine uterine PGF2alpha availability and eventual prepartum release in luteolytic amounts in dogs, canine-specific PGFS (AKR1C3) and 15-hydroxyprostaglandin dehydrogenase (HPGD) antibodies were generated and used to characterize the expression, cellular localization, and biochemical properties of PGFS (AKR1C3) and HPGD in the utero/placental compartments and corpus luteum throughout pregnancy and at prepartum luteolysis. PGFS (AKR1C3) expression was weak or absent in luteal samples. Uterine PGFS (AKR1C3) was up-regulated postimplantation and declined prepartum. The utero/placental expression of PGFS (AKR1C3) was identified in the superficial uterine glands throughout gestation and in the trophoblast cells within the feto-maternal contact zone during placentation, suggesting a possible role for PGFS (AKR1C3) in the trophoblast invasion. Utero-placental HPGD was up-regulated until postimplantation, lower at midgestation, and greatly suppressed at prepartum. Expression was routinely identified in the endometrial surface and glandular epithelia, and positive signals were also observed in the trophoblast cells at the feto-maternal contact zone. The biochemical activity of recombinant PGFS (AKR1C3) and HPGD was confirmed after its expression in a heterologous system. The colocalization of HPGD with PGFS (AKR1C3) expression suggests a modulatory role for HPGD as a gatekeeper of the supply of prostaglandin in the pregnant canine uterus. PMID:23677986

  3. Modulation of Alternaria infectoria Cell Wall Chitin and Glucan Synthesis by Cell Wall Synthase Inhibitors

    PubMed Central

    Fernandes, Chantal; Anjos, Jorge; Walker, Louise A.; Silva, Branca M. A.; Cortes, Luísa; Mota, Marta; Munro, Carol A.; Gow, Neil A. R.

    2014-01-01

    The present work reports the effects of caspofungin, a β-1,3-glucan synthase inhibitor, and nikkomycin Z, an inhibitor of chitin synthases, on two strains of Alternaria infectoria, a melanized fungus involved in opportunistic human infections and respiratory allergies. One of the strains tested, IMF006, bore phenotypic traits that conferred advantages in resisting antifungal treatment. First, the resting cell wall chitin content was higher and in response to caspofungin, the chitin level remained constant. In the other strain, IMF001, the chitin content increased upon caspofungin treatment to values similar to basal IMF006 levels. Moreover, upon caspofungin treatment, the FKS1 gene was upregulated in IMF006 and downregulated in IMF001. In addition, the resting β-glucan content was also different in both strains, with higher levels in IMF001 than in IMF006. However, this did not provide any advantage with respect to echinocandin resistance. We identified eight different chitin synthase genes and studied relative gene expression when the fungus was exposed to the antifungals under study. In both strains, exposure to caspofungin and nikkomycin Z led to modulation of the expression of class V and VII chitin synthase genes, suggesting its importance in the robustness of A. infectoria. The pattern of A. infectoria phagocytosis and activation of murine macrophages by spores was not affected by caspofungin. Monotherapy with nikkomycin Z and caspofungin provided only fungistatic inhibition, while a combination of both led to fungal cell lysis, revealing a strong synergistic action between the chitin synthase inhibitor and the β-glucan synthase inhibitor against this fungus. PMID:24614372

  4. Selective heterocyclic amidine inhibitors of human inducible nitric oxide synthase.

    PubMed

    Moormann, A E; Metz, S; Toth, M V; Moore, W M; Jerome, G; Kornmeier, C; Manning, P; Hansen, D W; Pitzele, B S; Webber, R K

    2001-10-01

    The potency and selectivity of a series of 5-hetero-2-iminohexahydroazepines were examined as inhibitors of the three human NOS isoforms. The effect of ring substitution of the 5-carbon for a heteroatom is presented. Potencies (IC(50)'s) for these inhibitors are in the low micromolar range for hi-NOS with some examples exhibiting a 500x selectivity versus hec-NOS.

  5. Ursolic acid from Plantago major, a selective inhibitor of cyclooxygenase-2 catalyzed prostaglandin biosynthesis.

    PubMed

    Ringbom, T; Segura, L; Noreen, Y; Perera, P; Bohlin, L

    1998-10-01

    A hexane extract of Plantago major was investigated by bioactivity-directed fractionation, using an in vitro cyclooxygenase-2 (COX-2) catalyzed prostaglandin biosynthesis inhibition assay, and resulted in the isolation of ursolic acid (1). This triterpenoid showed a significant COX-2 inhibitory effect, directly on the enzyme activity, with an IC50 value of 130 microM and a COX-2/COX-1 selectivity ratio of 0.6. The structural isomer oleanolic acid (2) was found to be less active than 1, with an IC50 value of 295 microM, but showed a similar selectivity ratio (0.8). Furthermore, no significant inhibition on COX-2 or COX-1 was observed by the triterpenoid, 18beta-glycyrrhetinic acid (3). The direct inhibitory effect of 1 and 2 on COX-2 catalyzed prostaglandin biosynthesis increased with preincubation, indicating a time-dependent inhibition, while the effect on COX-1 was found to be independent of preincubation time.

  6. Small-molecule inhibitor binding to an N-acyl-homoserine lactone synthase.

    PubMed

    Chung, Jiwoung; Goo, Eunhye; Yu, Sangheon; Choi, Okhee; Lee, Jeehyun; Kim, Jinwoo; Kim, Hongsup; Igarashi, Jun; Suga, Hiroaki; Moon, Jae Sun; Hwang, Ingyu; Rhee, Sangkee

    2011-07-19

    Quorum sensing (QS) controls certain behaviors of bacteria in response to population density. In gram-negative bacteria, QS is often mediated by N-acyl-L-homoserine lactones (acyl-HSLs). Because QS influences the virulence of many pathogenic bacteria, synthetic inhibitors of acyl-HSL synthases might be useful therapeutically for controlling pathogens. However, rational design of a potent QS antagonist has been thwarted by the lack of information concerning the binding interactions between acyl-HSL synthases and their ligands. In the gram-negative bacterium Burkholderia glumae, QS controls virulence, motility, and protein secretion and is mediated by the binding of N-octanoyl-L-HSL (C8-HSL) to its cognate receptor, TofR. C8-HSL is synthesized by the acyl-HSL synthase TofI. In this study, we characterized two previously unknown QS inhibitors identified in a focused library of acyl-HSL analogs. Our functional and X-ray crystal structure analyses show that the first inhibitor, J8-C8, binds to TofI, occupying the binding site for the acyl chain of the TofI cognate substrate, acylated acyl-carrier protein. Moreover, the reaction byproduct, 5'-methylthioadenosine, independently binds to the binding site for a second substrate, S-adenosyl-L-methionine. Closer inspection of the mode of J8-C8 binding to TofI provides a likely molecular basis for the various substrate specificities of acyl-HSL synthases. The second inhibitor, E9C-3oxoC6, competitively inhibits C8-HSL binding to TofR. Our analysis of the binding of an inhibitor and a reaction byproduct to an acyl-HSL synthase may facilitate the design of a new class of QS-inhibiting therapeutic agents.

  7. Human thromboxane synthase: comparative modeling and docking evaluation with the competitive inhibitors Dazoxiben and Ozagrel.

    PubMed

    Sathler, Plínio Cunha; Santana, Marcos; Lourenço, André Luiz; Rodrigues, Carlos Rangel; Abreu, Paula; Cabral, Lúcio Mendes; Castro, Helena Carla

    2014-08-01

    Thromboxane synthase (TXAS) is a P450 epoxygenase that synthesizes thromboxane A2 (TXA2), a potent mediator of platelet aggregation, vasoconstriction and bronchoconstriction. This enzyme plays an important role in several human diseases, including myocardial infarction, stroke, septic shock, asthma and cancer. Despite of the increasing interest on developing TXAS inhibitors, the structure and activity of TXAS are still not totally elucidated. In this study, we used a comparative molecular modeling approach to construct a reliable model of TXAS and analyze its interactions with Dazoxiben and Ozagrel, two competitive inhibitors. Our results were compatible with experimental published data, showing feasible cation-π interaction between the iron atom of the heme group of TXAS and the basic nitrogen atom of the imidazolyl group of those inhibitors. In the absence of the experimental structure of thromboxane synthase, this freely available model may be useful for designing new antiplatelet drugs for diseases related with TXA2.

  8. Design, synthesis, and enzyme kinetics of novel benzimidazole and quinoxaline derivatives as methionine synthase inhibitors.

    PubMed

    Elshihawy, Hosam; Helal, Mohamed A; Said, Mohamed; Hammad, Mohamed A

    2014-01-01

    Methionine synthase catalyzes the transfer of a methyl group from 5-methyltetrahydrofolate to homocysteine, producing methionine and tetrahydrofolate. Benzimidazole and deazatetrahydrofolates derivatives have been shown to inhibit methionine synthase by competing with the substrate 5-methyltetrahydrofolate. In this study, a novel series of substituted benzimidazoles and quinoxalines were designed and assessed for inhibitory activity against purified rat liver methionine synthase using a radiometric enzyme assay. Compounds 3g, 3j, and 5c showed the highest activity against methionine synthase (IC₅₀: 20 μM, 18 μM, 9 μM, respectively). Kinetic analysis of these compounds using Lineweaver-Burk plots revealed characteristics of mixed inhibition for 3g and 5c; and uncompetitive inhibition for 3j. Docking study into a homology model of the rat methionine synthase gave insights into the molecular determinants of the activity of this class of compounds. The identification of these drug-like inhibitors could lead the design of the next generation modulators of methionine synthase.

  9. Selective Acetamidine-Based Nitric Oxide Synthase Inhibitors: Synthesis, Docking, and Biological Studies.

    PubMed

    Maccallini, Cristina; Montagnani, Monica; Paciotti, Roberto; Ammazzalorso, Alessandra; De Filippis, Barbara; Di Matteo, Mauro; Di Silvestre, Sara; Fantacuzzi, Marialuigia; Giampietro, Letizia; Potenza, Maria A; Re, Nazzareno; Pandolfi, Assunta; Amoroso, Rosa

    2015-06-11

    N-[(3-Aminomethyl)benzyl]acetamidine derivatives were synthesized and in vitro evaluated as inhibitors of the inducible isoform of nitric oxide synthase (iNOS). Because of the high potency of action and the excellent selectivity over the endothelial nitric oxide synthase (eNOS), compound 10 was ex vivo evaluated on isolated and perfused resistance arteries. The results confirm that compound 10 selectively inhibits the iNOS, without affecting the endothelial isoform. The outcome of the docking studies showed that the hydrophobic interaction is the driving force of the binding process, especially for iNOS, where the binding pocket is characterized by a significant lipophilic region. PMID:26101565

  10. Selective Acetamidine-Based Nitric Oxide Synthase Inhibitors: Synthesis, Docking, and Biological Studies

    PubMed Central

    2015-01-01

    N-[(3-Aminomethyl)benzyl]acetamidine derivatives were synthesized and in vitro evaluated as inhibitors of the inducible isoform of nitric oxide synthase (iNOS). Because of the high potency of action and the excellent selectivity over the endothelial nitric oxide synthase (eNOS), compound 10 was ex vivo evaluated on isolated and perfused resistance arteries. The results confirm that compound 10 selectively inhibits the iNOS, without affecting the endothelial isoform. The outcome of the docking studies showed that the hydrophobic interaction is the driving force of the binding process, especially for iNOS, where the binding pocket is characterized by a significant lipophilic region. PMID:26101565

  11. Structures, mechanisms and inhibitors of undecaprenyl diphosphate synthase: a cis-prenyltransferase for bacterial peptidoglycan biosynthesis.

    PubMed

    Teng, Kuo-Hsun; Liang, Po-Huang

    2012-08-01

    Isoprenoids are an intensive group of compounds made from isopentenyl diphosphate (IPP), catalyzed by prenyltransferases such as farnesyl diphosphate (FPP) cyclases, squalene synthase, protein farnesyltransferases and geranylgeranyltransferases, aromatic prenyltransferases as well as a group of prenyltransferases (cis- and trans-types) catalyzing consecutive condensation reactions of FPP with specific numbers of IPP to generate linear products with designate chain lengths. These prenyltransferases play significant biological functions and some of them are drug targets. In this review, structures, mechanisms, and inhibitors of a cis-prenyltransferase, undecaprenyl diphosphate synthase (UPPS) that mediates bacterial peptidoglycan biosynthesis, are summarized for comparison with the most related trans-prenyltransferases and other prenyltransferases.

  12. Oxidized low-density lipoprotein-induced periodontal inflammation is associated with the up-regulation of cyclooxygenase-2 and microsomal prostaglandin synthase 1 in human gingival epithelial cells

    SciTech Connect

    Nagahama, Yu; Obama, Takashi; Usui, Michihiko; Kanazawa, Yukari; Iwamoto, Sanju; Suzuki, Kazushige; Miyazaki, Akira; Yamaguchi, Tomohiro; Yamamoto, Matsuo; Itabe, Hiroyuki

    2011-10-07

    Highlights: {yields} OxLDL-induced responses in human gingival epithelial cells were studied. {yields} OxLDL enhanced the production of IL-8, IL-1{beta} and PGE{sub 2} in Ca9-22 cells. {yields} An NF-{kappa}B inhibitor suppressed the expression of COX-2 and mPGES1 induced by oxLDL. {yields} Unlike the case in macrophages, oxLDL did not increase the CD36 level. -- Abstract: Periodontitis is characterized by chronic gingival tissue inflammation, and inflammatory mediators such as IL-8 and prostaglandin E{sub 2} (PGE{sub 2}) are associated with disease progression. Previously we showed that oxidatively modified low-density lipoprotein (oxLDL) was present in gingival crevicular fluid. In this study, the role of oxLDL in the gingival epithelial cell inflammatory response was further investigated using Ca9-22 cells and primary human oral keratinocytes (HOK). Treatment of Ca9-22 cells and HOK with oxLDL induced an up-regulation of IL-8 and the PGE{sub 2}-producing enzymes, cyclooxygenase-2 and microsomal PGE{sub 2} synthase-1. These responses induced by oxLDL were significantly suppressed by a nuclear factor-kappa B (NF-{kappa}B) inhibitor. However, unlike the result in macrophages, oxLDL did not lead to an increase in CD36 expression in these two cells. These results suggest that oxLDL elicits gingival epithelial cell inflammatory responses through an activation of the NF-{kappa}B pathway. These data suggest a mechanistic link between periodontal disease and lipid metabolism-related disorders, including atherosclerosis.

  13. Lipocalin-type prostaglandin D synthase as a marker for the proliferative potential of melanocyte-lineage cells in the human skin.

    PubMed

    Shimanuki, Miwa; Takeda, Kazuhisa; Kawaguchi, Masakazu; Suzuki, Tamio; Shibahara, Shigeki

    2012-08-01

    Melanocytes in the human epidermis actively produce and secrete various substances, thereby contributing to the maintenance of the skin homeostasis. Lipocalin-type prostaglandin D synthase (L-PGDS) that catalyzes the formation of prostaglandin D(2) (PGD(2) ) may be one of such secreted molecules. Once secreted, L-PGDS functions as a transporter for lipophilic ligands, including all-trans retinoic acid (RA). L-PGDS, therefore, may possess pleiotropic functions in the skin through PGD(2) and RA. We aimed to identify the cell types that express L-PGDS in human skin and to explore the role of L-PGDS in the growth potential of melanocyte-lineage cells. Immunohistochemical analysis for L-PGDS expression was performed with the tissue sections that were prepared from five malignant melanomas, six nevus cell nevi and one Spitz nevus. Normal skin tissues adjacent to the excised melanoma tissues were also analyzed. L-PGDS is expressed in epidermal melanocytes but its expression is undetectable in keratinocytes. Moreover, L-PGDS is undetectable in most benign nevus cells, which may reflect the marginally accelerated proliferation of nevus cells. In contrast, L-PGDS is overexpressed in malignant melanomas, although the frequency of L-PGDS-positive cells was variable (15-50%), depending on the specimens. Lastly, RNA interference analysis against human L-PGDS was performed with short interfering RNA. Knockdown of L-PGDS expression with short interfering RNA in cultured cells suggests that L-PGDS may restrict cell proliferation through RA. In conclusion, L-PGDS expression may contribute to the restricted proliferation of epidermal melanocytes, but conversely its overexpression may reflect the dysregulated proliferation of melanoma cells.

  14. Reduced T-cell-dependent humoral immune response in microsomal prostaglandin E synthase-1 null mice is mediated by non-hematopoietic cells

    PubMed Central

    Kojima, Fumiaki; Frolov, Andrey; Matnani, Rahul; Woodward, Jerold G.; Crofford, Leslie J.

    2013-01-01

    Microsomal prostaglandin E synthase-1 (mPGES-1) is an inducible enzyme that specifically catalyzes the conversion of prostaglandin (PG)H2 to PGE2. We showed that mPGES-1 null mice had a significantly reduced incidence and severity of collagen-induced arthritis (CIA) compared to wild-type (WT) mice associated with a marked reduction in antibodies to type II collagen. In the present study, we further elucidated the role of mPGES-1 in the humoral immune response. Basal levels of serum IgM and IgG were significantly reduced in mPGES-1 null mice. Compared with WT mice, mPGES-1 null mice exhibited a significant reduction of hapten-specific serum antibodies in response to immunization with the T-cell dependent antigen DNP-KLH. Immunization with the T-cell independent type-1 antigen TNP-LPS or the T-cell independent type-2 antigen DNP-Ficoll revealed minimal differences between strains. Germinal center formation in the spleens of mPGES-1 null and WT mice were similar after immunization with DNP-KLH. To determine if the effect of mPGES-1 and PGE2 was localized to hematopoietic or non-hematopoietic cells, we generated bone marrow chimeras. We demonstrated that mPGES-1 deficiency in non-hematopoietic cells was the critical factor for reduced T-cell dependent antibody production. We conclude that mPGES-1 and PGE2-dependent phenotypic changes of non-hematopoietic/mesenchymal stromal cells play a key role in T-cell dependent humoral immune responses in vivo. These findings may have relevance to the pathogenesis of rheumatoid arthritis and other autoimmune inflammatory diseases associated with autoantibody formation. PMID:24127557

  15. Mammalian class Sigma glutathione S-transferases: catalytic properties and tissue-specific expression of human and rat GSH-dependent prostaglandin D2 synthases.

    PubMed Central

    Jowsey, I R; Thomson, A M; Flanagan, J U; Murdock, P R; Moore, G B; Meyer, D J; Murphy, G J; Smith, S A; Hayes, J D

    2001-01-01

    GSH-dependent prostaglandin D(2) synthase (PGDS) enzymes represent the only vertebrate members of class Sigma glutathione S-transferases (GSTs) identified to date. Complementary DNA clones encoding the orthologous human and rat GSH-dependent PGDS (hPGDS and rPGDS, respectively) have been expressed in Escherichia coli, and the recombinant proteins isolated by affinity chromatography. The purified enzymes were both shown to catalyse specifically the isomerization of prostaglandin (PG) H(2) to PGD(2). Each transferase also exhibited GSH-conjugating and GSH-peroxidase activities. The ability of hPGDS to catalyse the conjugation of aryl halides and isothiocyanates with GSH was found to be less than that of the rat enzyme. Whilst there is no difference between the enzymes with respect to their K(m) values for 1-chloro-2,4-dinitrobenzene, marked differences were found to exist with respect to their K(m) for GSH (8 mM versus 0.3 mM for hPGDS and rPGDS, respectively). Using molecular modelling techniques, amino acid substitutions have been identified in the N-terminal domain of these enzymes that lie outside the proposed GSH-binding site, which may explain these catalytic differences. The tissue-specific expression of PGDS also varies significantly between human and rat; amongst the tissues examined, variation in expression between the two species was most apparent in spleen and bone marrow. Differences in catalytic properties and tissue-specific expression of hPGDS and rPGDS appears to reflect distinct physiological roles for class Sigma GST between species. The evolution of divergent functions for the hPGDS and rPGDS is discussed in the context of the orthologous enzyme from chicken. PMID:11672424

  16. Natural fatty acid synthase inhibitors as potent therapeutic agents for cancers: A review.

    PubMed

    Zhang, Jia-Sui; Lei, Jie-Ping; Wei, Guo-Qing; Chen, Hui; Ma, Chao-Ying; Jiang, He-Zhong

    2016-09-01

    Context Fatty acid synthase (FAS) is the only mammalian enzyme to catalyse the synthesis of fatty acid. The expression level of FAS is related to cancer progression, aggressiveness and metastasis. In recent years, research on natural FAS inhibitors with significant bioactivities and low side effects has increasingly become a new trend. Herein, we present recent research progress on natural fatty acid synthase inhibitors as potent therapeutic agents. Objective This paper is a mini overview of the typical natural FAS inhibitors and their possible mechanism of action in the past 10 years (2004-2014). Method The information was collected and compiled through major databases including Web of Science, PubMed, and CNKI. Results Many natural products induce cancer cells apoptosis by inhibiting FAS expression, with fewer side effects than synthetic inhibitors. Conclusion Natural FAS inhibitors are widely distributed in plants (especially in herbs and foods). Some natural products (mainly phenolics) possessing potent biological activities and stable structures are available as lead compounds to synthesise promising FAS inhibitors.

  17. Crystallization and preliminary X-ray diffraction analysis of mouse prostaglandin F2α synthase, AKR1B3

    PubMed Central

    Takashima, Yasuhide; Hatanaka, Seika; Mizohata, Eiichi; Nagata, Nanae; Fukunishi, Yoshifumi; Matsumura, Hiroyoshi; Urade, Yoshihiro; Inoue, Tsuyoshi

    2011-01-01

    Aldo-keto reductase 1B3 (AKR1B3) catalyzes the NADPH-dependent reduction of prostaglandin H2 (PGH2), which is a common intermediate of various prostanoids, to form PGF2α. AKR1B3 also reduces PGH2 to PGD2 in the absence of NADPH. AKR1B3 produced in Escherichia coli was crystallized in complex with NADPH by the sitting-drop vapour-diffusion method. The crystal was tetragonal, belonging to space group P41212 or P43212, with unit-cell parameters a = b = 107.62, c = 120.76 Å. X-ray diffraction data were collected to 2.4 Å resolution at 100 K using a synchrotron-radiation source. PMID:22139184

  18. Haplofungins, new inositol phosphorylceramide synthase inhibitors, from Lauriomyces bellulus SANK 26899 II. Structure elucidation.

    PubMed

    Ohnuki, Takashi; Yano, Tatsuya; Takatsu, Toshio

    2009-10-01

    Eight new inositol phosphorylceramide synthase inhibitors: haplofungin A, B, C, D, E, F, G and H, were discovered in a culture broth of the fungus Lauriomyces bellulus SANK 26899. The planar structures for these haplofungins were elucidated by various spectroscopic analyses and a GC/MS analysis of their degradation products. All eight compounds were found to comprise an arabinonic acid moiety linked through an ester bond to a modified long alkyl chain.

  19. CJ-15,183, a new inhibitor of squalene synthase produced by a fungus, Aspergillus aculeatus.

    PubMed

    Watanabe, S; Hirai, H; Ishiguro, M; Kambara, T; Kojima, Y; Matsunaga, T; Nishida, H; Suzuki, Y; Sugiura, A; Harwood, H J; Huang, L H; Kojima, N

    2001-11-01

    A new squalene synthase (SSase) inhibitor, CJ-15,183 (I) was isolated from the fermentation broth of a fungus, Aspergillus aculeatus CL38916. The compound potently inhibited rat liver and Candida albicans microsomal SSases and also inhibited the human enzyme. It also showed antifungal activities against filamentous fungi and a yeast. The structure was determined to be an aliphatic tetracarboxylic acid compound consisting of an alkyl gamma-lactone, malic acid and isocitric acid moieties by spectroscopic studies.

  20. Discovery and Characterization of a Class of Pyrazole Inhibitors of Bacterial Undecaprenyl Pyrophosphate Synthase.

    PubMed

    Concha, Nestor; Huang, Jianzhong; Bai, Xiaopeng; Benowitz, Andrew; Brady, Pat; Grady, LaShadric C; Kryn, Luz Helena; Holmes, David; Ingraham, Karen; Jin, Qi; Pothier Kaushansky, Laura; McCloskey, Lynn; Messer, Jeffrey A; O'Keefe, Heather; Patel, Amish; Satz, Alexander L; Sinnamon, Robert H; Schneck, Jessica; Skinner, Steve R; Summerfield, Jennifer; Taylor, Amy; Taylor, J David; Evindar, Ghotas; Stavenger, Robert A

    2016-08-11

    Undecaprenyl pyrophosphate synthase (UppS) is an essential enzyme in bacterial cell wall synthesis. Here we report the discovery of Staphylococcus aureus UppS inhibitors from an Encoded Library Technology screen and demonstrate binding to the hydrophobic substrate site through cocrystallography studies. The use of bacterial strains with regulated uppS expression and inhibitor resistant mutant studies confirmed that the whole cell activity was the result of UppS inhibition, validating UppS as a druggable antibacterial target. PMID:27379833

  1. 15-Deoxy-{delta}{sup 12,14}-prostaglandin J{sub 2}-induced down-regulation of endothelial nitric oxide synthase in association with HSP70 induction

    SciTech Connect

    Hwang, Jinah; Lee, Hyun-Il; Chang, Young-Sun; Lee, Soo Jae; Kim, Kwang Pyo; Park, Sang Ick . E-mail: parksi@nih.go.kr

    2007-05-25

    A natural ligand of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), 15-deoxy-{delta}{sup 12,14}-prostaglandin J{sub 2} (15d-PGJ{sub 2}), decreases endothelial nitric oxide synthase (eNOS) expression by an unknown mechanism. Here we found that 15d-PGJ{sub 2}-induced eNOS reduction is inversely associated with heat shock protein 70 (HSP70) induction in endothelial cells. Treatment of cells with 15d-PGJ{sub 2} decreased eNOS protein expression in a concentration- and time-dependent manner, but independently of PPAR{gamma} with no effect on mRNA levels. Although 15d-PGJ{sub 2} elicited endothelial apoptosis, inhibition of both pan-caspases and cathepsins failed to reverse reduction of eNOS protein. Interestingly, we observed that 15d-PGJ{sub 2} induced HSP70 in a dose-dependent manner. Immunoprecipitation and heat shock treatment demonstrated that eNOS reduction was strongly related to HSP70 induction. Cellular fractionation revealed that treatment with 15d-PGJ{sub 2} increased eNOS distribution 2.5-fold from soluble to insoluble fractions. These findings provide new insights into mechanisms whereby eNOS regulation by 15d-PGJ{sub 2} is related to HSP70 induction.

  2. Modulation of endothelin-1 in normal human keratinocytes by UVA1/B radiations, prostaglandin E2 and peptidase inhibitors.

    PubMed

    Pernet, I; Mayoux, C; Trompezinski, S; Schmitt, D; Viac, J

    2000-12-01

    In the skin, keratinocytes synthesize and secrete endothelin-(ET-1), a potent vasoconstrictor peptide which acts also as a growth factor for most skin cells. The aim of the study was to test the effects of UVA1 and the associations UVA1/B on the expression of ET-1 in normal human keratinocytes and to determine whether exogenously added prostaglandin E2 (PGE2) regulated ET-1 expression. As ET-1 is susceptible to degradation, we also evaluated whether ET-1 secretion was modulated by peptidase inhibitors. Our results showed that UVA1 (365 nm) did not modify the levels of preproET-1 mRNA and protein. Moreover, the associations UVA1+UVB or UVB+UVA1 down-regulated the overexpression of secreted ET-1 induced by UVB alone. PGE2 at 10(-5) M reduced the expression of ET-1 at the mRNA and protein levels but did not exert any significant modification at lower concentrations from 10(-10) to 10(6) M. Phosphoramidon, an endothelin converting enzyme (ECE) inhibitor, drastically decreased the amount of ET-1 accumulating in the culture medium in basal conditions or after UVB irradiation. Conversely, thiorphan, a specific inhibitor of neutral endopeptidase (NEP), rather increased the levels of ET-1 secretion mainly after UVB irradiation. Taken together, the results showed that normal human keratinocytes secrete and partly degrade ET-1 through ECE and NEP pathways and pointed out a differential regulation of ET-1 by UVB and UVA1 radiations without any noticeable role for PGE2.

  3. Hydroxybenzaldoximes are d-GAP-competitive inhibitors of E. coli 1-deoxy-d-xylulose-5-phosphate synthase

    PubMed Central

    Bartee, David; Morris, Francine; Al-khouja, Amer

    2015-01-01

    1-Deoxy-d-xylulose 5-phosphate (DXP) synthase is the first enzyme in the methylerythritol phosphate pathway to essential isoprenoids in pathogenic bacteria and apicomplexan parasites. In bacterial pathogens, DXP lies at a metabolic branchpoint, serving also as a precursor in the biosynthesis of vitamins B1 and B6 which are critical for central metabolism. Toward identifying novel bisubstrate analog inhibitors that exploit the large active site and distinct mechanism of DXP synthase, a library of aryl mixed oximes was prepared and evaluated. Trihydroxybenzaldoximes emerged as reversible, low micromolar inhibitors, competitive against d-glyceraldehyde 3-phosphate (d-GAP) and either uncompetitive or noncompetitive against pyruvate. Hydroxybenzaldoximes are the first class of d-GAP-competitive DXP synthase inhibitors offering new tools for mechanistic studies of DXP synthase and a new direction for the development of antimicrobial agents targeting isoprenoid biosynthesis. PMID:26174207

  4. Hydroxybenzaldoximes Are D-GAP-Competitive Inhibitors of E. coli 1-Deoxy-D-Xylulose-5-Phosphate Synthase.

    PubMed

    Bartee, David; Morris, Francine; Al-Khouja, Amer; Freel Meyers, Caren L

    2015-08-17

    1-Deoxy-D-xylulose 5-phosphate (DXP) synthase is the first enzyme in the methylerythritol phosphate pathway to essential isoprenoids in pathogenic bacteria and apicomplexan parasites. In bacterial pathogens, DXP lies at a metabolic branch point, serving also as a precursor in the biosynthesis of vitamins B1 and B6, which are critical for central metabolism. In an effort to identify new bisubstrate analogue inhibitors that exploit the large active site and distinct mechanism of DXP synthase, a library of aryl mixed oximes was prepared and evaluated. Trihydroxybenzaldoximes emerged as reversible, low-micromolar inhibitors, competitive against D-glyceraldehyde 3-phosphate (D-GAP) and either uncompetitive or noncompetitive against pyruvate. Hydroxybenzaldoximes are the first class of D-GAP-competitive DXP synthase inhibitors, offering new tools for mechanistic studies of DXP synthase and a new direction for the development of antimicrobial agents targeting isoprenoid biosynthesis.

  5. Evaluation of Improved Glycogen Synthase Kinase-3α Inhibitors in Models of Acute Myeloid Leukemia

    PubMed Central

    Neumann, Theresa; Benajiba, Lina; Göring, Stefan; Stegmaier, Kimberly; Schmidt, Boris

    2016-01-01

    The challenge for Glycogen Synthase Kinase-3 (GSK-3) inhibitor design lies in achieving high selectivity for one isoform over the other. The therapy of certain diseases, such as acute myeloid leukemia (AML) may require α-isoform specific targeting. The scorpion shaped GSK-3 inhibitors developed by our group achieved the highest GSK-3α selectivity reported so far, but suffered from insufficient aqueous solubility. This work presents the solubility-driven optimization of our isoform-selective inhibitors using a scorpion shaped lead. Among 15 novel compounds, compound 27 showed high activity against GSK-3α/β with the highest GSK-3α selectivity reported to date. Compound 27 was profiled for bioavailability and toxicity in a zebrafish embryo phenotype assay. Selective GSK-3α targeting in AML cell lines was achieved with compound 27, resulting in a strong differentiation phenotype and colony formation impairment, confirming the potential of GSK-3α inhibition in AML therapy. PMID:26496242

  6. Efficient synthesis of apricoxib, CS-706, a selective cyclooxygenase-2 inhibitor, and evaluation of inhibition of prostaglandin E2 production in inflammatory breast cancer cells.

    PubMed

    Mandal, Pijus K; Freiter, Eric M; Bagsby, Allison L; Robertson, Fredika M; McMurray, John S

    2011-10-15

    An efficient synthesis of apricoxib (CS-706), a selective cyclooxygenase inhibitor, was developed using copper catalyzed homoallylic ketone formation from methyl 4-ethoxybenzoate followed by ozonolysis to an aldehyde, and condensation with sulfanilamide. This method provided multi-gram access of aprocoxib in good yield. Apricoxib exhibited potency equal to celecoxib at inhibition of prostaglandin E2 synthesis in two inflammatory breast cancer cell lines. PMID:21903394

  7. Role of zinc in isoform-selective inhibitor binding to neuronal nitric oxide synthase .

    PubMed

    Delker, Silvia L; Xue, Fengtian; Li, Huiying; Jamal, Joumana; Silverman, Richard B; Poulos, Thomas L

    2010-12-28

    In previous studies [Delker, S. L., et al. (2010), J. Am. Chem. Soc. 132, 5437-5442], we determined the crystal structures of neuronal nitric oxide synthase (nNOS) in complex with nNOS-selective chiral pyrrolidine inhibitors, designed to have an aminopyridine group bound over the heme where it can electrostatically interact with the conserved active site Glu residue. However, in addition to the expected binding mode with the (S,S)-cis inhibitors, an unexpected "flipped" orientation was observed for the (R,R)-cis enantiomers. In the flipped mode, the aminopyridine extends out of the active site where it interacts with one heme propionate. This prompted us to design and synthesize symmetric "double-headed" inhibitors with an aminopyridine at each end of a bridging ring structure [Xue, F., Delker, S. L., Li, H., Fang, J., Jamal, J., Martásek, P., Roman, L. J., Poulos, T. L., and Silverman, R. B. Symmetric double-headed aminopyridines, a novel strategy for potent and membrane-permeable inhibitors of neuronal nitric oxide synthase. J. Med. Chem. (submitted for publication)]. One aminopyridine should interact with the active site Glu and the other with the heme propionate. Crystal structures of these double-headed aminopyridine inhibitors in complexes with nNOS show unexpected and significant protein and heme conformational changes induced by inhibitor binding that result in removal of the tetrahydrobiopterin (H(4)B) cofactor and creation of a new Zn(2+) site. These changes are due to binding of a second inhibitor molecule that results in the displacement of H(4)B and the placement of the inhibitor pyridine group in position to serve as a Zn(2+) ligand together with Asp, His, and a chloride ion. Binding of the second inhibitor molecule and generation of the Zn(2+) site do not occur in eNOS. Structural requirements for creation of the new Zn(2+) site in nNOS were analyzed in detail. These observations open the way for the potential design of novel inhibitors selective

  8. ATP Synthase and the Actions of Inhibitors Utilized To Study Its Roles in Human Health, Disease, and Other Scientific Areas

    PubMed Central

    Hong, Sangjin; Pedersen, Peter L.

    2008-01-01

    Summary: ATP synthase, a double-motor enzyme, plays various roles in the cell, participating not only in ATP synthesis but in ATP hydrolysis-dependent processes and in the regulation of a proton gradient across some membrane-dependent systems. Recent studies of ATP synthase as a potential molecular target for the treatment of some human diseases have displayed promising results, and this enzyme is now emerging as an attractive molecular target for the development of new therapies for a variety of diseases. Significantly, ATP synthase, because of its complex structure, is inhibited by a number of different inhibitors and provides diverse possibilities in the development of new ATP synthase-directed agents. In this review, we classify over 250 natural and synthetic inhibitors of ATP synthase reported to date and present their inhibitory sites and their known or proposed modes of action. The rich source of ATP synthase inhibitors and their known or purported sites of action presented in this review should provide valuable insights into their applications as potential scaffolds for new therapeutics for human and animal diseases as well as for the discovery of new pesticides and herbicides to help protect the world's food supply. Finally, as ATP synthase is now known to consist of two unique nanomotors involved in making ATP from ADP and Pi, the information provided in this review may greatly assist those investigators entering the emerging field of nanotechnology. PMID:19052322

  9. Nonpolyglutamatable antifolates as inhibitors of thymidylate synthase (TS) and potential antitumour agents.

    PubMed

    Bavetsias, V; Jackman, A L

    1998-08-01

    Thymidylate synthase (TS), an enzyme that catalyses the conversion of dUMP to dTMP, has been the focus of interest as a target in cancer chemotherapy for more than two decades. Over the last 10 years much research has been devoted to the design and development of nonpolyglutamatable inhibitors of TS as antitumour agents, mainly to over-come resistance due to unfavourable expression of folylpolyglutamate synthetase (FPGS). Lipophilic inhibitors of the enzyme were expected not to depend on the reduced folate carrier transporter (RFC) for cellular uptake, thus avoiding resistance due to an impaired RFC. Compounds of this type can be classified in three groups: A: nonclassical lipophilic inhibitors of TS, mainly folate-based analogues lacking the glutamate side chain; B: folate-based analogues in which the glutamate side chain has been modified in such a way that polyglutamation is precluded; and C: nonpolyglutamatable glutamate-containing inhibitors of TS. Compounds of group A included 5- or 6-substituted quinazolin-4-ones, benzo[flquinazolines, imidazotetrahydroquinoline- and benz[cd]indole-based inhibitors. The second group is mainly related to a series of g-linked dipeptide derivatives of ICIl98583, or analogues of this inhibitor where the glutamate residue was replaced with a range of a-amino acids. The third group is concerned with some 7-substituted derivatives of ICI198583 and the pyrrolo[3, 2-d]pyrimidine-based inhibitor 175. A large number of structurally diverse nonpolyglutamatable inhibitors of TS were synthesised some of which were potent inhibitors of the enzyme (human or E. coli) and in vitro cell growth. Three compounds, i.e. 49 (AG 337), 83 (AG 331), 123 (ZD9331) have reached the stage of clinical evaluation.

  10. Allosteric inhibitors of inducible nitric oxide synthase dimerization discovered via combinatorial chemistry

    PubMed Central

    McMillan, Kirk; Adler, Marc; Auld, Douglas S.; Baldwin, John J.; Blasko, Eric; Browne, Leslie J.; Chelsky, Daniel; Davey, David; Dolle, Ronald E.; Eagen, Keith A.; Erickson, Shawn; Feldman, Richard I.; Glaser, Charles B.; Mallari, Cornell; Morrissey, Michael M.; Ohlmeyer, Michael H. J.; Pan, Gonghua; Parkinson, John F.; Phillips, Gary B.; Polokoff, Mark A.; Sigal, Nolan H.; Vergona, Ronald; Whitlow, Marc; Young, Tish A.; Devlin, James J.

    2000-01-01

    Potent and selective inhibitors of inducible nitric oxide synthase (iNOS) (EC 1.14.13.39) were identified in an encoded combinatorial chemical library that blocked human iNOS dimerization, and thereby NO production. In a cell-based iNOS assay (A-172 astrocytoma cells) the inhibitors had low-nanomolar IC50 values and thus were >1,000-fold more potent than the substrate-based direct iNOS inhibitors 1400W and N-methyl-l-arginine. Biochemical studies confirmed that inhibitors caused accumulation of iNOS monomers in mouse macrophage RAW 264.7 cells. High affinity (Kd ≈ 3 nM) of inhibitors for isolated iNOS monomers was confirmed by using a radioligand binding assay. Inhibitors were >1,000-fold selective for iNOS versus endothelial NOS dimerization in a cell-based assay. The crystal structure of inhibitor bound to the monomeric iNOS oxygenase domain revealed inhibitor–heme coordination and substantial perturbation of the substrate binding site and the dimerization interface, indicating that this small molecule acts by allosterically disrupting protein–protein interactions at the dimer interface. These results provide a mechanism-based approach to highly selective iNOS inhibition. Inhibitors were active in vivo, with ED50 values of <2 mg/kg in a rat model of endotoxin-induced systemic iNOS induction. Thus, this class of dimerization inhibitors has broad therapeutic potential in iNOS-mediated pathologies. PMID:10677491

  11. Substituted Pyrrolo[2,3-d]pyrimidines as Cryptosporidium hominis Thymidylate Synthase Inhibitors

    PubMed Central

    Kumar, Vidya P.; Frey, Kathleen M.; Wang, Yiqiang; Jain, Hitesh K.; Gangjee, Aleem; Anderson, Karen S.

    2013-01-01

    Cryptosporidiosis, a gastrointestinal disease caused by a protozoan Cryptosporidium hominis is often fatal in immunocompromised individuals. There is little clinical data to show that the existing treatment by nitazoxanide and paromomycin is effective in immunocompromised individuals1, 2. Thymidylate synthase (TS) and dihydrofolate reductase (DHFR) are essential enzymes in the folate biosynthesis pathway and are well established as drug targets in cancer and malaria. A novel series of classical antifolates, 2-amino-4-oxo-5-substituted pyrrolo[2,3-d]pyrimidines have been evaluated as Cryptosporidium hominis thymidylate synthase (ChTS) inhibitors. Crystal structure in complex with the most potent compound, a 2’-chlorophenyl with a sulfur bridge with a Ki of 8.83 ± 0.67 nM is discussed in terms of several Van de Waals, hydrophobic and hydrogen bond interactions with the protein residues and the substrate analog 5-fluorodeoxyuridine monophosphate. Of these interactions, two interactions with the non-conserved residues (A287 and S290) offer an opportunity to develop ChTS specific inhibitors. Compound 6 serves as a lead compound for analog design and its crystal structure provides clues for the design of ChTS specific inhibitors. PMID:23927969

  12. Structure-Based Inhibitors Exhibit Differential Activities against Helicobacter pylori and Escherichia coli Undecaprenyl Pyrophosphate Synthases

    PubMed Central

    Kuo, Chih-Jung; Guo, Rey-Ting; Lu, I-Lin; Liu, Hun-Ge; Wu, Su-Ying; Ko, Tzu-Ping; Wang, Andrew H.-J.; Liang, Po-Huang

    2008-01-01

    Helicobacter pylori colonizes the human gastric epithelium and causes diseases such as gastritis, peptic ulcers, and stomach cancer. Undecaprenyl pyrophosphate synthase (UPPS), which catalyzes consecutive condensation reactions of farnesyl pyrophosphate with eight isopentenyl pyrophosphate to form lipid carrier for bacterial peptidoglycan biosynthesis, represents a potential target for developing new antibiotics. In this study, we solved the crystal structure of H. pylori UPPS and performed virtual screening of inhibitors from a library of 58,635 compounds. Two hits were found to exhibit differential activities against Helicobacter pylori and Escherichia coli UPPS, giving the possibility of developing antibiotics specially targeting pathogenic H. pylori without killing the intestinal E. coli. PMID:18382620

  13. Human Lipocalin-Type Prostaglandin D Synthase-Based Drug Delivery System for Poorly Water-Soluble Anti-Cancer Drug SN-38.

    PubMed

    Nakatsuji, Masatoshi; Inoue, Haruka; Kohno, Masaki; Saito, Mayu; Tsuge, Syogo; Shimizu, Shota; Ishida, Atsuko; Ishibashi, Osamu; Inui, Takashi

    2015-01-01

    Lipocalin-type prostaglandin D synthase (L-PGDS) is a member of the lipocalin superfamily, which is composed of secretory transporter proteins, and binds a wide variety of small hydrophobic molecules. Using this function, we have reported the feasibility of using L-PGDS as a novel drug delivery vehicle for poorly water-soluble drugs. In this study, we show the development of a drug delivery system using L-PGDS, one that enables the direct clinical use of 7-ethyl-10-hydroxy-camptothecin (SN-38), a poorly water-soluble anti-cancer drug. In the presence of 2 mM L-PGDS, the concentration of SN-38 in PBS increased 1,130-fold as compared with that in PBS. Calorimetric experiments revealed that L-PGDS bound SN-38 at a molecular ratio of 1:3 with a dissociation constant value of 60 μM. The results of an in vitro growth inhibition assay revealed that the SN-38/L-PGDS complexes showed high anti-tumor activity against 3 human cancer cell lines, i.e., Colo201, MDA-MB-231, and PC-3 with a potency similar to that of SN-38 used alone. The intravenous administration of SN-38/L-PGDS complexes to mice bearing Colo201 tumors showed a pronounced anti-tumor effect. Intestinal mucositis, which is one of the side effects of this drug, was not observed in mice administered SN-38/L-PGDS complexes. Taken together, L-PGDS enables the direct usage of SN-38 with reduced side effects.

  14. Hematopoietic prostaglandin D2 synthase controls the onset and resolution of acute inflammation through PGD2 and 15-deoxyDelta12 14 PGJ2.

    PubMed

    Rajakariar, Ravindra; Hilliard, Mark; Lawrence, Toby; Trivedi, Seema; Colville-Nash, Paul; Bellingan, Geoff; Fitzgerald, Desmond; Yaqoob, Muhammad M; Gilroy, Derek W

    2007-12-26

    Hematopoietic prostaglandin D(2) synthase (hPGD(2)S) metabolizes cyclooxygenase (COX)-derived PGH(2) to PGD(2) and 15-deoxyDelta(12-14) PGJ(2) (15d-PGJ(2)). Unlike COX, the role of hPGD(2)S in host defense is ambiguous. PGD(2) can be either pro- or antiinflammatory depending on disease etiology, whereas the existence of 15d-PGJ(2) and its relevance to pathophysiology remain controversial. Herein, studies on hPGD(2)S KO mice reveal that 15d-PGJ(2) is synthesized in a self-resolving peritonitis, detected by using liquid chromatography-tandem MS. Together with PGD(2) working on its DP1 receptor, 15d-PGJ(2) controls the balance of pro- vs. antiinflammatory cytokines that regulate leukocyte influx and monocyte-derived macrophage efflux from the inflamed peritoneal cavity to draining lymph nodes leading to resolution. Specifically, inflammation in hPGD(2)S KOs is more severe during the onset phase arising from a substantial cytokine imbalance resulting in enhanced polymorphonuclear leukocyte and monocyte trafficking. Moreover, resolution is impaired, characterized by macrophage and surprisingly lymphocyte accumulation. Data from this work place hPGD(2)S at the center of controlling the onset and the resolution of acute inflammation where it acts as a crucial checkpoint controller of cytokine/chemokine synthesis as well as leukocyte influx and efflux. Here, we provide definitive proof that 15d-PGJ(2) is synthesized during mammalian inflammatory responses, and we highlight DP1 receptor activation as a potential antiinflammatory strategy. PMID:18077391

  15. TIS10, a phorbol ester tumor promoter-inducible mRNA from Swiss 3T3 cells, encodes a novel prostaglandin synthase/cyclooxygenase homologue.

    PubMed

    Kujubu, D A; Fletcher, B S; Varnum, B C; Lim, R W; Herschman, H R

    1991-07-15

    TIS10 is a primary response gene whose cDNA was cloned as a result of its rapid, superinducible expression in Swiss 3T3 cells in response to 12-O-Tetradecanoylphorbol-13-acetate. The 5'-untranslated region of the 3.9-kilobase TIS10 message contains only 124 nucleotides, whereas the 3'-untranslated region is almost 2 kilobases in length. Within this long 3' region, there are multiple repeats of the sequence ATTTA, a sequence often present in rapidly degraded mRNA species. Primer extension revealed that the TIS10 cDNA begins 16 base pairs downstream of the transcription start site for the TIS10 gene. The TIS10 cDNA encodes a predicted protein of 604 amino acids. A computer search identified striking similarities between the predicted TIS10 protein product and the murine, sheep, and human prostaglandin synthase/cyclooxygenase proteins. The TIS10 protein has many of the same conserved amino acids that are thought to be important for cyclooxygenase function. TIS10 mRNA is undetectable by Northern analysis in quiescent 3T3 cells. The TIS10 gene is rapidly and transiently induced by forskolin and serum, as well as by 12-O-tetradecanoylphorbol-13-acetate, in Swiss 3T3 cells. These agents elicit far more dramatic changes in TIS10 mRNA levels than in cyclooxygenase mRNA levels. The expression of the TIS10 gene appears to be highly cell type-restricted in cultured cell lines; of 12 cell lines tested under superinducing conditions, only the rodent embryonic Swiss 3T3 and Rat1 cell lines expressed TIS10 gene.

  16. CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil

    NASA Astrophysics Data System (ADS)

    Almqvist, Helena; Axelsson, Hanna; Jafari, Rozbeh; Dan, Chen; Mateus, André; Haraldsson, Martin; Larsson, Andreas; Molina, Daniel Martinez; Artursson, Per; Lundbäck, Thomas; Nordlund, Pär

    2016-03-01

    Target engagement is a critical factor for therapeutic efficacy. Assessment of compound binding to native target proteins in live cells is therefore highly desirable in all stages of drug discovery. We report here the first compound library screen based on biophysical measurements of intracellular target binding, exemplified by human thymidylate synthase (TS). The screen selected accurately for all the tested known drugs acting on TS. We also identified TS inhibitors with novel chemistry and marketed drugs that were not previously known to target TS, including the DNA methyltransferase inhibitor decitabine. By following the cellular uptake and enzymatic conversion of known drugs we correlated the appearance of active metabolites over time with intracellular target engagement. These data distinguished a much slower activation of 5-fluorouracil when compared with nucleoside-based drugs. The approach establishes efficient means to associate drug uptake and activation with target binding during drug discovery.

  17. CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil.

    PubMed

    Almqvist, Helena; Axelsson, Hanna; Jafari, Rozbeh; Dan, Chen; Mateus, André; Haraldsson, Martin; Larsson, Andreas; Martinez Molina, Daniel; Artursson, Per; Lundbäck, Thomas; Nordlund, Pär

    2016-01-01

    Target engagement is a critical factor for therapeutic efficacy. Assessment of compound binding to native target proteins in live cells is therefore highly desirable in all stages of drug discovery. We report here the first compound library screen based on biophysical measurements of intracellular target binding, exemplified by human thymidylate synthase (TS). The screen selected accurately for all the tested known drugs acting on TS. We also identified TS inhibitors with novel chemistry and marketed drugs that were not previously known to target TS, including the DNA methyltransferase inhibitor decitabine. By following the cellular uptake and enzymatic conversion of known drugs we correlated the appearance of active metabolites over time with intracellular target engagement. These data distinguished a much slower activation of 5-fluorouracil when compared with nucleoside-based drugs. The approach establishes efficient means to associate drug uptake and activation with target binding during drug discovery. PMID:27010513

  18. Development Of Nitric Oxide Synthase Inhibitors for Neurodegeneration and Neuropathic Pain

    PubMed Central

    Mukherjee, Paramita; Cinelli, Maris A.; Kang, Soosung; Silverman, Richard B.

    2014-01-01

    Nitric oxide (NO) is an important signaling molecule in the human body, playing a crucial role in cell and neuronal communication, regulation of blood pressure, and in immune activation. However, overproduction of NO by the neuronal isoform of nitric oxide synthase (nNOS)is one of the fundamental causes underlying neurodegenerative disorders and neuropathic pain. Therefore, developing small molecules for selective inhibition of nNOS over related isoforms(eNOS and iNOS) is therapeutically desirable. The aims of this review focus on the regulation and dysregulation of NO signaling, the role of NO in neurodegeneration and pain, the structure and mechanism of nNOS, and the use of this information to design selective inhibitors of this enzyme. Structure-based drug design, the bioavailability and pharmacokinetics of these inhibitors, and extensive target validation through animal studies are addressed. PMID:24549364

  19. Fatty acid synthase inhibitors of phenolic constituents isolated from Garcinia mangostana.

    PubMed

    Jiang, He Zhong; Quan, Xiao Fang; Tian, Wei Xi; Hu, Jiang Miao; Wang, Peng Cheng; Huang, Sheng Zhuo; Cheng, Zhong Quan; Liang, Wen Juan; Zhou, Jun; Ma, Xiao Feng; Zhao, You Xing

    2010-10-15

    Natural inhibitors of fatty acid synthase (FAS) are emerging as potential therapeutic agents to treat cancer and obesity. The bioassay-guided chemical investigation of the hulls of Garcinia mangostana led to the isolation of 13 phenolic compounds (1-13) mainly including xanthone and benzophenone, in which compounds 7, 8, 9, 10, and 11 were isolated from this plant for the first time and compound 9 was a new natural product. These isolates possess strong inhibitory activity of FAS with the IC(50) values ranging from 1.24 to 91.07 μM. The study indicates that two types of natural products, xanthones and benzophenones, could be considered as promising FAS inhibitors.

  20. CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil

    PubMed Central

    Almqvist, Helena; Axelsson, Hanna; Jafari, Rozbeh; Dan, Chen; Mateus, André; Haraldsson, Martin; Larsson, Andreas; Molina, Daniel Martinez; Artursson, Per; Lundbäck, Thomas; Nordlund, Pär

    2016-01-01

    Target engagement is a critical factor for therapeutic efficacy. Assessment of compound binding to native target proteins in live cells is therefore highly desirable in all stages of drug discovery. We report here the first compound library screen based on biophysical measurements of intracellular target binding, exemplified by human thymidylate synthase (TS). The screen selected accurately for all the tested known drugs acting on TS. We also identified TS inhibitors with novel chemistry and marketed drugs that were not previously known to target TS, including the DNA methyltransferase inhibitor decitabine. By following the cellular uptake and enzymatic conversion of known drugs we correlated the appearance of active metabolites over time with intracellular target engagement. These data distinguished a much slower activation of 5-fluorouracil when compared with nucleoside-based drugs. The approach establishes efficient means to associate drug uptake and activation with target binding during drug discovery. PMID:27010513

  1. Lysine-specific demethylase 1-mediated demethylation of histone H3 lysine 9 contributes to interleukin 1β-induced microsomal prostaglandin E synthase 1 expression in human osteoarthritic chondrocytes

    PubMed Central

    2014-01-01

    Introduction Microsomal prostaglandin E synthase 1 (mPGES-1) catalyzes the terminal step in the biosynthesis of PGE2, a critical mediator in the pathophysiology of osteoarthritis (OA). Histone methylation plays an important role in epigenetic gene regulation. In this study, we investigated the roles of histone H3 lysine 9 (H3K9) methylation in interleukin 1β (IL-1β)-induced mPGES-1 expression in human chondrocytes. Methods Chondrocytes were stimulated with IL-1β, and the expression of mPGES-1 mRNA was evaluated using real-time RT-PCR. H3K9 methylation and the recruitment of the histone demethylase lysine-specific demethylase 1 (LSD1) to the mPGES-1 promoter were evaluated using chromatin immunoprecipitation assays. The role of LSD1 was further evaluated using the pharmacological inhibitors tranylcypromine and pargyline and small interfering RNA (siRNA)-mediated gene silencing. The LSD1 level in cartilage was determined by RT-PCR and immunohistochemistry. Results The induction of mPGES-1 expression by IL-1β correlated with decreased levels of mono- and dimethylated H3K9 at the mPGES-1 promoter. These changes were concomitant with the recruitment of the histone demethylase LSD1. Treatment with tranylcypromine and pargyline, which are potent inhibitors of LSD1, prevented IL-1β-induced H3K9 demethylation at the mPGES-1 promoter and expression of mPGES-1. Consistently, LSD1 gene silencing with siRNA prevented IL-1β-induced H3K9 demethylation and mPGES-1 expression, suggesting that LSD1 mediates IL-1β-induced mPGES-1 expression via H3K9 demethylation. We show that the level of LSD1 was elevated in OA compared to normal cartilage. Conclusion These results indicate that H3K9 demethylation by LSD1 contributes to IL-1β-induced mPGES-1 expression and suggest that this pathway could be a potential target for pharmacological intervention in the treatment of OA and possibly other arthritic conditions. PMID:24886859

  2. A Small-Molecule Screening Platform for the Discovery of Inhibitors of Undecaprenyl Diphosphate Synthase.

    PubMed

    Czarny, Tomasz L; Brown, Eric D

    2016-07-01

    The bacterial cell wall has long been a celebrated target for antibacterial drug discovery due to its critical nature in bacteria and absence in mammalian systems. At the heart of the cell wall biosynthetic pathway lies undecaprenyl phosphate (Und-P), the lipid-linked carrier upon which the bacterial cell wall is built. This study exploits recent insights into the link between late-stage wall teichoic acid inhibition and Und-P production, in Gram-positive organisms, to develop a cell-based small-molecule screening platform that enriches for inhibitors of undecaprenyl diphosphate synthase (UppS). Screening a chemical collection of 142,000 small molecules resulted in the identification of 6 new inhibitors of UppS. To date, inhibitors of UppS have generally shown off-target effects on membrane potential due to their physical-chemical characteristics. We demonstrate that MAC-0547630, one of the six inhibitors identified, exhibits selective, nanomolar inhibition against UppS without off-target effects on membrane potential. Such characteristics make it a unique chemical probe for exploring the inhibition of UppS in bacterial cell systems. PMID:27626101

  3. Increase of 20-HETE synthase after brain ischemia in rats revealed by PET study with 11C-labeled 20-HETE synthase-specific inhibitor

    PubMed Central

    Kawasaki, Toshiyuki; Marumo, Toshiyuki; Shirakami, Keiko; Mori, Tomoko; Doi, Hisashi; Suzuki, Masaaki; Watanabe, Yasuyoshi; Chaki, Shigeyuki; Nakazato, Atsuro; Ago, Yukio; Hashimoto, Hitoshi; Matsuda, Toshio; Baba, Akemichi; Onoe, Hirotaka

    2012-01-01

    20-Hydroxyeicosatetraenoic acid (20-HETE), an arachidonic acid metabolite known to be produced after cerebral ischemia, has been implicated in ischemic and reperfusion injury by mediating vasoconstriction. To develop a positron emission tomography (PET) probe for 20-HETE synthase imaging, which might be useful for monitoring vasoconstrictive processes in patients with brain ischemia, we synthesized a 11C-labeled specific 20-HETE synthase inhibitor, N′(4-dimethylaminohexyloxy)phenyl imidazole ([11C]TROA). Autoradiographic study showed that [11C]TROA has high-specific binding in the kidney and liver consistent with the previously reported distribution of 20-HETE synthase. Using transient middle cerebral artery occlusion in rats, PET study showed significant increases in the binding of [11C]TROA in the ipsilateral hemisphere of rat brains after 7 and 10 days, which was blocked by co-injection of excess amounts of TROA (10 mg/kg). The increased [11C]TROA binding on the ipsilateral side returned to basal levels within 14 days. In addition, quantitative real-time PCR revealed that increased expression of 20-HETE synthase was only shown on the ipsilateral side on day 7. These results indicate that [11C]TROA might be a useful PET probe for imaging of 20-HETE synthase in patients with cerebral ischemia. PMID:22669478

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

    PubMed Central

    Sprenger, Janina; Carey, Jannette; Svensson, Bo; Wengel, Verena

    2016-01-01

    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. PMID:27661085

  5. Recent Advances in the Development of Undecaprenyl Pyrophosphate Synthase Inhibitors as Potential Antibacterials.

    PubMed

    Jukic, Marko; Rozman, Kaja; Gobec, Stanislav

    2016-01-01

    Expanding antibiotic use in clinical practice and emergence of bacterial resistance are fueling research efforts for the development of novel antibacterials. Underexploited or completely novel mechanistic approaches and biological targets are of especial interest. Undecaprenyl pyrophosphate synthase (UppS) is an essential enzyme in the biosynthesis of the bacterial cell wall. Although UppS is a validated target, no selective inhibitors occur in materia medica. Nevertheless, several native substrate analogues have been reported and used in enzyme kinetics studies or as pharmacological probes. The majority of small-molecule UppS inhibitors belong to the well-known class of bisphosphonates that are primarily used for treatment of bone resorption disorders. The most potent compound of this class has an IC50 of 0.59 µM. Inherently, the selectivity and suitability of such compounds for antimicrobial drug design can be questioned. Therefore, highthroughput and virtual screenings for non-bisphosphonate inhibitors were performed, and nanomolar inhibitors of UppS were identified, some with antimicrobial activities towards clinically relevant strains. The reported scaffolds belong to tetramic and tetronic acids with IC50 in the 100-nM range, and to dihydropyridines with IC50 down to 40 nM, all with antibacterial activity. Aryl-diketo acids are also potent inhibitors with MRSA antimicrobial activity, with the allosteric inhibitor methylisoxazole-4-carboxamide (IC50, 50 nM) active on several pathogenic Streptococcus pneumoniae strains. Clomiphene is a well-known oestrogen receptor modulator, and it has been reported to inhibit UppS. Although conclusions on the structure activity relationships cannot be drawn from all these data, these compound series represent an important contribution to the field of antibiotics. PMID:26718796

  6. Epidermal growth factor receptor (EGFR) and prostaglandin-endoperoxide synthase 2 (PTGS2) are prognostic biomarkers for patients with resected colorectal cancer liver metastases

    PubMed Central

    Goos, J A C M; Hiemstra, A C; Coupé, V M H; Diosdado, B; Kooijman, W; Delis-Van Diemen, P M; Karga, C; Beliën, J A M; Menke-van der Houven van Oordt, C W; Geldof, A A; Meijer, G A; Hoekstra, O S; Fijneman, R J A; van Grieken, N C T; Perk, L R; van den Tol, M P; te Velde, E A; Windhorst, A D; Baas, J; Rijken, A M; van Beek, M W; Pijpers, H J; Bril, H; Stockmann, H B A C; Zwijnenburg, A; Bosscha, K; van den Brule, A J; Hoekstra, C J; van der Linden, J C; Borel Rinkes, I H; van Diest, P J; van Hillegersberg, R; Kranenburg, O; Lam, M G; Snoeren, N; Liem, I H; Roumen, R M; Vening, W

    2014-01-01

    Background: Resection of colorectal cancer liver metastasis (CRCLM) with curative intent has long-term benefit in ∼40% of cases. Prognostic biomarkers are needed to improve clinical management and reduce futile surgeries. Expression of epidermal growth factor receptor (EGFR) and prostaglandin-endoperoxide synthase 2 (PTGS2; also known as cyclooxygenase-2) has been associated with carcinogenesis and survival. We investigated the prognostic value of EGFR and PTGS2 expression in patients with resected CRCLM. Methods: Formalin-fixed paraffin-embedded CRCLM tissue and corresponding primary tumour specimens from a multi-institutional cohort of patients who underwent liver resection between 1990 and 2010 were incorporated into tissue microarrays (TMAs). TMAs were stained for EGFR and PTGS2 by immunohistochemistry. The hazard rate ratio (HRR) for the association between expression in CRCLM and overall survival was calculated using a 500-fold cross-validation procedure. Results: EGFR and PTGS2 expression could be evaluated in 323 and 351 patients, respectively. EGFR expression in CRCLM was associated with poor prognosis (HRR 1.54; P<0.01) with a cross-validated HRR of 1.47 (P=0.03). PTGS2 expression was also associated with poor prognosis (HRR 1.60; P<0.01) with a cross-validated HRR of 1.63 (P<0.01). Expression of EGFR and PTGS2 remained prognostic after multivariate analysis with standard clinicopathological variables (cross-validated HRR 1.51; P=0.02 and cross-validated HRR 1.59; P=0.01, respectively). Stratification for the commonly applied systemic therapy regimens demonstrated prognostic value for EGFR and PTGS2 only in the subgroup of patients who were not treated with systemic therapy (HRR 1.78; P<0.01 and HRR 1.64; P=0.04, respectively), with worst prognosis when both EGFR and PTGS2 were highly expressed (HRR 3.08; P<0.01). Expression of PTGS2 in CRCLM was correlated to expression in patient-matched primary tumours (P=0.02, 69.2% concordance). Conclusions: EGFR

  7. Glycogen Synthase Kinase-3 Inhibitors as Potent Therapeutic Agents for the Treatment of Parkinson Disease.

    PubMed Central

    2012-01-01

    Parkinson's disease (PD) is a devastating neurodegenerative disorder characterized by degeneration of the nigrostriatal dopaminergic pathway. Because the current therapies only lead to temporary, limited improvement and have severe side effects, new approaches to treat PD need to be developed. To discover new targets for potential therapeutic intervention, a chemical genetic approach involving the use of small molecules as pharmacological tools has been implemented. First, a screening of an in-house chemical library on a well-established cellular model of PD was done followed by a detailed pharmacological analysis of the hits. Here, we report the results found for the small heterocyclic derivative called SC001, which after different enzymatic assays was revealed to be a new glycogen synthase kinase-3 (GSK-3) inhibitor with IC50 = 3.38 ± 0.08 μM. To confirm that GSK-3 could be a good target for PD, the evaluation of a set of structurally diverse GSK-3 inhibitors as neuroprotective agents for PD was performed. Results show that inhibitors of GSK-3 have neuroprotective effects in vitro representing a new pharmacological option for the disease-modifying treatment of PD. Furthermore, we show that SC001 is able to cross the blood–brain barrier, protects dopaminergic neurons, and reduces microglia activation in in vivo models of Parkinson disease, being a good candidate for further drug development. PMID:23421686

  8. Quinazolinones, Quinazolinthiones, and Quinazolinimines as Nitric Oxide Synthase Inhibitors: Synthetic Study and Biological Evaluation.

    PubMed

    Camacho, M Encarnación; Chayah, Mariem; García, M Esther; Fernández-Sáez, Nerea; Arias, Fabio; Gallo, Miguel A; Carrión, M Dora

    2016-08-01

    The synthesis of different compounds with a quinazolinone, quinazolinthione, or quinazolinimine skeleton and their in vitro biological evaluation as inhibitors of inducible and neuronal nitric oxide synthase (iNOS and nNOS) isoforms are described. These derivatives were obtained from substituted 2-aminobenzylamines, using diverse cyclization procedures. Furthermore, the diamines were synthesized by two routes: A conventional pathway and an efficient one-pot synthesis in a continuous-flow hydrogenator. The structures of these heterocycles were confirmed by (1) H and (13) C nuclear magnetic resonance and high-resolution mass spectroscopy data. The structure-activity relationships of the target molecules are discussed in terms of the effects of both the R radical and the X heteroatom in the 2-position. In general, the assayed compounds behave as better iNOS than nNOS inhibitors, with the quinazolinone 11e being the most active inhibitor of all tested compounds and the most iNOS/nNOS selective one. PMID:27328401

  9. Triazolyl phenyl disulfides: 8-Amino-7-oxononanoate synthase inhibitors as potential herbicides.

    PubMed

    Hahn, Hoh-Gyu; Choi, Jung-Sup; Lim, Hee Kyung; Lee, Kee-In; Hwang, In Taek

    2015-11-01

    The chemical validation of a potential herbicide target was investigated with 8-amino-7-oxononanoate synthase (AONS, also known as 7-keto-8-aminopelargonate synthase, KAPAS) and triazolyl phenyl disulfide derivatives in vitro and in vivo. AONS activity was completely inhibited by these synthesized compounds, with an IC50 of 48 to 592μM in vitro. Forty five-day old Arabidopsis thaliana plants were completely killed by representative compound KHG23844 {N-(2-fluorophenyl)-3-(phenyldisulphanyl)-1H-1,2,4-triazole-1-carboxamide} at the application rate of 250gha(-1) of foliar treatment in greenhouse conditions. Foliar application of 1000gha(-1) KHG23844 induced 2.3-fold higher l-alanine accumulation in the treated A. thaliana plants. Foliar supplement of 1mM biotin at 1 and 2days before KHG23844 application effectively recovered the growth inhibition of A. thaliana plant treated with KHG23844. The results strongly suggested that representative compound KHG23844 and its derivatives are potential AONS inhibitors.

  10. Effect of nitric oxide synthase inhibitor on diaphragmatic function after resistive loading.

    PubMed

    Bisnett, T; Anzueto, A; Andrade, F H; Rodney, G G; Napier, W R; Levine, S M; Maxwell, L C; Mureeba, P; Derdak, S D; Grisham, M B; Jenkinson, S G

    1998-01-01

    We studied the effect of a nitric oxide synthase inhibitor, Nomega-Nitro-L-arginine-methyl-ester (L-NAME), on in vitro diphragmatic function both at rest (control) or after inspiratory resistive loading (IRL). Sprague-Dawley rats were anesthetized, instrumented, and then the following experimental groups: (1) controls; (2) L-NAME (100 mg/kg/body weight intravenously alone); (3) IRL alone; and (4) L-NAME + IRL. The IRL protocol consisted of applying a variable resistor to the inspiratory limb of a two-way valve at 70% of maximal airway pressure until apnea. After the experiment, the animals were sacrificed and diaphragmatic strips were obtained for activity of constitutive nitric oxide synthase (cNOS) and measurements of in vitro contractile properties: tetanic (Po) and twitch tensions (Pt). cNOS activity was significantly decreased in the L-NAME and L-NAME + IRL groups (P < or = 0.05) as compared with control and IRL groups. L-NAME alone did not affect Po or Pt. However, in both IRL groups, with and without was a significant decrease in Po and Pt. This reduction was comparable in both groups. In summary, our data showed that L-NAME resulted in a significant decrease cNOS activity, but in vitro contractility was impaired. PMID:11253784

  11. Visualizing inducible nitric-oxide synthase in living cells with a heme-binding fluorescent inhibitor.

    PubMed

    Panda, Koustubh; Chawla-Sarkar, Mamta; Santos, Cecile; Koeck, Thomas; Erzurum, Serpil C; Parkinson, John F; Stuehr, Dennis J

    2005-07-19

    The study of nitric-oxide synthase (NOS) physiology is constrained by the lack of suitable probes to detect NOS in living cells or animals. Here, we characterized a fluorescent inducible NOS (iNOS) inhibitor called PIF (pyrimidine imidazole FITC) and examined its utility for microscopic imaging of iNOS in living cells. PIF binding to iNOS displayed high affinity, isoform selectivity, and heme specificity, and was essentially irreversible. PIF was used to successfully image iNOS expressed in RAW264.7 cells, HEK293T cells, human A549 epithelial cells, and freshly obtained human lung epithelium. PIF was used to estimate a half-life for iNOS of 1.8 h in HEK293T cells. Our work reveals that fluorescent probes like PIF will be valuable for studying iNOS cell biology and in understanding the pathophysiology of diseases that involve dysfunctional iNOS expression.

  12. Competitive inhibition of nitric oxide synthase by p-aminobenzamidine, a serine proteinase inhibitor.

    PubMed

    Venturini, G; Menegatti, E; Ascenzi, P

    1997-03-01

    p-Aminobenzamidine competitively inhibits bovine trypsin, human and bovine thrombin, and human plasmin, all of which act on substrates containing preferentially the L-arginyl side chain at their P1 position. Considering the structural and functional similarity between p-aminobenzamidine and the L-arginyl side chain in trypsin-like serine proteinases, we investigated the interaction of p-aminobenzamidine with mouse brain nitric oxide synthase (NOS), which uses L-arginine as the substrate for generating NO and L-citrulline. p-Aminobenzamidine is a competitive NOS inhibitor (Ki = 1.2 x 10(-4) M, at pH 7.5 and 37.0 degrees C), but not an NO precursor. Therefore, p-aminobenzamidine affects the NO production and the trypsin-like serine proteinase action. PMID:9125158

  13. Pharmacology and clinical pharmacology of methylarginines used as inhibitors of nitric oxide synthases.

    PubMed

    Kittel, Anja; Maas, Renke

    2014-01-01

    The methylarginines asymmetric dimethylarginine (ADMA) and monomethylarginine (L-NMMA) are endogenously formed inhibitors of nitric oxide synthases (NOS), which have extensively been investigated as risk markers and used as pharmacological tools to study the L-arginine-nitric oxide (NO) pathway in vitro and in vivo. It is the aim of the present review to summarize the clinical and experimental data on the pharmacological properties that are of relevance when planning and conducting experiments and clinical studies involving methylarginines. Key pharmacodynamic and pharmacokinetic data including IC50 values of ADMA and L-NMMA for NOS isoforms and transport proteins, as well as metabolism by dimethylarginine dimethylaminohydrolases (DDAH1 and DDAH2) and alanine-glyoxylate aminotransferase 2 (AGXT2) are discussed.

  14. The structure of nitric oxide synthase oxygenase domain and inhibitor complexes.

    PubMed

    Crane, B R; Arvai, A S; Gachhui, R; Wu, C; Ghosh, D K; Getzoff, E D; Stuehr, D J; Tainer, J A

    1997-10-17

    The nitric oxide synthase oxygenase domain (NOSox) oxidizes arginine to synthesize the cellular signal and defensive cytotoxin nitric oxide (NO). Crystal structures determined for cytokine-inducible NOSox reveal an unusual fold and heme environment for stabilization of activated oxygen intermediates key for catalysis. A winged beta sheet engenders a curved alpha-beta domain resembling a baseball catcher's mitt with heme clasped in the palm. The location of exposed hydrophobic residues and the results of mutational analysis place the dimer interface adjacent to the heme-binding pocket. Juxtaposed hydrophobic O2- and polar L-arginine-binding sites occupied by imidazole and aminoguanidine, respectively, provide a template for designing dual-function inhibitors and imply substrate-assisted catalysis.

  15. Thiolactomycin-based β-Ketoacyl-AcpM Synthase A (KasA) Inhibitors

    PubMed Central

    Kapilashrami, Kanishk; Bommineni, Gopal R.; Machutta, Carl A.; Kim, Pilho; Lai, Cheng-Tsung; Simmerling, Carlos; Picart, Francis; Tonge, Peter J.

    2013-01-01

    Thiolactomycin (TLM) is a natural product inhibitor of KasA, the β-ketoacyl synthase A from Mycobacterium tuberculosis. To improve the affinity of TLM for KasA, a series of TLM analogs have been synthesized based on interligand NOEs between TLM and a pantetheine analog when both are bound simultaneously to the enzyme. Kinetic binding data reveal that position 3 of the thiolactone ring is a suitable position for elaboration of the TLM scaffold, and the structure-activity relationship studies provide information on the molecular features that govern time-dependent inhibition in this enzyme system. These experiments also exemplify the utility of transient one-dimensional NOE spectroscopy for obtaining interligand NOEs compared with traditional steady state two-dimensional NOESY spectroscopy. PMID:23306195

  16. Contrasting effects of peroxisome-proliferator-activated receptor (PPAR)γ agonists on membrane-associated prostaglandin E2 synthase-1 in IL-1β-stimulated rat chondrocytes: evidence for PPARγ-independent inhibition by 15-deoxy-Δ12,14prostaglandin J2

    PubMed Central

    Bianchi, Arnaud; Moulin, David; Sebillaud, Sylvie; Koufany, Meriem; Galteau, Marie-Madeleine; Netter, Patrick; Terlain, Bernard; Jouzeau, Jean-Yves

    2005-01-01

    Microsomal prostaglandin E synthase (mPGES)-1 is a newly identified inducible enzyme of the arachidonic acid cascade with a key function in prostaglandin (PG)E2 synthesis. We investigated the kinetics of inducible cyclo-oxygenase (COX)-2 and mPGES-1 expression with respect to the production of 6-keto-PGF1α and PGE2 in rat chondrocytes stimulated with 10 ng/ml IL-1β, and compared their modulation by peroxisome-proliferator-activated receptor (PPAR)γ agonists. Real-time PCR analysis showed that IL-1β induced COX-2 expression maximally (37-fold) at 12 hours and mPGES-1 expression maximally (68-fold) at 24 hours. Levels of 6-keto-PGF1α and PGE2 peaked 24 hours after stimulation with IL-1β; the induction of PGE2 was greater (11-fold versus 70-fold, respectively). The cyclopentenone 15-deoxy-Δ12,14prostaglandin J2 (15d-PGJ2) decreased prostaglandin synthesis in a dose-dependent manner (0.1 to 10 μM), with more potency on PGE2 level than on 6-keto-PGF1α level (-90% versus -66% at 10 μM). A high dose of 15d-PGJ2 partly decreased COX-2 expression but decreased mPGES-1 expression almost completely at both the mRNA and protein levels. Rosiglitazone was poorly effective on these parameters even at 10 μM. Inhibitory effects of 10 μM 15d-PGJ2 were neither reduced by PPARγ blockade with GW-9662 nor enhanced by PPARγ overexpression, supporting a PPARγ-independent mechanism. EMSA and TransAM® analyses demonstrated that mutated IκBα almost completely suppressed the stimulating effect of IL-1β on mPGES-1 expression and PGE2 production, whereas 15d-PGJ2 inhibited NF-κB transactivation. These data demonstrate the following in IL-1-stimulated rat chondrocytes: first, mPGES-1 is rate limiting for PGE2 synthesis; second, activation of the prostaglandin cascade requires NF-κB activation; third, 15d-PGJ2 strongly inhibits the synthesis of prostaglandins, in contrast with rosiglitazone; fourth, inhibition by 15d-PGJ2 occurs independently of PPARγ through inhibition of

  17. Molecular Docking Analysis of Selected Clinacanthus nutans Constituents as Xanthine Oxidase, Nitric Oxide Synthase, Human Neutrophil Elastase, Matrix Metalloproteinase 2, Matrix Metalloproteinase 9 and Squalene Synthase Inhibitors

    PubMed Central

    Narayanaswamy, Radhakrishnan; Isha, Azizul; Wai, Lam Kok; Ismail, Intan Safinar

    2016-01-01

    Background: Clinacanthus nutans (Burm. f.) Lindau has gained popularity among Malaysians as a traditional plant for anti-inflammatory activity. Objective: This prompted us to carry out the present study on a selected 11 constituents of C. nutans which are clinacoside A–C, cycloclinacoside A1, shaftoside, vitexin, orientin, isovitexin, isoorientin, lupeol and β-sitosterol. Materials and Methods: Selected 11 constituents of C. nutans were evaluated on the docking behavior of xanthine oxidase (XO), nitric oxide synthase (NOS), human neutrophil elastase (HNE), matrix metalloproteinase (MMP 2 and 9), and squalene synthase (SQS) using Discovery Studio Version 3.1. Also, molecular physicochemical, bioactivity, absorption, distribution, metabolism, excretion, and toxicity (ADMET), and toxicity prediction by computer assisted technology analyzes were also carried out. Results: The molecular physicochemical analysis revealed that four ligands, namely clinacoside A–C and cycloclinacoside A1 showed nil violations and complied with Lipinski's rule of five. As for the analysis of bioactivity, all the 11 selected constituents of C. nutans exhibited active score (>0) toward enzyme inhibitors descriptor. ADMET analysis showed that the ligands except orientin and isoorientin were predicted to have Cytochrome P4502D6 inhibition effect. Docking studies and binding free energy calculations revealed that clinacoside B exhibited the least binding energy for the target enzymes except for XO and SQS. Isovitexin and isoorientin showed the potentials in the docking and binding with all of the six targeted enzymes, whereas vitexin and orientin docked and bound with only NOS and HNE. Conclusion: This present study has paved a new insight in understanding these 11 C. nutans ligands as potential inhibitors against XO, NOS, HNE, MMP 2, MMP 9, and SQS. SUMMARY Isovitexin and isoorientin (Clinacanthus nutans constituent) showed potentials in the docking and binding with all of the six targeted

  18. Synthesis and biological evaluation of several dephosphonated analogues of CMP-Neu5Ac as inhibitors of GM3-synthase.

    PubMed

    Rota, Paola; Cirillo, Federica; Piccoli, Marco; Gregorio, Antonio; Tettamanti, Guido; Allevi, Pietro; Anastasia, Luigi

    2015-10-01

    Previous studies demonstrated that reducing the GM3 content in myoblasts increased the cell resistance to hypoxic stress, suggesting that a pharmacological inhibition of the GM3 synthesis could be instrumental for the development of new treatments for ischemic diseases. Herein, the synthesis of several dephosphonated CMP-Neu5Ac congeners and their anti-GM3-synthase activity is reported. Biological activity testes revealed that some inhibitors almost completely blocked the GM3-synthase activity in vitro and reduced the GM3 content in living embryonic kidney 293A cells, eventually activating the epidermal growth factor receptor (EGFR) signaling cascade.

  19. Haplofungins, novel inositol phosphorylceramide synthase inhibitors, from Lauriomyces bellulus SANK 26899 I. Taxonomy, fermentation, isolation and biological activities.

    PubMed

    Ohnuki, Takashi; Yano, Tatsuya; Ono, Yasunori; Kozuma, Shiho; Suzuki, Toshihiro; Ogawa, Yasumasa; Takatsu, Toshio

    2009-10-01

    In the course of screening for antifungal agents, we have discovered eight novel compounds, haplofungin A, B, C, D, E, F, G and H, from a culture broth of the fungus strain Lauriomyces bellulus SANK 26899. Haplofungins are composed of an arabinonic acid moiety linked through an ester to a modified long alkyl chain and show potent inhibitory activities against fungal inositol phosphorylceramide (IPC) synthase. Haplofungin A inhibited the activity of IPC synthase from Saccharomyces cerevisiae with an IC(50) value of 0.0015 microg ml(-1). This inhibitor also suppressed the growth of Candida glabrata at the MIC value of 0.5 microg ml(-1).

  20. Hybrid inhibitor of peripheral cannabinoid-1 receptors and inducible nitric oxide synthase mitigates liver fibrosis

    PubMed Central

    Iyer, Malliga R.; Liu, Ziyi; Cao, Zongxian; Jourdan, Tony; Erdelyi, Katalin; Godlewski, Grzegorz; Szanda, Gergő; Liu, Jie; Park, Joshua K.; Mukhopadhyay, Bani; Rosenberg, Avi Z.; Liow, Jeih-San; Lorenz, Robin G.; Pacher, Pal; Innis, Robert B.

    2016-01-01

    Liver fibrosis, a consequence of chronic liver injury and a way station to cirrhosis and hepatocellular carcinoma, lacks effective treatment. Endocannabinoids acting via cannabinoid-1 receptors (CB1R) induce profibrotic gene expression and promote pathologies that predispose to liver fibrosis. CB1R antagonists produce opposite effects, but their therapeutic development was halted due to neuropsychiatric side effects. Inducible nitric oxide synthase (iNOS) also promotes liver fibrosis and its underlying pathologies, but iNOS inhibitors tested to date showed limited therapeutic efficacy in inflammatory diseases. Here, we introduce a peripherally restricted, orally bioavailable CB1R antagonist, which accumulates in liver to release an iNOS inhibitory leaving group. In mouse models of fibrosis induced by CCl4 or bile duct ligation, the hybrid CB1R/iNOS antagonist surpassed the antifibrotic efficacy of the CB1R antagonist rimonabant or the iNOS inhibitor 1400W, without inducing anxiety-like behaviors or CB1R occupancy in the CNS. The hybrid inhibitor also targeted CB1R-independent, iNOS-mediated profibrotic pathways, including increased PDGF, Nlrp3/Asc3, and integrin αvβ6 signaling, as judged by its ability to inhibit these pathways in cnr1–/– but not in nos2–/– mice. Additionally, it was able to slow fibrosis progression and to attenuate established fibrosis. Thus, dual-target peripheral CB1R/iNOS antagonists have therapeutic potential in liver fibrosis. PMID:27525312

  1. Hybrid inhibitor of peripheral cannabinoid-1 receptors and inducible nitric oxide synthase mitigates liver fibrosis

    PubMed Central

    Liu, Ziyi; Cao, Zongxian; Jourdan, Tony; Erdelyi, Katalin; Godlewski, Grzegorz; Szanda, Gergő; Liu, Jie; Park, Joshua K.; Mukhopadhyay, Bani; Rosenberg, Avi Z.; Liow, Jeih-San; Lorenz, Robin G.; Pacher, Pal; Innis, Robert B.; Kunos, George

    2016-01-01

    Liver fibrosis, a consequence of chronic liver injury and a way station to cirrhosis and hepatocellular carcinoma, lacks effective treatment. Endocannabinoids acting via cannabinoid-1 receptors (CB1R) induce profibrotic gene expression and promote pathologies that predispose to liver fibrosis. CB1R antagonists produce opposite effects, but their therapeutic development was halted due to neuropsychiatric side effects. Inducible nitric oxide synthase (iNOS) also promotes liver fibrosis and its underlying pathologies, but iNOS inhibitors tested to date showed limited therapeutic efficacy in inflammatory diseases. Here, we introduce a peripherally restricted, orally bioavailable CB1R antagonist, which accumulates in liver to release an iNOS inhibitory leaving group. In mouse models of fibrosis induced by CCl4 or bile duct ligation, the hybrid CB1R/iNOS antagonist surpassed the antifibrotic efficacy of the CB1R antagonist rimonabant or the iNOS inhibitor 1400W, without inducing anxiety-like behaviors or CB1R occupancy in the CNS. The hybrid inhibitor also targeted CB1R-independent, iNOS-mediated profibrotic pathways, including increased PDGF, Nlrp3/Asc3, and integrin αvβ6 signaling, as judged by its ability to inhibit these pathways in cnr1−/− but not in nos2−/− mice. Additionally, it was able to slow fibrosis progression and to attenuate established fibrosis. Thus, dual-target peripheral CB1R/iNOS antagonists have therapeutic potential in liver fibrosis. PMID:27525312

  2. Farnesyl pyrophosphate synthase inhibitor, ibandronate, improves endothelial function in spontaneously hypertensive rats

    PubMed Central

    HAN, JIE; JIANG, DONG-MEI; YE, YANG; DU, CHANG-QING; YANG, JIAN; HU, SHEN-JIANG

    2016-01-01

    Reactive oxygen species (ROS), originating predominantly from vascular smooth muscle cells (VSMCs), lead to vascular damage and endothelial dysfunction in rats with hypertension. The downstream signaling pathways of farnesyl pyrophosphate (FPP) synthase, Ras-related C3 botulinum toxin substrate 1 (Rac1) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, mediate the generation of ROS. The present study investigated the effect of the FPP synthase inhibitor, ibandronate, on ROS production, the possible beneficial effect on endothelial dysfunction and the underlying mechanisms in spontaneously hypertensive rats (SHRs). The SHRs were treated with ibandronate for 30 days. Endothelium-dependent and independent vasorelaxation were measured in isolated aortic rings. Additionally, VSMCs from the SHRs and Wistar-Kyoto (WKY) rats were cultured. The production of ROS and activation of NADPH oxidase were determined using fluorescence and chemiluminescence, respectively, in vivo and in vitro. Angiotensin II (Ang II) increased ROS production in the cultured VSMCs from the WKY rats and SHRs, in a concentration-dependent manner. The Ang II-induced responses were more marked in the SHR VSMCs, compare with those in the WKY VSMCs, however, the response decreased significantly following ibandronate pretreatment. Treatment with ibandronate significantly decreased the production of ROS, translocation of NADPH oxidase subunit p47phox, and activities of NADPH oxidase and Rac1 in the aorta and VSMCs, and improved the impaired endothelium-dependent vasodilation in the SHRs. Adding geranylgeraniol, but not farnesol or mevalonate, reversed the inhibitory effects of ibandronate. In addition, inhibiting geranylgeranyl-transferase mimicked the effect of ibandronate on the excess oxidative response. Ibandronate exerted cellular antioxidant effects through the Rac1/NADPH oxidase pathway. These effects may have contributed to the vasoprotective effects on the impaired endothelium in

  3. Circadian variation in the effects of nitric oxide synthase inhibitors on body temperature, feeding and activity in rats.

    PubMed

    Kamerman, Peter; Mitchell, Duncan; Laburn, Helen

    2002-02-01

    We have investigated whether there is circadian variation in the effects of nitric oxide synthase inhibitors on body temperature, physical activity and feeding. We used nocturnally active Sprague-Dawley rats, housed at approximately 24 degrees C with a 12:12 h light:dark cycle (lights on 07:00 hours) and provided with food and water ad libitum. Nitric oxide synthesis was inhibited by intraperitoneal injection of the unspecific nitric oxide synthase inhibitor N-nitro- L-arginine methyl ester ( L-NAME, 100, 50, 25, 10 mg/kg), or the relatively selective inducible nitric oxide synthase inhibitor aminoguanidine (100, 50 mg/kg), during the day ( approximately 09:00 hours) or night ( approximately 21:00 hours). Body temperature and physical activity were measured using radiotelemetry, while food intake was calculated by weighing each animal's food before as well as 12 and 24 h after each injection. We found that daytime injection of L-NAME and aminoguanidine had no effect on daytime body temperature. However, daytime injection of both drugs did decrease nocturnal food intake ( P<0.05) and activity ( P<0.05). When injected at night, L-NAME reduced night-time body temperature ( P<0.01), activity ( P<0.05) and food intake ( P<0.05) in a dose-dependent manner, but night-time injection of aminoguanidine inhibited only night-time activity ( P<0.05). The effects of nitric oxide synthase inhibition on body temperature, feeding and activity therefore are primarily a consequence of inhibiting constitutively expressed nitric oxide synthase, and are subject to circadian variation.

  4. Non-antibiotic quorum sensing inhibitors acting against N-acyl homoserine lactone synthase as druggable target

    PubMed Central

    Chang, Chien-Yi; Krishnan, Thiba; Wang, Hao; Chen, Ye; Yin, Wai-Fong; Chong, Yee-Meng; Tan, Li Ying; Chong, Teik Min; Chan, Kok-Gan

    2014-01-01

    N-acylhomoserine lactone (AHL)-based quorum sensing (QS) is important for the regulation of proteobacterial virulence determinants. Thus, the inhibition of AHL synthases offers non-antibiotics-based therapeutic potentials against QS-mediated bacterial infections. In this work, functional AHL synthases of Pseudomonas aeruginosa LasI and RhlI were heterologously expressed in an AHL-negative Escherichia coli followed by assessments on their AHLs production using AHL biosensors and high resolution liquid chromatography–mass spectrometry (LCMS). These AHL-producing E. coli served as tools for screening AHL synthase inhibitors. Based on a campaign of screening synthetic molecules and natural products using our approach, three strongest inhibitors namely are salicylic acid, tannic acid and trans-cinnamaldehyde have been identified. LCMS analysis further confirmed tannic acid and trans-cinnemaldehyde efficiently inhibited AHL production by RhlI. We further demonstrated the application of trans-cinnemaldehyde inhibiting Rhl QS system regulated pyocyanin production in P. aeruginosa up to 42.06%. Molecular docking analysis suggested that trans-cinnemaldehyde binds to the LasI and EsaI with known structures mainly interacting with their substrate binding sites. Our data suggested a new class of QS-inhibiting agents from natural products targeting AHL synthase and provided a potential approach for facilitating the discovery of anti-QS signal synthesis as basis of novel anti-infective approach. PMID:25430794

  5. Non-antibiotic quorum sensing inhibitors acting against N-acyl homoserine lactone synthase as druggable target.

    PubMed

    Chang, Chien-Yi; Krishnan, Thiba; Wang, Hao; Chen, Ye; Yin, Wai-Fong; Chong, Yee-Meng; Tan, Li Ying; Chong, Teik Min; Chan, Kok-Gan

    2014-01-01

    N-acylhomoserine lactone (AHL)-based quorum sensing (QS) is important for the regulation of proteobacterial virulence determinants. Thus, the inhibition of AHL synthases offers non-antibiotics-based therapeutic potentials against QS-mediated bacterial infections. In this work, functional AHL synthases of Pseudomonas aeruginosa LasI and RhlI were heterologously expressed in an AHL-negative Escherichia coli followed by assessments on their AHLs production using AHL biosensors and high resolution liquid chromatography-mass spectrometry (LCMS). These AHL-producing E. coli served as tools for screening AHL synthase inhibitors. Based on a campaign of screening synthetic molecules and natural products using our approach, three strongest inhibitors namely are salicylic acid, tannic acid and trans-cinnamaldehyde have been identified. LCMS analysis further confirmed tannic acid and trans-cinnemaldehyde efficiently inhibited AHL production by RhlI. We further demonstrated the application of trans-cinnemaldehyde inhibiting Rhl QS system regulated pyocyanin production in P. aeruginosa up to 42.06%. Molecular docking analysis suggested that trans-cinnemaldehyde binds to the LasI and EsaI with known structures mainly interacting with their substrate binding sites. Our data suggested a new class of QS-inhibiting agents from natural products targeting AHL synthase and provided a potential approach for facilitating the discovery of anti-QS signal synthesis as basis of novel anti-infective approach. PMID:25430794

  6. Identification of a Glycogen Synthase Kinase-3[beta] Inhibitor that Attenuates Hyperactivity in CLOCK Mutant Mice

    SciTech Connect

    Kozikowski, Alan P.; Gunosewoyo, Hendra; Guo, Songpo; Gaisina, Irina N.; Walter, Richard L.; Ketcherside, Ariel; McClung, Colleen A.; Mesecar, Andrew D.; Caldarone, Barbara

    2012-05-02

    Bipolar disorder is characterized by a cycle of mania and depression, which affects approximately 5 million people in the United States. Current treatment regimes include the so-called 'mood-stabilizing drugs', such as lithium and valproate that are relatively dated drugs with various known side effects. Glycogen synthase kinase-3{beta} (GSK-3{beta}) plays a central role in regulating circadian rhythms, and lithium is known to be a direct inhibitor of GSK-3{beta}. We designed a series of second generation benzofuran-3-yl-(indol-3-yl)maleimides containing a piperidine ring that possess IC{sub 50} values in the range of 4 to 680 nM against human GSK-3{beta}. One of these compounds exhibits reasonable kinase selectivity and promising preliminary absorption, distribution, metabolism, and excretion (ADME) data. The administration of this compound at doses of 10 to 25 mg kg{sup -1} resulted in the attenuation of hyperactivity in amphetamine/chlordiazepoxide-induced manic-like mice together with enhancement of prepulse inhibition, similar to the effects found for valproate (400 mg kg{sup -1}) and the antipsychotic haloperidol (1 mg kg{sup -1}). We also tested this compound in mice carrying a mutation in the central transcriptional activator of molecular rhythms, the CLOCK gene, and found that the same compound attenuates locomotor hyperactivity in response to novelty. This study further demonstrates the use of inhibitors of GSK-3{beta} in the treatment of manic episodes of bipolar/mood disorders, thus further validating GSK-3{beta} as a relevant therapeutic target in the identification of new therapies for bipolar patients.

  7. A suppressive effect of prostaglandin E2 on the expression of SERPINE1/plasminogen activator inhibitor-1 in human articular chondrocytes: An in vitro pilot study

    PubMed Central

    Masuko, Kayo; Murata, Minako; Suematsu, Naoya; Okamoto, Kazuki; Yudoh, Kazuo; Shimizu, Hiroyuki; Beppu, Moroe; Nakamura, Hiroshi; Kato, Tomohiro

    2009-01-01

    Prostaglandin E2 (PGE2) is expressed in articular joints with inflammatory arthropathy and may exert catabolic effects leading to cartilage degradation. As we observed in a preliminary experiment that PGE2 suppressed the expression of SERPINE1/plasminogen activator inhibitor (PAI)-1 mRNA in chondrocytes, we focused on the effect of PGE2 on PAI-1 in a panel of cultured chondrocytes obtained from osteoarthritic patients. Specifically, articular cartilage specimens were obtained from patients with osteoarthritis who underwent joint surgery. Isolated chondrocytes were cultured in vitro as a monolayer and stimulated with PGE2. Stimulated cells and culture supernatants were analyzed using Western blotting and enzyme-linked immunosorbent assay. The results confirmed that the in vitro PGE2 stimulation suppressed the expression of PAI-1 in the tested chondrocyte samples. The inhibitory effect was partly abrogated by an antagonist of EP4 receptor of PGE2, but not by an EP2 antagonist. Although PGE2 induced activations of mitogen-activated protein kinases (MAPK), blocking of the MAPK did not abrogate the suppressive effect of PGE2, implying a distinct signaling pathway. In summary, prostaglandin is suggested to modulate the plasminogen system in chondrocytes. Further elucidation of the interaction might open a new avenue to understand the degradative process of cartilage.

  8. Glucose-Modulated Mitochondria Adaptation in Tumor Cells: A Focus on ATP Synthase and Inhibitor Factor 1

    PubMed Central

    Domenis, Rossana; Bisetto, Elena; Rossi, Davide; Comelli, Marina; Mavelli, Irene

    2012-01-01

    Warburg’s hypothesis has been challenged by a number of studies showing that oxidative phosphorylation is repressed in some tumors, rather than being inactive per se. Thus, treatments able to shift energy metabolism by activating mitochondrial pathways have been suggested as an intriguing basis for the optimization of antitumor strategies. In this study, HepG2 hepatocarcinoma cells were cultivated with different metabolic substrates under conditions mimicking “positive” (activation/biogenesis) or “negative” (silencing) mitochondrial adaptation. In addition to the expected up-regulation of mitochondrial biogenesis, glucose deprivation caused an increase in phosphorylating respiration and a rise in the expression levels of the ATP synthase β subunit and Inhibitor Factor 1 (IF1). Hyperglycemia, on the other hand, led to a markedly decreased level of the transcriptional coactivator PGC-α suggesting down-regulation of mitochondrial biogenesis, although no change in mitochondrial mass and no impairment of phosphorylating respiration were observed. Moreover, a reduction in mitochondrial networking and in ATP synthase dimer stability was produced. No effect on β-ATP synthase expression was elicited. Notably, hyperglycemia caused an increase in IF1 expression levels, but it did not alter the amount of IF1 associated with ATP synthase. These results point to a new role of IF1 in relation to high glucose utilization by tumor cells, in addition to its well known effect upon mitochondrial ATP synthase regulation. PMID:22408432

  9. The neuronal nitric oxide synthase inhibitor, 7-nitroindazole, protects against methamphetamine-induced neurotoxicity in vivo.

    PubMed

    Itzhak, Y; Ali, S F

    1996-10-01

    The present study was undertaken to investigate whether the relatively selective neuronal nitric oxide synthase (NOS) inhibitor, 7-nitroindazole (7-NI), protects against methamphetamine (METH)-induced neurotoxicity. Male Swiss Webster mice received the following treatments (i.p.; q 3 h x 3): (a) vehicle/saline, (b) 7-NI (25 mg/kg)/saline, (c) vehicle/METH (5 mg/kg), and (d) 7-NI (25 mg/kg)/METH (5 mg/kg). On the second day, groups (a) and (b) received two vehicle injections, and groups (c) and (d) received two 7-NI injections (25 mg/kg, each). Administration of vehicle/METH resulted in 68, 44, and 55% decreases in the concentration of dopamine, 3,4-dihydroxyphenylacetic acid, and homovanillic acid, respectively, and a 48% decrease in the number of [3H]mazindol binding sites in the striatum compared with control values. Treatment with 7-NI (group d) provided full protection against the depletion of dopamine and its metabolites and the loss of dopamine transporter binding sites. Administration of 7-NI/saline (group b) affected neither the tissue concentration of dopamine and its metabolites nor the binding parameters of [3H] mazindol compared with control values. 7-NI had no significant effect on animals' body temperature, and it did not affect METH-induced hyperthermia. These findings indicate a role for nitric oxide in methamphetamine-induced neurotoxicity and also suggest that blockade of NOS may be beneficial for the management of Parkinson's disease.

  10. CNS-accessible Inhibitor of Glucosylceramide Synthase for Substrate Reduction Therapy of Neuronopathic Gaucher Disease

    PubMed Central

    Marshall, John; Sun, Ying; Bangari, Dinesh S; Budman, Eva; Park, Hyejung; Nietupski, Jennifer B; Allaire, Amy; Cromwell, Mary A; Wang, Bing; Grabowski, Gregory A; Leonard, John P; Cheng, Seng H

    2016-01-01

    Gaucher disease (GD) is caused by a deficiency of glucocerebrosidase and the consequent lysosomal accumulation of unmetabolized glycolipid substrates. Enzyme-replacement therapy adequately manages the visceral manifestations of nonneuronopathic type-1 Gaucher patients, but not the brain disease in neuronopathic types 2 and 3 GD. Substrate reduction therapy through inhibition of glucosylceramide synthase (GCS) has also been shown to effectively treat the visceral disease. Here, we evaluated the efficacy of a novel small molecule inhibitor of GCS with central nervous system (CNS) access (Genz-682452) to treat the brain disease. Treatment of the conduritol β epoxide-induced mouse model of neuronopathic GD with Genz-682452 reduced the accumulation of liver and brain glycolipids (>70% and >20% respectively), extent of gliosis, and severity of ataxia. In the genetic 4L;C* mouse model, Genz-682452 reduced the levels of substrate in the brain by >40%, the extent of gliosis, and paresis. Importantly, Genz-682452-treated 4L;C* mice also exhibited an ~30% increase in lifespan. Together, these data indicate that an orally available antagonist of GCS that has CNS access is effective at attenuating several of the neuropathologic and behavioral manifestations associated with mouse models of neuronopathic GD. Therefore, Genz-682452 holds promise as a potential therapeutic approach for patients with type-3 GD. PMID:26948439

  11. Effect of 7-nitroindazole, a neuronal nitric oxide synthase inhibitor, on behavioral and physiological parameters.

    PubMed

    Brožíčková, C; Mikulecká, A; Otáhal, J

    2014-01-01

    The role of brain derived nitric oxide in the physiology and behavior remains disputable. One of the reasons of the controversies might be systemic side effects of nitric oxide synthase inhibitors. Therefore, under nNOS inhibition by 7-nitroindazole (7-NI) we carried out recordings of blood gasses, blood pressure and spontaneous EEG in conscious adult rats. Locomotion and spontaneous behavior were assessed in an open field. In addition skilled walking and limb coordination were evaluated using a ladder rung walking test. The blood gas analysis revealed a significant increase in pCO(2) 180 min and 240 min after the application of 7-NI. The power and entropy decreased simultaneously with a shift of the mean frequency of the spontaneous EEG toward slow oscillations after 7-NI treatment. The thresholds of evoked potentials underwent a significant drop and a trend towards a slight increase in the I-O curve slope was observed. 7-NI significantly suppressed open field behavior expressed as distance moved, exploratory rearing and grooming. As for the ladder rung walking test the 7-NI treated animals had more errors in foot placement indicating impairment in limb coordination. Therefore our findings suggest that 7-NI increased cortical excitability and altered some physiological and behavioral parameters. PMID:24908089

  12. Stereocontrolled Synthesis of a Potential Transition-State Inhibitor of the Salicylate Synthase MbtI from Mycobacterium tuberculosis.

    PubMed

    Liu, Zheng; Liu, Feng; Aldrich, Courtney C

    2015-07-01

    Mycobactins are small-molecule iron chelators (siderophores) produced by Mycobacterium tuberculosis (Mtb) for iron mobilization. The bifunctional salicylate synthase MbtI catalyzes the first step of mycobactin biosynthesis through the conversion of the primary metabolite chorismate into salicylic acid via isochorismate. We report the design, synthesis, and biochemical evaluation of an inhibitor based on the putative transition state (TS) for the isochorismatase partial reaction of MbtI. The inhibitor mimics the hypothesized charge buildup at C-4 of chorismate in the TS as well as C-O bond formation at C-6. Another important design element of the inhibitor is replacement of the labile pyruvate side chain in chorismate with a stable C-linked propionate isostere. We developed a stereocontrolled synthesis of the highly functionalized cyclohexene inhibitor that features an asymmetric aldol reaction using a titanium enolate, diastereoselective Grignard addition to a tert-butanesulfinyl aldimine, and ring closing olefin metathesis as key steps. PMID:26035083

  13. Reversed-phase high-performance liquid chromatography method with fluorescence detection to screen nitric oxide synthases inhibitors.

    PubMed

    Maccallini, Cristina; Di Matteo, Mauro; Ammazzalorso, Alessandra; D'Angelo, Alessandra; De Filippis, Barbara; Di Silvestre, Sara; Fantacuzzi, Marialuigia; Giampietro, Letizia; Pandolfi, Assunta; Amoroso, Rosa

    2014-06-01

    Nitric oxide synthase (NOS) inhibitors are potential drug candidates due to the critical role of an excessive production of nitric oxide in a range of diseases. At present, the radiometric detection of L-[(3)H]-citrulline produced from L-[(3)H]-arginine during the enzymatic reaction is one of the most accepted methods to assess the in vitro activity of NOS inhibitors. Here we report a fast, easy, and cheap reversed-phase high-performance liquid chromatography method with fluorescence detection, based on the precolumn derivatization of L-citrulline with o-phthaldialdehyde/N-acetyl cysteine, for the in vitro screening of NOS inhibitors. To evaluate enzyme inhibition by the developed method, N-[3-(aminomethyl)benzyl]acetamidine, a potent and selective inhibitor of inducible NOS, was used as a test compound. The half maximal inhibitory concentration obtained was comparable to that derived by the well-established radiometric assay. PMID:24687974

  14. An EPSP synthase inhibitor joining shikimate 3-phosphate with glyphosate: synthesis and ligand binding studies.

    PubMed

    Marzabadi, M R; Gruys, K J; Pansegrau, P D; Walker, M C; Yuen, H K; Sikorski, J A

    1996-04-01

    A novel EPSP synthase inhibitor 4 has been designed and synthesized to probe the configurational details of glyphosate recognition in its herbicidal ternary complex with enzyme and shikimate 3-phosphate (S3P). A kinetic evaluation of the new 3-dephospho analog 12, as well as calorimetric and (31)P NMR spectroscopic studies of enzyme-bound 4, now provides a more precise quantitative definition for the molecular interactions of 4 with this enzyme. The very poor binding, relative to 4, displayed by the 3-dephospho analog 12 is indicative that 4 has a specific interaction with the S3P site. A comparison of Ki(calc) for 12 versus the Ki(app) for 4 indicates that the 3-phosphate group in 4 contributes about 4.8 kcal/mol to binding. This compares well with the 5.2 kcal/mol which the 3-phosphate group in S3P contributes to binding. Isothermal titration calorimetry demonstrates that 4 binds to free enzyme with an observed Kd of 0.53 +/- 0.04 microM. As such, 4 binds only 3-fold weaker than glyphosate and about 150-fold better than N-methylglyphosate. Consequently, 4 represents the most potent N-alkylglyphosate derivative identified to date. However, the resulting thermodynamic binding parameters clearly demonstrate that the formation of EPSPS x 4 is entropy driven like S3P. The binding characteristics of 4 are fully consistent with a primary interaction localized at the S3P subsite. Furthermore, (31)P NMR studies of enzyme-bound 4 confirm the expected interaction at the shikimate 3-phosphate site. However, the chemical shift observed for the phosphonate signal of EPSPS x 4 is in the opposite direction than that observed previously when glyphosate binds with enzyme and S3P. Therefore, when 4 occupies the S3P binding site, there is incomplete overlap at the glyphosate phosphonate subsite. As a glyphosate analog inhibitor, the potency of 4 most likely arises from predominant interactions which occur outside the normal glyphosate binding site. Consequently, 4 is best described

  15. Inhibition of matrix metalloproteinase-3 and -13 synthesis induced by IL-1beta in chondrocytes from mice lacking microsomal prostaglandin E synthase-1.

    PubMed

    Gosset, Marjolaine; Pigenet, Audrey; Salvat, Colette; Berenbaum, Francis; Jacques, Claire

    2010-11-15

    Joint destruction in arthritis is in part due to the induction of matrix metalloproteinase (MMP) expression and their inhibitors, especially MMP-13 and -3, which directly degrade the cartilage matrix. Although IL-1β is considered as the main catabolic factor involved in MMP-13 and -3 expression, the role of PGE(2) remains controversial. The goal of this study was to determine the role of PGE(2) on MMP synthesis in articular chondrocytes using mice lacking microsomal PGE synthase-1 (mPGES-1), which catalyses the rate-limiting step of PGE(2) synthesis. MMP-3 and MMP-13 mRNA and protein expressions were assessed by real-time RT-PCR, immunoblotting, and ELISA in primary cultures of articular chondrocytes from mice with genetic deletion of mPGES-1. IL-1β-induced PGE(2) synthesis was dramatically reduced in mPGES-1(-/-) and mPGES-1(+/-) compared with mPGES-1(+/+) chondrocytes. A total of 10 ng/ml IL-1β increased MMP-3 and MMP-13 mRNA, protein expression, and release in mPGES-1(+/+) chondrocytes in a time-dependent manner. IL-1β-induced MMP-3 and MMP-13 mRNA expression, protein expression, and release decreased in mPGES-1(-/-) and mPGES-1(+/-) chondrocytes compared with mPGES-1(+/+) chondrocytes from 8 up to 24 h. Otherwise, MMP inhibition was partially reversed by addition of 10 ng/ml PGE(2) in mPGES-1(-/-) chondrocytes. Finally, in mPGES-1(-/-) chondrocytes treated by forskolin, MMP-3 protein expression was significantly decreased compared with wild-type, suggesting that PGE(2) regulates MMP-3 expression via a signaling pathway dependent on cAMP. These results demonstrate that PGE(2) plays a key role in the induction of MMP-3 and MMP-13 in an inflammatory context. Therefore, mPGES-1 could be considered as a critical target to counteract cartilage degradation in arthritis.

  16. Induction of intrachromosomal homologous recombination in human cells by raltitrexed, an inhibitor of thymidylate synthase.

    PubMed

    Waldman, Barbara Criscuolo; Wang, Yibin; Kilaru, Kasturi; Yang, Zhengguan; Bhasin, Alaukik; Wyatt, Michael D; Waldman, Alan S

    2008-10-01

    Thymidylate deprivation brings about "thymineless death" in prokaryotes and eukaryotes. Although the precise mechanism for thymineless death has remained elusive, inhibition of the enzyme thymidylate synthase (TS), which catalyzes the de novo synthesis of TMP, has served for many years as a basis for chemotherapeutic strategies. Numerous studies have identified a variety of cellular responses to thymidylate deprivation, including disruption of DNA replication and induction of DNA breaks. Since stalled or collapsed replication forks and strand breaks are generally viewed as being recombinogenic, it is not surprising that a link has been demonstrated between recombination induction and thymidylate deprivation in bacteria and lower eukaryotes. A similar connection between recombination and TS inhibition has been suggested by studies done in mammalian cells, but the relationship between recombination and TS inhibition in mammalian cells had not been demonstrated rigorously. To gain insight into the mechanism of thymineless death in mammalian cells, in this work we undertook a direct investigation of recombination in human cells treated with raltitrexed (RTX), a folate analog that is a specific inhibitor of TS. Using a model system to study intrachromosomal homologous recombination in cultured fibroblasts, we provide definitive evidence that treatment with RTX can stimulate accurate recombination events in human cells. Gene conversions not associated with crossovers were specifically enhanced several-fold by RTX. Additional experiments demonstrated that recombination events provoked by a double-strand break (DSB) were not impacted by treatment with RTX, nor was error-prone DSB repair via nonhomologous end-joining. Our work provides evidence that thymineless death in human cells is not mediated by corruption of DSB repair processes and suggests that an increase in chromosomal recombination may be an important element of cellular responses leading to thymineless death.

  17. Identification of natural inhibitors of Entamoeba histolytica cysteine synthase from microbial secondary metabolites

    PubMed Central

    Mori, Mihoko; Jeelani, Ghulam; Masuda, Yui; Sakai, Kazunari; Tsukui, Kumiko; Waluyo, Danang; Tarwadi; Watanabe, Yoshio; Nonaka, Kenichi; Matsumoto, Atsuko; Ōmura, Satoshi; Nozaki, Tomoyoshi; Shiomi, Kazuro

    2015-01-01

    Amebiasis is a common worldwide diarrheal disease, caused by the protozoan parasite, Entamoeba histolytica. Metronidazole has been a drug of choice against amebiasis for decades despite its known side effects and low efficacy against asymptomatic cyst carriers. E. histolytica is also capable of surviving sub-therapeutic levels of metronidazole in vitro. Novel drugs with different mode of action are therefore urgently needed. The sulfur assimilatory de novo L-cysteine biosynthetic pathway is essential for various cellular activities, including the proliferation and anti-oxidative defense of E. histolytica. Since the pathway, consisting of two reactions catalyzed by serine acetyltransferase (SAT) and cysteine synthase (CS, O-acetylserine sulfhydrylase), does not exist in humans, it is a rational drug target against amebiasis. To discover inhibitors against the CS of E. histolytica (EhCS), the compounds of Kitasato Natural Products Library were screened against two recombinant CS isozymes: EhCS1 and EhCS3. Nine compounds inhibited EhCS1 and EhCS3 with IC50 values of 0.31–490 μM. Of those, seven compounds share a naphthoquinone moiety, indicating the structural importance of the moiety for binding to the active site of EhCS1 and EhCS3. We further screened >9,000 microbial broths for CS inhibition and purified two compounds, xanthofulvin and exophillic acid from fungal broths. Xanthofulvin inhibited EhCS1 and EhCS3. Exophillic acid showed high selectivity against EhCS1, but exhibited no inhibition against EhCS3. In vitro anti-amebic activity of the 11 EhCS inhibitors was also examined. Deacetylkinamycin C and nanaomycin A showed more potent amebicidal activity with IC50 values of 18 and 0.8 μM, respectively, in the cysteine deprived conditions. The differential sensitivity of trophozoites against deacetylkinamycin C in the presence or absence of L-cysteine in the medium and the IC50 values against EhCS suggest the amebicidal effect of deacetylkinamycin C is due to

  18. Glycogen Synthase Kinase-3 Inhibitor Protects Against Microvascular Hyperpermeability Following Hemorrhagic Shock

    PubMed Central

    Sawant, Devendra A.; Tharakan, Binu; Hunter, Felicia A.; Childs, Ed W.

    2015-01-01

    Background Hemorrhagic shock (HS)-induce microvascular hyperpermeability involves disruption of endothelial cell adherens junctions leading to increase in paracellular permeability. β-Catenin, an integral component of the adherens junctional complex and Wnt pathway, and caspase-3 via its apoptotic signaling regulate endothelial cell barrier integrity. We have hypothesized that inhibiting phosphorylation of β-catenin and caspase-3 activity using glycogen synthase kinase-3 (GSK-3) specific inhibitor SB216763, would attenuate microvascular hyperpermeability following HS. Methods In Sprague-Dawley rats, HS was induced by withdrawing blood to reduce mean arterial pressure to 40 mmHg for 60 minutes followed by resuscitation. Rats were given SB216763 (600 μg/kg) intravenously 10 minutes prior to shock. To study microvascular permeability, the rats were injected intravenously with FITC-albumin (50 mg/kg) and its flux across the mesenteric post-capillary venules was determined using intravital microscopy. In cell-culture studies, rat lung microvascular endothelial cell (RLMEC) monolayers grown on Transwell plates were pre-treated with SB216763 (5 μM) followed by BAK (5 μg/mL) and caspase-3 (5 μg/mL) protein transfection. FITC-albumin (5 mg/mL) flux across cell monolayers indicates change in monolayer permeability. Activity of canonical Wnt pathway was determined by luciferase assay. Caspase-3 enzyme activity was assayed fluorometrically. Results The HS group showed significant increase in FITC-albumin extravasation (p<0.05) compared with sham. SB216763 significantly decrease HS-induced FITC-albumin extravasation (p<0.05). Pre-treatment with SB216763, protected against a BAK-induced increase in RLMEC monolayer permeability and caspase-3 activity, but failed to show similar results with a caspase-3-induced increase in monolayer permeability. Wnt3a treatment showed an increase in β-catenin dependent TCF-mediated transcription. Conclusion Inhibiting phosphorylation of

  19. Synthesis and biological evaluation of novel phosphoramidate derivatives of coumarin as chitin synthase inhibitors and antifungal agents.

    PubMed

    Ji, Qinggang; Ge, Zhiqiang; Ge, Zhixing; Chen, Kaizhi; Wu, Hualong; Liu, Xiaofei; Huang, Yanrong; Yuan, Lvjiang; Yang, Xiaolan; Liao, Fei

    2016-01-27

    A series of novel phosphoramidate derivatives of coumarin have been designed and synthesized as chitin synthase (CHS) inhibitors. All the synthesized compounds have been screened for their chitin synthase inhibition activity and antimicrobial activity in vitro. The bioactive assay manifested that most of the target compounds exhibited good efficacy against CHS and a variety of clinically important fungal pathogens. In particular, compound 7t with IC50 of 0.08 mM against CHS displayed stronger efficiency than the reference Polyoxin B with IC50 of 0.16 mM. In addition, the apparent Ki values of compound 7t was 0.096 mM while the Km of Chitin synthase prepared from Candida tropicalis was 3.86 mM for UDP-N-acetylglucosamine, and the result of the Ki showed that the compounds was a non-competitive inhibitor of the CHS. As far as the antifungal activity is concerned, compounds 7o, 7r and 7t were highly active against Aspergillus flavus with MIC values in the range of 1 μg/mL to 2 μg/Ml while the results of antibacterial screening showed that these compounds have negligible actions to the tested bacteria. These results indicated that the design of these compounds as antifungal agents was rational.

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

    PubMed

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

    2012-12-14

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

  1. Inhibitors of phosphodiesterases PDE2, PDE3, and PDE4 do not increase the sinoatrial tachycardia of noradrenaline and prostaglandin PGE₁ in mice.

    PubMed

    Galindo-Tovar, Alejandro; Vargas, María Luisa; Kaumann, Alberto J

    2016-02-01

    Phosphodiesterases PDE2, PDE3, and PDE4 are expressed in murine sinoatrial cells. PDE3 and/or PDE4 reduce heart rate but apparently do not influence the tachycardia mediated through sinoatrial β1- and β2-adrenoceptors despite the high content of sinoatrial cAMP. The function of PDE2 is, however, uncertain. Prostaglandin PGE1 elicits sinoatrial tachycardia through EP receptors, but the control by phosphodiesterases is unknown. We investigated on spontaneously beating right atria of mice the effects of the PDE2 inhibitors Bay 60-7550 and EHNA on basal beating and the tachycardia produced by noradrenaline (3 nM) and PGE1 (1 μM). Bay 60-7550 (1 μM), but not EHNA (10 μM), increased basal sinoatrial beating. EHNA also failed to produce tachycardia in the presence of the adenosine deaminase inhibitor 2'-deoxycoformycin (10 μM), remaining inconclusive whether PDE2 reduces basal sinoatrial beating. Rolipram (10 μM) and cilostamide (300 nM) caused moderate tachycardia. The tachycardia evoked by Bay 60-7550 was similar in the absence and presence of rolipram. Noradrenaline elicited stable tachycardia that was not increased by Bay 60-7550. A stable tachycardia caused by PGE1 was not increased by the inhibitors of PDE2, PDE3, and PDE4. Unlike PDE3 and PDE4 which reduce murine basal sinoatrial beating, a possible effect of PDE2 needs further research. The stable tachycardia produced by noradrenaline and PGE1, together with the lack potentiation by the inhibitors of PDE2, PDE3, and PDE4, suggests that cAMP generated at the receptor compartments is hardly hydrolyzed by these phophodiesterases. Evidence from human volunteers is consistent with this proposal.

  2. Lipophilic Bisphosphonates as Dual Farnesyl/Geranylgeranyl Diphosphate Synthase Inhibitors: An X-ray and NMR Investigation

    SciTech Connect

    Zhang, Y.; Cao, R; Yin, F; Hudock, M; Guo, R; Song, Y; No, J; Bergan, K; Leon, A; et al,

    2009-01-01

    Considerable effort has focused on the development of selective protein farnesyl transferase (FTase) and protein geranylgeranyl transferase (GGTase) inhibitors as cancer chemotherapeutics. Here, we report a new strategy for anticancer therapeutic agents involving inhibition of farnesyl diphosphate synthase (FPPS) and geranylgeranyl diphosphate synthase (GGPPS), the two enzymes upstream of FTase and GGTase, by lipophilic bisphosphonates. Due to dual site targeting and decreased polarity, the compounds have activities far greater than do current bisphosphonate drugs in inhibiting tumor cell growth and invasiveness, both in vitro and in vivo. We explore how these compounds inhibit cell growth and how cell activity can be predicted based on enzyme inhibition data, and using X-ray diffraction, solid state NMR, and isothermal titration calorimetry, we show how these compounds bind to FPPS and/or GGPPS.

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

    PubMed

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

    2012-06-19

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

  4. Mechanistic analysis of a synthetic inhibitor of the Pseudomonas aeruginosa LasI quorum-sensing signal synthase

    PubMed Central

    Lidor, O.; Al-Quntar, A.; Pesci, E. C.; Steinberg, D.

    2015-01-01

    Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen responsible for many human infections. LasI is an acyl-homoserine lactone synthase that produces a quorum-sensing (QS) signal that positively regulates numerous P. aeruginosa virulence determinants. The inhibition of the LasI protein is therefore an attractive drug target. In this study, a novel in silico to in vitro complementation was applied to screen thiazolidinedione-type compounds for their ability to inhibit biofilm formation at concentrations not affecting bacterial growth. The compound (z)-5-octylidenethiazolidine-2, 4-dione (TZD-C8) was a strong inhibitor of biofilm formation and chosen for further study. Structural exploration of in silico docking predicted that the compound had high affinity for the LasI activity pocket. The TZD-C8 compound was also predicted to create hydrogen bonds with residues Arg30 and Ile107. Site-directed mutagenesis (SDM) of these two sites demonstrated that TZD-C8 inhibition was abolished in the lasI double mutant PAO-R30D, I107S. In addition, in vitro swarming motility and quorum sensing signal production were affected by TZD-C 8, confirming this compound alters the cell to cell signalling circuitry. Overall, this novel inhibitor of P. aeruginosa quorum sensing shows great promise and validates our mechanistic approach to discovering inhibitors of LuxI-type acyl-homoserine lactone synthases. PMID:26593271

  5. A novel bisphosphonate inhibitor of squalene synthase combined with a statin or a nitrogenous bisphosphonate in vitro.

    PubMed

    Wasko, Brian M; Smits, Jacqueline P; Shull, Larry W; Wiemer, David F; Hohl, Raymond J

    2011-11-01

    Statins and nitrogenous bisphosphonates (NBP) inhibit 3-hydroxy-3-methylglutaryl-coenzyme-A reductase (HMGCR) and farnesyl diphosphate synthase (FDPS), respectively, leading to depletion of farnesyl diphosphate (FPP) and disruption of protein prenylation. Squalene synthase (SQS) utilizes FPP in the first committed step from the mevalonate pathway toward cholesterol biosynthesis. Herein, we have identified novel bisphosphonates as potent and specific inhibitors of SQS, including the tetrasodium salt of 9-biphenyl-4,8-dimethyl-nona-3,7-dienyl-1,1-bisphosphonic acid (compound 5). Compound 5 reduced cholesterol biosynthesis and lead to a substantial intracellular accumulation of FPP without reducing cell viability in HepG2 cells. At high concentrations, lovastatin and zoledronate impaired protein prenylation and decreased cell viability, which limits their potential use for cholesterol depletion. When combined with lovastatin, compound 5 prevented lovastatin-induced FPP depletion and impairment of protein farnesylation. Compound 5 in combination with the NBP zoledronate completely prevented zoledronate-induced impairment of both protein farnesylation and geranylgeranylation. Cotreatment of cells with compound 5 and either lovastatin or zoledronate was able to significantly prevent the reduction of cell viability caused by lovastatin or zoledronate alone. The combination of an SQS inhibitor with an HMGCR or FDPS inhibitor provides a rational approach for reducing cholesterol synthesis while preventing nonsterol isoprenoid depletion.

  6. Sulfa and trimethoprim-like drugs - antimetabolites acting as carbonic anhydrase, dihydropteroate synthase and dihydrofolate reductase inhibitors.

    PubMed

    Capasso, Clemente; Supuran, Claudiu T

    2014-06-01

    Recent advances in microbial genomics, synthetic organic chemistry and X-ray crystallography provided opportunities to identify novel antibacterial targets for the development of new classes of antibiotics and to design more potent antimicrobial compounds derived from existing antibiotics in clinical use for decades. The antimetabolites, sulfa drugs and trimethoprim (TMP)-like agents, are inhibitors of three families of enzymes. One family belongs to the carbonic anhydrases, which catalyze a simple but physiologically relevant reaction in all life kingdoms, carbon dioxide hydration to bicarbonate and protons. The other two enzyme families are involved in the synthesis of tetrahydrofolate (THF), i.e. dihydropteroate synthase (DHPS) and dihydrofolate reductase. The antibacterial agents belonging to the THF and DHPS inhibitors were developed decades ago and present significant bacterial resistance problems. However, the molecular mechanisms of drug resistance both to sulfa drugs and TMP-like inhibitors were understood in detail only recently, when several X-ray crystal structures of such enzymes in complex with their inhibitors were reported. Here, we revue the state of the art in the field of antibacterials based on inhibitors of these three enzyme families.

  7. Discovery of Potential Inhibitors of Aldosterone Synthase from Chinese Herbs Using Pharmacophore Modeling, Molecular Docking, and Molecular Dynamics Simulation Studies

    PubMed Central

    Lu, Fang; Qiao, Liansheng; Chen, Xi; Li, Gongyu

    2016-01-01

    Aldosterone synthase (CYP11B2) is a key enzyme for the biosynthesis of aldosterone, which plays a significant role for the regulation of blood pressure. Excess aldosterone can cause the dysregulation of the renin-angiotensin-aldosterone system (RAAS) and lead to hypertension. Therefore, research and development of CYP11B2 inhibitor are regarded as a novel approach for the treatment of hypertension. In this study, the pharmacophore models of CYP11B2 inhibitors were generated and the optimal model was used to identify potential CYP11B2 inhibitors from the Traditional Chinese Medicine Database (TCMD, Version 2009). The hits were further refined by molecular docking and the interactions between compounds and CYP11B2 were analyzed. Compounds with high Fitvalue, high docking score, and expected interactions with key residues were selected as potential CYP11B2 inhibitors. Two most promising compounds, ethyl caffeate and labiatenic acid, with high Fitvalue and docking score were reserved for molecular dynamics (MD) study. All of them have stability of ligand binding which suggested that they might perform the inhibitory effect on CYP11B2. This study provided candidates for novel drug-like CYP11B2 inhibitors by molecular simulation methods for the hypertension treatment. PMID:27781210

  8. Interaction among beta-endorphin, nitric oxide and prostaglandins during ovulation in rats.

    PubMed

    Faletti, A G; Mohn, C; Farina, M; Lomniczi, A; Rettori, V

    2003-04-01

    The aim of this study was to investigate the relationship between beta-endorphin and nitric oxide (NO) during the ovulatory process in rats. Immature rats were treated with equine chorionic gonadotrophin-hCG to induce ovulation. An intrabursal injection of beta-endorphin stimulated nitric oxide synthase (NOS) activity. This effect was completely reversed when naltrexone was co-injected with beta-endorphin. The stimulatory action of beta-endorphin on NOS activity was studied to determine whether it was exerted via prostaglandins. Treatment with prostaglandin E(2) (PGE(2)) completely reversed the beta-endorphin-induced stimulation of NOS activity. Moreover, intrabursal injection of meloxicam, an inhibitor of cyclooxygenase 2, increased NOS activity, but this effect was not altered by co-injection with beta-endorphin. The presence of both endothelial NOS (eNOS) and inducible NOS (iNOS) in the ovary at 10 h after hCG treatment was studied by western blot analysis. Local administration of beta-endorphin inhibited the expression of eNOS protein, whereas expression of iNOS protein was not detectable. Ovarian beta-endorphin content was diminished at 10 h after hCG injection. Treatment with prostaglandin synthesis inhibitors in vivo augmented the ovarian beta-endorphin content. In conclusion, these results indicate that beta-endorphin stimulates the activity of ovarian NOS indirectly by inhibiting prostaglandin production. PMID:12683918

  9. Analogs of the antituberculous agent pyrazinamide are competitive inhibitors of NADPH binding to M. tuberculosis fatty acid synthase I.

    PubMed

    Sayahi, Halimah; Pugliese, Kaitlin M; Zimhony, Oren; Jacobs, William R; Shekhtman, Alexander; Welch, John T

    2012-11-01

    Analogs of pyrazinamide (=pyrazine-2-carboxamide; PZA), an essential component of short-course antituberculous chemotherapy, such as 5-chloropyrazinamide (5-Cl-PZA) act as competitive inhibitors of NADPH binding to purified mycobacterial fatty acid synthase I (FAS I) as shown by Saturation Transfer Difference (STD) NMR studies. In addition, pyrazinoic acid esters (POE) and 5-Cl-POE reversibly bind to FAS I with the relatively greater affinity of longer-chain esters for FAS I, clear from the STD amplification factors. The competitive binding of PZA and 5-Cl-PZA clearly illustrates that both agents bind FAS. In contrast to PZA, at low NADPH concentrations 5-Cl-PZA is a cooperative inhibitor of NADPH binding.

  10. Discovery and in Vivo Evaluation of Potent Dual CYP11B2 (Aldosterone Synthase) and CYP11B1 Inhibitors.

    PubMed

    Meredith, Erik L; Ksander, Gary; Monovich, Lauren G; Papillon, Julien P N; Liu, Qian; Miranda, Karl; Morris, Patrick; Rao, Chang; Burgis, Robin; Capparelli, Michael; Hu, Qi-Ying; Singh, Alok; Rigel, Dean F; Jeng, Arco Y; Beil, Michael; Fu, Fumin; Hu, Chii-Whei; LaSala, Daniel

    2013-12-12

    Aldosterone is a key signaling component of the renin-angiotensin-aldosterone system and as such has been shown to contribute to cardiovascular pathology such as hypertension and heart failure. Aldosterone synthase (CYP11B2) is responsible for the final three steps of aldosterone synthesis and thus is a viable therapeutic target. A series of imidazole derived inhibitors, including clinical candidate 7n, have been identified through design and structure-activity relationship studies both in vitro and in vivo. Compound 7n was also found to be a potent inhibitor of 11β-hydroxylase (CYP11B1), which is responsible for cortisol production. Inhibition of CYP11B1 is being evaluated in the clinic for potential treatment of hypercortisol diseases such as Cushing's syndrome. PMID:24900631

  11. Arsenic toxicity induced endothelial dysfunction and dementia: Pharmacological interdiction by histone deacetylase and inducible nitric oxide synthase inhibitors

    SciTech Connect

    Sharma, Bhupesh Sharma, P.M.

    2013-11-15

    Arsenic toxicity has been reported to damage all the major organs including the brain and vasculature. Dementia including Alzheimer's disease (AD) and vascular dementia (VaD) are posing greater risk to the world population as it is now increasing at a faster rate. We have investigated the role of sodium butyrate, a selective histone deacetylase (HDAC) inhibitor and aminoguanidine, a selective inducible nitric oxide synthase (iNOS) inhibitor in pharmacological interdiction of arsenic toxicity induced vascular endothelial dysfunction and dementia in rats. Arsenic toxicity was done by administering arsenic drinking water to rats. Morris water-maze (MWM) test was used for assessment of learning and memory. Endothelial function was assessed using student physiograph. Oxidative stress (aortic superoxide anion, serum and brain thiobarbituric acid reactive species, brain glutathione) and nitric oxide levels (serum nitrite/nitrate) were also measured. Arsenic treated rats have shown impairment of endothelial function, learning and memory, reduction in serum nitrite/nitrate and brain GSH levels along with increase in serum and brain TBARS. Sodium butyrate as well as aminoguanidine significantly convalesce arsenic induced impairment of learning, memory, endothelial function, and alterations in various biochemical parameters. It may be concluded that arsenic induces endothelial dysfunction and dementia, whereas, sodium butyrate, a HDAC inhibitor as well as aminoguanidine, a selective iNOS inhibitor may be considered as potential agents for the management of arsenic induced endothelial dysfunction and dementia. - Highlights: • As has induced endothelial dysfunction (Edf) and vascular dementia (VaD). • As has increased oxidative stress, AChE activity and decreased serum NO. • Inhibitors of HDAC and iNOS have attenuated As induced Edf and VaD. • Both the inhibitors have attenuated As induced biochemical changes. • Inhibitor of HDAC and iNOS has shown good potential in

  12. NOpiates: Novel Dual Action Neuronal Nitric Oxide Synthase Inhibitors with μ-Opioid Agonist Activity

    PubMed Central

    2012-01-01

    A novel series of benzimidazole designed multiple ligands (DMLs) with activity at the neuronal nitric oxide synthase (nNOS) enzyme and the μ-opioid receptor was developed. Targeting of the structurally dissimilar heme-containing enzyme and the μ-opioid GPCR was predicated on the modulatory role of nitric oxide on μ-opioid receptor function. Structure–activity relationship studies yielded lead compound 24 with excellent nNOS inhibitory activity (IC50 = 0.44 μM), selectivity over both endothelial nitric oxide synthase (10-fold) and inducible nitric oxide synthase (125-fold), and potent μ-opioid binding affinity, Ki = 5.4 nM. The functional activity as measured in the cyclic adenosine monosphospate secondary messenger assay resulted in full agonist activity (EC50 = 0.34 μM). This work represents a novel approach in the development of new analgesics for the treatment of pain. PMID:24900459

  13. Phosphorylation of inhibitor-2 and activation of MgATP-dependent protein phosphatase by rat skeletal muscle glycogen synthase kinase

    SciTech Connect

    Hegazy, M.G.; Reimann, E.M.; Thysseril, T.J.; Schlender, K.K.

    1986-05-01

    Rat skeletal muscle contains a glycogen synthase kinase (GSK-M) which is not stimulated by Ca/sup 2 +/ or cAMP. This kinase has an apparent Mr of 62,000 and uses ATP but not GTP as a phosphoryl donor. GSK-M phosphorylated glycogen synthase at sites 2 and 3. It phosphorylated ATP-citrate lyase and activated MgATP-dependent phosphatase in the presence of ATP but not GTP. As expected, the kinase also phosphorylated phosphatase inhibitor 2 (I-2). Phosphatase incorporation reached approximately 0.3 mol/mol of I-2. Phosphopeptide maps were obtained by digesting /sup 32/P-labeled I-2 with trypsin and separating the peptides by reversed phase HPLC. Two partially separated /sup 32/P-labeled peaks were obtained when I-2 was phosphorylated with either GSK-M or glycogen synthase kinase 3 (GSK-3) and these peptides were different from those obtained when I-2 was phosphorylated with the catalytic subunit of cAMP-dependent protein kinase (CSU) or casein kinase II (CK-II). When I-2 was phosphorylated with GSK-M or GSK-3 and cleaved by CNBr, a single radioactive peak was obtained. Phosphoamino acid analysis showed that I-2 was phosphorylated by GSK-M or GSK-3 predominately in Thr whereas CSU and CK-II phosphorylated I-2 exclusively in Ser. These results indicate that GSK-M is similar to GSK-3 and to ATP-citrate lyase kinase. However, it appears to differ in Mr from ATP-citrate lyase kinase and it differs from GSK-3 in that it phosphorylates glycogen synthase at site 2 and it does not use GTP as a phosphoryl donor.

  14. 7-Nitroindazole, a nitric oxide synthase inhibitor, has anxiolytic-like properties in exploratory models of anxiety.

    PubMed

    Volke, V; Soosaar, A; Kõks, S; Bourin, M; Männistö, P T; Vasar, E

    1997-06-01

    The action of the novel nitric oxide synthase (NOS) inhibitor 7-nitroindazole (7-NI) was studied in different exploratory models of anxiety. In the rat plus-maze test, 7-NI potently increased time spent on open arms and percentage of open arm visits in a dose dependent manner with the minimal effective dose of 40 mg/kg. 7-NI caused an anxiolytic-like effect in the rat social interaction test. The minimal dose increasing social interaction time was 20 mg/kg. However, the drug also produced a clear sedative effect occurring even at smaller doses (10 mg/kg) in the open field test. 7-NI also showed an anxiolytic-like profile in the mouse light-dark compartment test and in the elevated plus-maze test, but the doses required were higher (80-120 mg/kg) than in rat models. Also, the sedative effect occurred at these doses in open field. We failed to demonstrate any effect of L-arginine either in the rat elevated plus-maze test or in the open field test at doses up to 600 mg/kg IP. These results indicate that there are no major interspecies differences between rats and mice in respect of action of 7-NI. The clear anxiolytic-like action of the nitric oxide synthase inhibitor in four different models shows that nitric oxide is involved in the process of anxiety and that NOS could be a new target in developing anxiolytic drugs.

  15. Protective effects of a squalene synthase inhibitor, lapaquistat acetate (TAK-475), on statin-induced myotoxicity in guinea pigs

    SciTech Connect

    Nishimoto, Tomoyuki; Ishikawa, Eiichiro; Anayama, Hisashi; Hamajyo, Hitomi; Nagai, Hirofumi; Hirakata, Masao; Tozawa, Ryuichi

    2007-08-15

    High-dose statin treatment has been recommended as a primary strategy for aggressive reduction of LDL cholesterol levels and protection against coronary artery disease. The effectiveness of high-dose statins may be limited by their potential for myotoxic side effects. There is currently little known about the molecular mechanisms of statin-induced myotoxicity. Previously we showed that T-91485, an active metabolite of the squalene synthase inhibitor lapaquistat acetate (lapaquistat: a previous name is TAK-475), attenuated statin-induced cytotoxicity in human skeletal muscle cells [Nishimoto, T., Tozawa, R., Amano, Y., Wada, T., Imura, Y., Sugiyama, Y., 2003a. Comparing myotoxic effects of squalene synthase inhibitor, T-91485, and 3-hydroxy-3-methylglutaryl coenzyme A. Biochem. Pharmacol. 66, 2133-2139]. In the current study, we investigated the effects of lapaquistat administration on statin-induced myotoxicity in vivo. Guinea pigs were treated with either high-dose cerivastatin (1 mg/kg) or cerivastatin together with lapaquistat (30 mg/kg) for 14 days. Treatment with cerivastatin alone decreased plasma cholesterol levels by 45% and increased creatine kinase (CK) levels by more than 10-fold (a marker of myotoxicity). The plasma CK levels positively correlated with the severity of skeletal muscle lesions as assessed by histopathology. Co-administration of lapaquistat almost completely prevented the cerivastatin-induced myotoxicity. Administration of mevalonolactone (100 mg/kg b.i.d.) prevented the cerivastatin-induced myotoxicity, confirming that this effect is directly related to HMG-CoA reductase inhibition. These results strongly suggest that cerivastatin-induced myotoxicity is due to depletion of mevalonate derived isoprenoids. In addition, squalene synthase inhibition could potentially be used clinically to prevent statin-induced myopathy.

  16. Stereocontrolled Synthesis of a Potential Transition-State Inhibitor of the Salicylate Synthase MbtI from Mycobacterium tuberculosis

    PubMed Central

    Liu, Zheng; Liu, Feng; Aldrich, Courtney C.

    2015-01-01

    Mycobactins are small-molecule iron chelators (siderophores) produced by Mycobacterium tuberculosis (Mtb) for iron mobilization. The bifunctional salicylate synthase MbtI catalyzes the first step of mycobactin biosynthesis through the conversion of the primary metabolite chorismate into salicylic acid via isochorismate. We report the design, synthesis and biochemical evaluation of an inhibitor based on the putative transition-state (TS) for the isochorismatase partial reaction of MbtI. The inhibitor mimics the hypothesized charge build-up at C-4 of chorismate in the TS as well as C-O bond-formation at C-6. Another important design element of the inhibitor is replacement of the labile pyruvate side-chain in chorismate with a stable C-linked propionate isostere. We developed a stereocontrolled synthesis of the highly functionalized cyclohexene inhibitor that features an asymmetric aldol reaction using a titanium enolate, diastereoselective Grignard addition to a tert-butanesulfinyl aldimine, and ring closing olefin metathesis as key steps. PMID:26035083

  17. Novel 2,4-Disubstituted Pyrimidines as Potent, Selective, and Cell-Permeable Inhibitors of Neuronal Nitric Oxide Synthase

    PubMed Central

    2014-01-01

    Selective inhibition of neuronal nitric oxide synthase (nNOS) is an important therapeutic approach to target neurodegenerative disorders. However, the majority of the nNOS inhibitors developed are arginine mimetics and, therefore, suffer from poor bioavailability. We designed a novel strategy to combine a more pharmacokinetically favorable 2-imidazolylpyrimidine head with promising structural components from previous inhibitors. In conjunction with extensive structure–activity studies, several highly potent and selective inhibitors of nNOS were discovered. X-ray crystallographic analysis reveals that these type II inhibitors utilize the same hydrophobic pocket to gain strong inhibitory potency (13), as well as high isoform selectivity. Interestingly, select compounds from this series (9) showed good permeability and low efflux in a Caco-2 assay, suggesting potential oral bioavailability, and exhibited minimal off-target binding to 50 central nervous system receptors. Furthermore, even with heme-coordinating groups in the molecule, modifying other pharmacophoric fragments minimized undesirable inhibition of cytochrome P450s from human liver microsomes. PMID:25489882

  18. Three-dimensional structures of Plasmodium falciparum spermidine synthase with bound inhibitors suggest new strategies for drug design

    SciTech Connect

    Sprenger, Janina; Svensson, Bo; Hålander, Jenny; Carey, Jannette; Persson, Lo; Al-Karadaghi, Salam

    2015-03-01

    In this work, X-ray crystallography was used to examine ligand complexes of spermidine synthase from the malaria parasite Plasmodium falciparum (PfSpdS). The enzymes of the polyamine-biosynthesis pathway have been proposed to be promising drug targets in the treatment of malaria. Spermidine synthase (SpdS; putrescine aminopropyltransferase) catalyzes the transfer of the aminopropyl moiety from decarboxylated S-adenosylmethionine to putrescine, leading to the formation of spermidine and 5′-methylthioadenosine (MTA). In this work, X-ray crystallography was used to examine ligand complexes of SpdS from the malaria parasite Plasmodium falciparum (PfSpdS). Five crystal structures were determined of PfSpdS in complex with MTA and the substrate putrescine, with MTA and spermidine, which was obtained as a result of the enzymatic reaction taking place within the crystals, with dcAdoMet and the inhibitor 4-methylaniline, with MTA and 4-aminomethylaniline, and with a compound predicted in earlier in silico screening to bind to the active site of the enzyme, benzimidazol-(2-yl)pentan-1-amine (BIPA). In contrast to the other inhibitors tested, the complex with BIPA was obtained without any ligand bound to the dcAdoMet-binding site of the enzyme. The complexes with the aniline compounds and BIPA revealed a new mode of ligand binding to PfSpdS. The observed binding mode of the ligands, and the interplay between the two substrate-binding sites and the flexible gatekeeper loop, can be used in the design of new approaches in the search for new inhibitors of SpdS.

  19. Simulated microgravity upregulates an endothelial vasoconstrictor prostaglandin

    NASA Technical Reports Server (NTRS)

    Sangha, D. S.; Han, S.; Purdy, R. E.

    2001-01-01

    Endothelial nitric oxide contributes to the vascular hyporesponsiveness to norepinephrine (NE) observed in carotid arteries from rats exposed to simulated microgravity. The goal of the present study was to determine whether a cyclooxygenase product of arachidonic acid also influences vascular responsiveness in this setting. Microgravity was simulated in rats by hindlimb unweighting (HU). After 20 days of HU, carotid arteries were isolated from control and HU-treated rats, and vascular rings were mounted in tissue baths for the measurement of isometric contraction. Two cyclooxygenase inhibitors, indomethacin and ibuprofen, and the selective thromboxane A(2) prostanoid-receptor antagonist, SQ-29548, had no effect on the contraction to NE in control vessels but markedly reduced contraction to NE in HU vessels. When the endothelium was removed, indomethacin no longer had any effect on the NE-induced contraction in HU vessels. In endothelium-intact vessels in the presence of indomethacin, the addition of the nitric oxide synthase inhibitor, N(G)-L-nitro-arginine methyl ester, to the medium bathing HU vessels increased the contraction to NE to the level of that of the control vessels. These results indicate that HU treatment induced two endothelial changes in carotid artery that opposed each other. Nitric oxide activity was increased and was responsible for the vascular hyporesponsiveness to NE. The activity of a vasoconstrictor prostaglandin was also increased, and attenuated the vasodilating effect of nitric oxide.

  20. Lysophosphatidic acid-mediated signal-transduction pathways involved in the induction of the early-response genes prostaglandin G/H synthase-2 and Egr-1: a critical role for the mitogen-activated protein kinase p38 and for Rho proteins.

    PubMed Central

    Reiser, C O; Lanz, T; Hofmann, F; Hofer, G; Rupprecht, H D; Goppelt-Struebe, M

    1998-01-01

    During inflammatory processes of the kidney, lesions of the glomerulus lead to aggregation of thrombocytes and infiltration of macrophages, which can release bioactive mediators. One of these important signalling molecules is lysophosphatidic acid (LPA). Incubation of rat mesangial cells with LPA induced mRNA and protein expression of the early-response genes pghs-2 (for prostaglandin G/H synthase-2/cyclo-oxygenase-2) and egr-1. As shown by antisense experiments, induction of egr-1 was related to the strong mitogenic effect of LPA. LPA-mediated gene expression was inhibited by pertussis toxin, indicating coupling to G-proteins of the Gi family. Specific inhibition of proteins of the small G-protein subfamily Rho with toxin B from Clostridium difficile led to changes in mesangial cell morphology without induction of apoptosis. LPA-mediated expression of pghs-2 and egr-1 was reduced to base-line levels by toxin B, indicating a role for Rho proteins in LPA-mediated gene induction. Of the two mitogen-activated protein kinase (MAPK) pathways investigated, the MAPK kinase-extracellular signal-regulated kinase pathway was involved in the induction of both pghs-2 and egr-1 mRNA expression, as shown by the inhibitory effect of PD98059. Activation of the MAPK p38, however, was only related to pghs-2 expression, whereas egr-1 expression was not affected by treatment of mesangial cells with the specific inhibitor SB203580. Taken together our data provide evidence that LPA-mediated activation of MAPK kinase and Rho proteins leads to the induction of the functionally distinct early-response genes pghs-2 and egr-1, whereas activation of MAPK p38 revealed considerable differences between the regulation of these two genes. PMID:9494074

  1. NADPH Oxidase NOX5-S and Nuclear Factor κB1 Mediate Acid-Induced Microsomal Prostaglandin E Synthase-1 Expression in Barrett’s Esophageal Adenocarcinoma Cells

    PubMed Central

    Zhou, Xiaoxu; Li, Dan; Resnick, Murray B.; Wands, Jack

    2013-01-01

    The mechanisms of progression from Barrett’s esophagus (BE) to esophageal adenocarcinoma (EA) are not known. Cycloxygenase-2 (COX-2)-derived prostaglandin E2 (PGE2) has been shown to be important in esophageal tumorigenesis. We have shown that COX-2 mediates acid-induced PGE2 production. The prostaglandin E synthase (PGES) responsible for acid-induced PGE2 production in BE, however, is not known. We found that microsomal PGES1 (mPGES1), mPGES2, and cytosolic PGES (cPGES) were present in FLO EA cells. Pulsed acid treatment significantly increased mPGES1 mRNA and protein levels but had little or no effect on mPGES2 or cPGES mRNA. Knockdown of mPGES1 by mPGES1 small interfering RNA (siRNA) blocked acid-induced increase in PGE2 production and thymidine incorporation. Knockdown of NADPH oxidase, NOX5-S, a variant lacking calcium-binding domains, by NOX5 siRNA significantly inhibited acid-induced increase in mPGES1 expression, thymidine incorporation, and PGE2 production. Overexpression of NOX5-S significantly increased the luciferase activity in FLO cells transfected with a nuclear factor κB (NF-κB) in vivo activation reporter plasmid pNF-κB-Luc. Knockdown of NF-κB1 p50 by p50 siRNA significantly decreased acid-induced increase in mPGES1 expression, thymidine incorporation, and PGE2 production. Two novel NF-κB binding elements, GGAGTCTCCC and CGGGACACCC, were identified in the mPGES1 gene promoter. We conclude that mPGES1 mediates acid-induced increase in PGE2 production and cell proliferation. Acid-induced mPGES1 expression depends on activation of NOX5-S and NF-κB1 p50. Microsomal PGES1 may be a potential target to prevent or treat EA. PMID:23439561

  2. Identification of novel scaffold of benzothiazepinones as non-ATP competitive glycogen synthase kinase-3β inhibitors through virtual screening.

    PubMed

    Zhang, Peng; Hu, Hai-Rong; Huang, Zhao-Hui; Lei, Jia-Yi; Chu, Yong; Ye, De-Yong

    2012-12-01

    Glycogen synthase kinase-3β (GSK-3β) is an important serine/threonine kinase that has been proved as a key target for neurodegenerative diseases and diabetes. Up to date, most of known inhibitors are bound to the ATP-binding pocket of GSK-3β, which might lead widespread effects due to the high homology between kinases. Recently, some of its non-ATP competitive inhibitors had been confirmed having therapeutical effects owing to their high selectivity. This finding opens a new pathway to study hopeful drugs for treatment of these diseases. However, it is still a challenge nowadays on how to efficiently find non-ATP competitors. Here, we successfully discovered a novel scaffold of benzothiazepinones (BTZs) as selective non-ATP competitive GSK-3β inhibitors through virtual screening approach. A 3D receptor model of substrate binding site of GSK-3β was constructed and applied to screen against drug-like Maybridge database through Autodock program. BTZ compounds were top ranked as efficient hits and were then synthesized for further screening. Among them, the representative compound 4j showed activity to GSK-3β (IC(50): 25 μM) in non-ATP competitive mechanism, and nearly no inhibitory effect on other 10 related protein kinases. Overall, the results point out that BTZ compounds might be useful in treatment of Alzheimer's disease and diabetes mellitus as novel GSK-3β inhibitors. It also suggests, on the other hand, that virtual screening would provide a valuable tool in combination with in vitro assays for the identification of novel selective and potent inhibitors.

  3. A nanotherapy strategy significantly enhances anticryptosporidial activity of an inhibitor of bifunctional thymidylate synthase-dihydrofolate reductase from Cryptosporidium.

    PubMed

    Mukerjee, Anindita; Iyidogan, Pinar; Castellanos-Gonzalez, Alejandro; Cisneros, José A; Czyzyk, Daniel; Ranjan, Amalendu Prakash; Jorgensen, William L; White, A Clinton; Vishwanatha, Jamboor K; Anderson, Karen S

    2015-01-01

    Cryptosporidiosis, a gastrointestinal disease caused by protozoans of the genus Cryptosporidium, is a common cause of diarrheal diseases and often fatal in immunocompromised individuals. Bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) from Cryptosporidium hominis (C. hominis) has been a molecular target for inhibitor design. C. hominis TS-DHFR inhibitors with nM potency at a biochemical level have been developed however drug delivery to achieve comparable antiparasitic activity in Cryptosporidium infected cell culture has been a major hurdle for designing effective therapies. Previous mechanistic and structural studies have identified compound 906 as a nM C. hominis TS-DHFR inhibitor in vitro, having μM antiparasitic activity in cell culture. In this work, proof of concept studies are presented using a nanotherapy approach to improve drug delivery and the antiparasitic activity of 906 in cell culture. We utilized PLGA nanoparticles that were loaded with 906 (NP-906) and conjugated with antibodies to the Cryptosporidium specific protein, CP2, on the nanoparticle surface in order to specifically target the parasite. Our results indicate that CP2 labeled NP-906 (CP2-NP-906) reduces the level of parasites by 200-fold in cell culture, while NP-906 resulted in 4.4-fold decrease. Moreover, the anticryptosporidial potency of 906 improved 15 to 78-fold confirming the utility of the antibody conjugated nanoparticles as an effective drug delivery strategy.

  4. Three-dimensional structures of Plasmodium falciparum spermidine synthase with bound inhibitors suggest new strategies for drug design

    PubMed Central

    Sprenger, Janina; Svensson, Bo; Hålander, Jenny; Carey, Jannette; Persson, Lo; Al-Karadaghi, Salam

    2015-01-01

    The enzymes of the polyamine-biosynthesis pathway have been proposed to be promising drug targets in the treatment of malaria. Spermidine synthase (SpdS; putrescine aminopropyltransferase) catalyzes the transfer of the aminopropyl moiety from decarboxylated S-adenosylmethionine to putrescine, leading to the formation of spermidine and 5′-methylthio­adenosine (MTA). In this work, X-ray crystallography was used to examine ligand complexes of SpdS from the malaria parasite Plasmodium falciparum (PfSpdS). Five crystal structures were determined of PfSpdS in complex with MTA and the substrate putrescine, with MTA and spermidine, which was obtained as a result of the enzymatic reaction taking place within the crystals, with dcAdoMet and the inhibitor 4-methylaniline, with MTA and 4-aminomethyl­aniline, and with a compound predicted in earlier in silico screening to bind to the active site of the enzyme, benzimidazol-(2-yl)pentan-1-amine (BIPA). In contrast to the other inhibitors tested, the complex with BIPA was obtained without any ligand bound to the dcAdoMet-binding site of the enzyme. The complexes with the aniline compounds and BIPA revealed a new mode of ligand binding to PfSpdS. The observed binding mode of the ligands, and the interplay between the two substrate-binding sites and the flexible gatekeeper loop, can be used in the design of new approaches in the search for new inhibitors of SpdS. PMID:25760598

  5. Transmembrane BAX Inhibitor Motif Containing (TMBIM) Family Proteins Perturbs a trans-Golgi Network Enzyme, Gb3 Synthase, and Reduces Gb3 Biosynthesis*

    PubMed Central

    Yamaji, Toshiyuki; Nishikawa, Kiyotaka; Hanada, Kentaro

    2010-01-01

    Globotriaosylceramide (Gb3) is a well known receptor for Shiga toxin (Stx), produced by enterohemorrhagic Escherichia coli and Shigella dysenteriae. The expression of Gb3 also affects several diseases, including cancer metastasis and Fabry disease, which prompted us to look for factors involved in its metabolism. In the present study, we isolated two cDNAs that conferred resistance to Stx-induced cell death in HeLa cells by expression cloning: ganglioside GM3 synthase and the COOH terminus region of glutamate receptor, ionotropic, N-methyl-d-asparate-associated protein 1 (GRINA), a member of the transmembrane BAX inhibitor motif containing (TMBIM) family. Overexpression of the truncated form, named GRINA-C, and some members of the full-length TMBIM family, including FAS inhibitory molecule 2 (FAIM2), reduced Gb3, and lactosylceramide was accumulated instead. The change of glycolipid composition was restored by overexpression of Gb3 synthase, suggesting that the synthase is affected by GRINA-C and FAIM2. Interestingly, the mRNA level of Gb3 synthase was unchanged. Rather, localization of the synthase as well as TGN46, a trans-Golgi network marker, was perturbed to form punctate structures, and degradation of the synthase in lysosomes was enhanced. Furthermore, GRINA-C was associated with Gb3 synthase. These observations may demonstrate a new type of posttranscriptional regulation of glycosyltransferases. PMID:20837469

  6. Transmembrane BAX inhibitor motif containing (TMBIM) family proteins perturbs a trans-Golgi network enzyme, Gb3 synthase, and reduces Gb3 biosynthesis.

    PubMed

    Yamaji, Toshiyuki; Nishikawa, Kiyotaka; Hanada, Kentaro

    2010-11-12

    Globotriaosylceramide (Gb3) is a well known receptor for Shiga toxin (Stx), produced by enterohemorrhagic Escherichia coli and Shigella dysenteriae. The expression of Gb3 also affects several diseases, including cancer metastasis and Fabry disease, which prompted us to look for factors involved in its metabolism. In the present study, we isolated two cDNAs that conferred resistance to Stx-induced cell death in HeLa cells by expression cloning: ganglioside GM3 synthase and the COOH terminus region of glutamate receptor, ionotropic, N-methyl-D-asparate-associated protein 1 (GRINA), a member of the transmembrane BAX inhibitor motif containing (TMBIM) family. Overexpression of the truncated form, named GRINA-C, and some members of the full-length TMBIM family, including FAS inhibitory molecule 2 (FAIM2), reduced Gb3, and lactosylceramide was accumulated instead. The change of glycolipid composition was restored by overexpression of Gb3 synthase, suggesting that the synthase is affected by GRINA-C and FAIM2. Interestingly, the mRNA level of Gb3 synthase was unchanged. Rather, localization of the synthase as well as TGN46, a trans-Golgi network marker, was perturbed to form punctate structures, and degradation of the synthase in lysosomes was enhanced. Furthermore, GRINA-C was associated with Gb3 synthase. These observations may demonstrate a new type of posttranscriptional regulation of glycosyltransferases.

  7. Inhibitors of the salicylate synthase (MbtI) from Mycobacterium tuberculosis discovered by high-throughput screening.

    PubMed

    Vasan, Mahalakshmi; Neres, João; Williams, Jessica; Wilson, Daniel J; Teitelbaum, Aaron M; Remmel, Rory P; Aldrich, Courtney C

    2010-12-01

    A simple steady-state kinetic high-throughput assay was developed for the salicylate synthase MbtI from Mycobacterium tuberculosis, which catalyzes the first committed step of mycobactin biosynthesis. The mycobactins are small-molecule iron chelators produced by M. tuberculosis, and their biosynthesis has been identified as a promising target for the development of new antitubercular agents. The assay was miniaturized to a 384-well plate format and high-throughput screening was performed at the National Screening Laboratory for the Regional Centers of Excellence in Biodefense and Emerging Infectious Diseases (NSRB). Three classes of compounds were identified comprising the benzisothiazolones (class I), diarylsulfones (class II), and benzimidazole-2-thiones (class III). Each of these compound series was further pursued to investigate their biochemical mechanism and structure-activity relationships. Benzimidazole-2-thione 4 emerged as the most promising inhibitor owing to its potent reversible inhibition. PMID:21053346

  8. Inhibitors of the Salicylate Synthase (MbtI) from Mycobacterium tuberculosis Discovered by High-Throughput Screening

    PubMed Central

    Vasan, Mahalakshmi; Neres, João; Williams, Jessica; Wilson, Daniel J.; Teitelbaum, Aaron M.; Remmel, Rory P.; Aldrich, Courtney C.

    2010-01-01

    A simple steady-state kinetic high-throughput assay was developed for the salicylate synthase MbtI from Mycobacterium tuberculosis, which catalyzes the first committed step of mycobactin biosynthesis. The mycobactins are small-molecule iron chelators produced by M. tuberculosis, and their biosynthesis has been identified as a promising target for the development of new antitubercular agents. The assay was miniaturized to a 384-well plate format and high-throughput screening was performed at the National Screening Laboratory for the Regional Centers of Excellence in Biodefense and Emerging Infectious Diseases (NSRB). Three classes of compounds were identified comprising the benzisothiazolones (class I), diarylsulfones (class II), and benzimidazole-2-thiones (class III). Each of these compound series was further pursued to investigate their biochemical mechanism and structure–activity relationships. Benzimidazole-2-thione 4 emerged as the most promising inhibitor owing to its potent reversible inhibition. PMID:21053346

  9. Synthesis of benzimidazole based thiadiazole and carbohydrazide conjugates as glycogen synthase kinase-3β inhibitors with anti-depressant activity.

    PubMed

    Khan, Imran; Tantray, Mushtaq A; Hamid, Hinna; Alam, Mohammad Sarwar; Kalam, Abul; Dhulap, Abhijeet

    2016-08-15

    A series of benzimidazole based thiadiazole and carbohydrazide conjugates have been synthesized and evaluated for inhibition of glycogen synthase kinase-3β and anti-depressant effect. Compounds 4f, 4j, 5b, 5g and 5i were found to be the most potent inhibitors of GSK-3β in vitro amongst the twenty-five benzimidazole based thiadiazole and carbohydrazide conjugates synthesized. Compound 5i was also found to exhibit significant antidepressant activity in vivo at 50mg/kg, when compared to fluoxetine, a known antidepressant drug. The molecular docking studies revealed multiple hydrogen bond interactions by the synthesized compounds with various amino acid residues, viz, ASP-133, LYS-183, PRO-136, VAL-135, TYR-134, or LYS-60 at the GSK-3β receptor site. PMID:27406796

  10. Protective effect of (±)α-tocopherol on brominated diphenyl ether-47-stimulated prostaglandin pathways in human extravillous trophoblasts in vitro.

    PubMed

    Park, Hae-Ryung; Loch-Caruso, Rita

    2015-10-01

    Brominated diphenyl ether (BDE)-47 is a prevalent flame retardant chemical found in human tissues and is linked to adverse pregnancy outcomes in humans. Because dysregulation of the prostaglandin pathway is implicated in adverse pregnancy outcomes, the present study investigates BDE-47 induction of prostaglandin synthesis in a human extravillous trophoblast cell line, HTR-8/SVneo, examining the hypothesis that BDE-47 increases generation of reactive oxygen species (ROS) to stimulate the prostaglandin response. Treatment with 20 μM BDE-47 significantly increased mRNA expression of prostaglandin-endoperoxide synthase 2 (PTGS2) at 4, 12 and 24 h, and 24-h treatment significantly increased cyclooxygenase (COX)-2 cellular protein expression and prostaglandin E2 (PGE2) concentration in culture medium. The BDE-47-stimulated PGE2 release was inhibited by the COX inhibitors indomethacin and NS398, implicating COX activity. Exposure to 20 μM BDE-47 significantly increased ROS generation as measured by carboxydichlorofluorescein fluorescence, and this response was blocked by cotreatment with the peroxyl radical scavenger (±)-α-tocopherol. (±)-α-Tocopherol cotreatment suppressed BDE-47-stimulated increases of PGE2 release without significant effects on COX-2 mRNA and protein expression, implicating a role for ROS in post-translational regulation of COX activity. Because prostaglandins regulate trophoblast functions necessary for placentation and pregnancy, further investigation is warranted of BDE-47 impacts on trophoblast responses. PMID:26026498

  11. Protective Effect of (±)α-Tocopherol on Brominated Diphenyl Ether-47-Stimulated Prostaglandin Pathways in Human Extravillous Trophoblasts In Vitro

    PubMed Central

    Park, Hae-Ryung; Loch-Caruso, Rita

    2015-01-01

    Brominated diphenyl ether (BDE)-47 is a prevalent flame retardant chemical found in human tissues and is linked to adverse pregnancy outcomes in humans. Because dysregulation of the prostaglandin pathway is implicated in adverse pregnancy outcomes, the present study investigates BDE-47 induction of prostaglandin synthesis in a human extravillous trophoblast cell line, HTR-8/SVneo, examining the hypothesis that BDE-47 increases generation of reactive oxygen species (ROS) to stimulate the prostaglandin response. Treatment with 20 μM BDE-47 significantly increased mRNA expression of prostaglandin-endoperoxide synthase 2 (PTGS2) at 4, 12 and 24 h, and 24-h treatment significantly increased cyclooxygenase (COX)-2 cellular protein expression and prostaglandin E2 (PGE2) concentration in culture medium. The BDE-47-stimulated PGE2 release was inhibited by the COX inhibitors indomethacin and NS398, implicating COX activity. Exposure to 20 μM BDE-47 significantly increased ROS generation as measured by carboxydichlorofluorescein fluorescence, and this response was blocked by cotreatment with the peroxyl radical scavenger (±)-α-tocopherol. (±)-α-Tocopherol cotreatment suppressed BDE-47-stimulated increases of PGE2 release without significant effects on COX-2 mRNA and protein expression, implicating a role for ROS in post-translational regulation of COX activity. Because prostaglandins regulate trophoblast functions necessary for placentation and pregnancy, further investigation is warranted of BDE-47 impacts on trophoblast responses. PMID:26026498

  12. Coordinate Functional Regulation between Microsomal Prostaglandin E Synthase-1 (mPGES-1) and Peroxisome Proliferator-activated Receptor γ (PPARγ) in the Conversion of White-to-brown Adipocytes*

    PubMed Central

    García-Alonso, Verónica; López-Vicario, Cristina; Titos, Esther; Morán-Salvador, Eva; González-Périz, Ana; Rius, Bibiana; Párrizas, Marcelina; Werz, Oliver; Arroyo, Vicente; Clària, Joan

    2013-01-01

    Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated nuclear receptor and a master regulator of adipogenesis. Microsomal prostaglandin E (PGE) synthase-1 (mPGES-1) is an inducible enzyme that couples with cyclooxygenase-2 for the biosynthesis of PGE2. In this study we demonstrate the existence of a coordinate functional interaction between PPARγ and mPGES-1 in controlling the process of pre-adipocyte differentiation in white adipose tissue (WAT). Adipocyte-specific PPARγ knock-out mice carrying an aP2 promoter-driven Cre recombinase transgene showed a blunted response to the adipogenic effects of a high fat diet. Pre-adipocytes from these knock-out mice showed loss of PPARγ and were resistant to rosiglitazone-induced WAT differentiation. In parallel, WAT from these mice showed increased expression of uncoupling protein 1, a mitochondrial enzyme that dissipates chemical energy as heat. Adipose tissue from mice lacking PPARγ also showed mPGES-1 up-regulation and increased PGE2 levels. In turn, PGE2 suppressed PPARγ expression and blocked rosiglitazone-induced pre-adipocyte differentiation toward white adipocytes while directly elevating uncoupling protein 1 expression and pre-adipocyte differentiation into mature beige/brite adipocytes. Consistently, pharmacological mPGES-1 inhibition directed pre-adipocyte differentiation toward white adipocytes while suppressing differentiation into beige/brite adipocytes. This browning effect was reproduced in knockdown experiments using a siRNA directed against mPGES-1. The effects of PGE2 on pre-adipocyte differentiation were not seen in mice lacking PPARγ in adipose tissue and were not mirrored by other eicosanoids (i.e. leukotriene B4). Taken together, these findings identify PGE2 as a key regulator of white-to-brown adipogenesis and suggest the existence of a coordinate regulation of adipogenesis between PPARγ and mPGES-1. PMID:23943621

  13. Neuronal nitric oxide synthase inhibitor, 7-nitroindazole, delays motor dysfunction and spinal motoneuron degeneration in the wobbler mouse.

    PubMed

    Ikeda, K; Iwasaki, Y; Kinoshita, M

    1998-09-18

    Gene mutations of superoxide dismutase (SOD) have been discovered in familial amyotrophic lateral sclerosis (ALS). Neuronal nitric oxide synthase (NOS), endothelial NOS and 3-nitrotyrosine immunoreactivities are selectively increased in the spinal motoneurons of sporadic ALS. Other study suggests that 3-nitrotyrosine immunoreactivity is enhanced in the spinal motoneurons of sporadic and familial ALS patients. The hypothesis is postulated that increased production of radical species, such as superoxide and peroxynitrite, may cause motoneuron degeneration in ALS. There are increased amounts of nitric oxide and SOD hypoactivities in the brain and spinal cord of wobbler mice. NOS is also induced in the vacuolated spinal motoneurons or axons in this animal. Free radicals might contribute to the pathogenesis of wobbler mouse motoneuron disease. Lecithinized SOD treatment has retarded the progression of this disease. This evidence allowed us to determine whether NOS inhibitors delay progression of wobbler mouse motoneuron disease. After clinical diagnosis at age 3-4 weeks, wobbler mice were injected with intraperitoneal non-selective NOS inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME, 50 mg/kg), two doses of neuronal NOS inhibitor, 7-nitroindazole (5 or 50 mg/kg) or a vehicle solution, daily for 4 weeks in a blind fashion. In comparison with vehicle, 7-nitroindazole-treated mice potentiated grip strength and attenuated deformities in the forelimbs. 7-Nitroindazole treatment increased the biceps muscle weight, reduced denervation muscle atrophy, and suppressed degeneration of spinal motoneurons. To a lesser degree, L-NAME-treated mice displayed slowed progression of disease. The present studies indicate that neuronal NOS inhibitor may be a candidate for promising therapy in lower motoneuron disease or motor neuropathy. PMID:9804111

  14. Immunolocalization, gene expression, and enzymatic activity of cyclooxygenases, prostaglandin e2-9-ketoreductase, and nitric oxide synthases in Mediterranean buffalo (Bubalus bubalis) corpora lutea during diestrus.

    PubMed

    Parillo, F; Catone, G; Maranesi, M; Gobbetti, A; Gasparrini, B; Russo, M; Boiti, C; Zerani, M

    2012-12-01

    Immunopresence, gene expression, and enzymatic activity of cyclooxygenase 1 (COX1), COX2, PGE2-9-ketoreductase (PGE2-9-K), endothelial (eNOS), and inducible nitric oxide synthases (iNOS), and hormone in vitro production were examined in early, mid, late, and regressive buffalo corpora lutea (CL). COX1 immunosignals were detected in the cytoplasm of small luteal cells, COX2 in large luteal cells, and PGE2-9-K in all luteal cells. COX2 and PGE2-9-K immunosignals were greater in late CL. Immunopresence of both NOS types were evidenced in the nuclei and cytoplasm of all luteal cells, as well as in the nuclei of endothelial cells, during all stages studied. The eNOS and iNOS immunosignals increased during the early stage. COX1 transcripts were lower in late and regressive CL, COX2 in late, PGE2-9-K higher in regressive, and iNOS higher in early and lower in regressive CL. COX1 enzymatic activity was lower in regressive CL, COX2 increased in mid and late stages, and PGE2-9-K was higher in late CL. Endothelial NOS activity was higher during mid and late stages and lower in regressive, whereas iNOS was greater in late and lower in early. Progesterone in vitro release was higher in mid and lower in late phase, while PGF2α synthesis was higher in late CL and lower in regressive, and PGE2 was higher during regressive stage. These results support the idea that COX, NOS, and PGE2-9-K regulate buffalo CL life span. In particular, regressive CL seems involved in the development of the contralateral early CL, through the production of the luteotrophic PGE2. PMID:22865504

  15. Characterization of acetohydroxyacid synthase I from Escherichia coli K-12 and identification of its inhibitors.

    PubMed

    Pham, Ngoc Chien; Moon, Ji-Young; Cho, Jun-Haeng; Lee, Soo-Jae; Park, Joon-Shik; Kim, Dong-Eun; Park, Yoonkyung; Yoon, Moon-Young

    2010-01-01

    The first step in branched-chain amino acid biosynthesis is catalyzed by acetohydroxyacid synthase (EC 2.2.1.6). This reaction involves decarboxylation of pyruvate followed by condensation with either an additional pyruvate molecule or with 2-oxobutyrate. The enzyme requires three cofactors, thiamine diphosphate (ThDP), a divalent ion, and flavin adenine dinucleotide (FAD). Escherichia coli contains three active isoenzymes, and acetohydroxyacid synthase I (AHAS I) large subunit is encoded by the ilvB gene. In this study, the ilvB gene from E. coli K-12 was cloned into expression vector pETDuet-1, and was expressed in E. coli BL21 (DH3). The purified protein was identified on a 12% SDS-PAGE gel as a single band with a mass of 65 kDa. The optimum temperature, buffer, and pH for E. coli K-12 AHAS I were 37 °C, potassium phosphate buffer, and 7.5. Km values for E. coli K-12 AHAS I binding to pyruvate, Mg(+2), ThDP, and FAD were 4.15, 1.26, 0.2 mM, and 0.61 µM respectively. Inhibition of purified AHAS I protein was determined with herbicides and new compounds.

  16. Identification and development of mPGES-1 inhibitors: where we are at?

    PubMed Central

    Chang, Hui-Hua; Meuillet, Emmanuelle J

    2011-01-01

    Microsomal prostaglandin E synthase-1 (mPGES-1) is the terminal synthase responsible for the synthesis of the pro-tumorigenic prostaglandin E2 (PGE2). mPGES-1 is overexpressed in a wide variety of cancers. Since its discovery in 1997 by Bengt Samuelsson and collaborators, the enzyme has been the object of over 200 peer-reviewed articles. Although today mPGES-1 is considered a validated and promising therapeutic target for anticancer drug discovery, challenges in inhibitor design and selectivity are such that up to this date there are only a few published records of small-molecule inhibitors targeting the enzyme and exhibiting some in vivo anticancer activity. This review summarizes the structures, and the in vitro and in vivo activities of these novel mPGES-1 inhibitors. Challenges that have been encountered are also discussed. PMID:22023034

  17. The Design and Synthesis of Potent and Selective Inhibitors of Trypanosoma brucei Glycogen Synthase Kinase 3 for the Treatment of Human African Trypanosomiasis

    PubMed Central

    2014-01-01

    Glycogen synthase kinase 3 (GSK3) is a genetically validated drug target for human African trypanosomiasis (HAT), also called African sleeping sickness. We report the synthesis and biological evaluation of aminopyrazole derivatives as Trypanosoma brucei GSK3 short inhibitors. Low nanomolar inhibitors, which had high selectivity over the off-target human CDK2 and good selectivity over human GSK3β enzyme, have been prepared. These potent kinase inhibitors demonstrated low micromolar levels of inhibition of the Trypanosoma brucei brucei parasite grown in culture. PMID:25198388

  18. Phenyl Ether- and Aniline-Containing 2-Aminoquinolines as Potent and Selective Inhibitors of Neuronal Nitric Oxide Synthase.

    PubMed

    Cinelli, Maris A; Li, Huiying; Pensa, Anthony V; Kang, Soosung; Roman, Linda J; Martásek, Pavel; Poulos, Thomas L; Silverman, Richard B

    2015-11-12

    Excess nitric oxide (NO) produced by neuronal nitric oxide synthase (nNOS) is implicated in neurodegenerative disorders. As a result, inhibition of nNOS and reduction of NO levels is desirable therapeutically, but many nNOS inhibitors are poorly bioavailable. Promising members of our previously reported 2-aminoquinoline class of nNOS inhibitors, although orally bioavailable and brain-penetrant, suffer from unfavorable off-target binding to other CNS receptors, and they resemble known promiscuous binders. Rearranged phenyl ether- and aniline-linked 2-aminoquinoline derivatives were therefore designed to (a) disrupt the promiscuous binding pharmacophore and diminish off-target interactions and (b) preserve potency, isoform selectivity, and cell permeability. A series of these compounds was synthesized and tested against purified nNOS, endothelial NOS (eNOS), and inducible NOS (iNOS) enzymes. One compound, 20, displayed high potency, selectivity, and good human nNOS inhibition, and retained some permeability in a Caco-2 assay. Most promisingly, CNS receptor counterscreening revealed that this rearranged scaffold significantly reduces off-target binding.

  19. Intracellular quantitative detection of human thymidylate synthase engagement with an unconventional inhibitor using tetracysteine-diarsenical-probe technology.

    PubMed

    Ponterini, Glauco; Martello, Andrea; Pavesi, Giorgia; Lauriola, Angela; Luciani, Rosaria; Santucci, Matteo; Pelà, Michela; Gozzi, Gaia; Pacifico, Salvatore; Guerrini, Remo; Marverti, Gaetano; Costi, Maria Paola; D'Arca, Domenico

    2016-01-01

    Demonstrating a candidate drug's interaction with its target protein in live cells is of pivotal relevance to the successful outcome of the drug discovery process. Although thymidylate synthase (hTS) is an important anticancer target protein, the efficacy of the few anti-hTS drugs currently used in clinical practice is limited by the development of resistance. Hence, there is an intense search for new, unconventional anti-hTS drugs; there are approximately 1600 ongoing clinical trials involving hTS-targeting drugs, both alone and in combination protocols. We recently discovered new, unconventional peptidic inhibitors of hTS that are active against cancer cells and do not result in the overexpression of hTS, which is a known molecular source of resistance. Here, we propose an adaptation of the recently proposed tetracysteine-arsenic-binding-motif technology to detect and quantitatively characterize the engagement of hTS with one such peptidic inhibitor in cell lysates. This new model can be developed into a test for high-throughput screening studies of intracellular target-protein/small-molecule binding. PMID:27250901

  20. Potentiation by nitric oxide synthase inhibitor and calcium channel blocker of aspartame-induced antinociception in the mouse formalin test.

    PubMed

    Abdollahi, M; Nikfar, S; Abdoli, N

    2001-04-01

    By applying a 12 day regimen of the non-calorific sweetener, aspartame, in combination with representative compounds of the calcium channel blocker and nitric oxide synthase inhibitor, we tried to investigate using a formalin-test in mice the relative role of aspartame on pain and its mechanism of action. Verapamil (2, 3.5, 5, 7.5 mg/kg) induced significant (P < 0.01) antinociception in both phases of the formalin test. L-Nitro-arginine-methyl-ester (L-NAME) at the doses used, induced significant (P < 0.01) antinociception in early phase (1, 2, 5, 10 mg/kg) and late phase (5, 10 mg/kg). Twelve days of treatment in animals by aspartame (0.16% w/v) significantly induced antinociception in both phases of the formalin test. Both verapamil (5 mg/kg) and L-NAME (10 mg/kg) significantly (P < 0.01) potentiated aspartame-induced antinociception in both phases of formalin test. The present findings support the hypothesis that the activation of NMDA receptors by aspartame modulates pain-related behaviour via a nitric oxide/cGMP/glutamate release cascade. It is concluded that aspartame would be a good analgesic agent if it would be used in combination with a calcium channel blocker or NOS inhibitor.

  1. Intracellular quantitative detection of human thymidylate synthase engagement with an unconventional inhibitor using tetracysteine-diarsenical-probe technology

    PubMed Central

    Ponterini, Glauco; Martello, Andrea; Pavesi, Giorgia; Lauriola, Angela; Luciani, Rosaria; Santucci, Matteo; Pelà, Michela; Gozzi, Gaia; Pacifico, Salvatore; Guerrini, Remo; Marverti, Gaetano; Costi, Maria Paola; D’Arca, Domenico

    2016-01-01

    Demonstrating a candidate drug’s interaction with its target protein in live cells is of pivotal relevance to the successful outcome of the drug discovery process. Although thymidylate synthase (hTS) is an important anticancer target protein, the efficacy of the few anti-hTS drugs currently used in clinical practice is limited by the development of resistance. Hence, there is an intense search for new, unconventional anti-hTS drugs; there are approximately 1600 ongoing clinical trials involving hTS-targeting drugs, both alone and in combination protocols. We recently discovered new, unconventional peptidic inhibitors of hTS that are active against cancer cells and do not result in the overexpression of hTS, which is a known molecular source of resistance. Here, we propose an adaptation of the recently proposed tetracysteine-arsenic-binding-motif technology to detect and quantitatively characterize the engagement of hTS with one such peptidic inhibitor in cell lysates. This new model can be developed into a test for high-throughput screening studies of intracellular target-protein/small-molecule binding. PMID:27250901

  2. In silico deconstruction of ATP-competitive inhibitors of glycogen synthase kinase-3β.

    PubMed

    Bisignano, Paola; Lambruschini, Chiara; Bicego, Manuele; Murino, Vittorio; Favia, Angelo D; Cavalli, Andrea

    2012-12-21

    Fragment-based methods have emerged in the last two decades as alternatives to traditional high throughput screenings for the identification of chemical starting points in drug discovery. One arguable yet popular assumption about fragment-based design is that the fragment binding mode remains conserved upon chemical expansion. For instance, the question of the binding conservation upon fragmentation of a molecule is still unclear. A number of papers have challenged this hypothesis by means of experimental techniques, with controversial results, "underlining" the idea that a simple generalization, maybe, is not possible. From a computational standpoint, the issue has been rarely addressed and mostly to test novel protocols on limited data sets. To fill this gap, we here report on a computational retrospective study concerned with the in silico deconstruction of leadlike compounds, active on the pharmaceutically relevant enzyme glycogen synthase kinase-3β.

  3. Glycogen Synthase Kinase 3 Inhibitors in the Next Horizon for Alzheimer's Disease Treatment

    PubMed Central

    Martinez, Ana; Gil, Carmen; Perez, Daniel I.

    2011-01-01

    Glycogen synthase kinase 3 (GSK-3), a proline/serine protein kinase ubiquitously expressed and involved in many cellular signaling pathways, plays a key role in the pathogenesis of Alzheimer's disease (AD) being probably the link between β-amyloid and tau pathology. A great effort has recently been done in the discovery and development of different new molecules, of synthetic and natural origin, able to inhibit this enzyme, and several kinetics mechanisms of binding have been described. The small molecule called tideglusib belonging to the thiadiazolidindione family is currently on phase IIb clinical trials for AD. The potential risks and benefits of this new kind of disease modifying drugs for the future therapy of AD are discussed in this paper. PMID:21760986

  4. Glycogen synthase kinase 3 inhibitors in the next horizon for Alzheimer's disease treatment.

    PubMed

    Martinez, Ana; Gil, Carmen; Perez, Daniel I

    2011-01-01

    Glycogen synthase kinase 3 (GSK-3), a proline/serine protein kinase ubiquitously expressed and involved in many cellular signaling pathways, plays a key role in the pathogenesis of Alzheimer's disease (AD) being probably the link between β-amyloid and tau pathology. A great effort has recently been done in the discovery and development of different new molecules, of synthetic and natural origin, able to inhibit this enzyme, and several kinetics mechanisms of binding have been described. The small molecule called tideglusib belonging to the thiadiazolidindione family is currently on phase IIb clinical trials for AD. The potential risks and benefits of this new kind of disease modifying drugs for the future therapy of AD are discussed in this paper.

  5. Crystal Structures of mPGES-1 Inhibitor Complexes Form a Basis for the Rational Design of Potent Analgesic and Anti-Inflammatory Therapeutics.

    PubMed

    Luz, John Gately; Antonysamy, Stephen; Kuklish, Steven L; Condon, Bradley; Lee, Matthew R; Allison, Dagart; Yu, Xiao-Peng; Chandrasekhar, Srinivasan; Backer, Ryan; Zhang, Aiping; Russell, Marijane; Chang, Shawn S; Harvey, Anita; Sloan, Ashley V; Fisher, Matthew J

    2015-06-11

    Microsomal prostaglandin E synthase 1 (mPGES-1) is an α-helical homotrimeric integral membrane inducible enzyme that catalyzes the formation of prostaglandin E2 (PGE2) from prostaglandin H2 (PGH2). Inhibition of mPGES-1 has been proposed as a therapeutic strategy for the treatment of pain, inflammation, and some cancers. Interest in mPGES-1 inhibition can, in part, be attributed to the potential circumvention of cardiovascular risks associated with anti-inflammatory cyclooxygenase 2 inhibitors (coxibs) by targeting the prostaglandin pathway downstream of PGH2 synthesis and avoiding suppression of antithrombotic prostacyclin production. We determined the crystal structure of mPGES-1 bound to four potent inhibitors in order to understand their structure-activity relationships and provide a framework for the rational design of improved molecules. In addition, we developed a light-scattering-based thermal stability assay to identify molecules for crystallographic studies. PMID:25961169

  6. The Fatty Acid Synthase Inhibitor Platensimycin Improves Insulin Resistance without Inducing Liver Steatosis in Mice and Monkeys

    PubMed Central

    Nawrocki, Andrea R.; Zhou, Dan; Wu, Margaret; Previs, Stephen; Miller, Corey; Liu, Haiying; Hines, Catherine D. G.; Madeira, Maria; Cao, Jin; Herath, Kithsiri; Wang, Liangsu; Kelley, David E.; Li, Cai

    2016-01-01

    Objectives Platensimycin (PTM) is a natural antibiotic produced by Streptomyces platensis that selectively inhibits bacterial and mammalian fatty acid synthase (FAS) without affecting synthesis of other lipids. Recently, we reported that oral administration of PTM in mouse models (db/db and db/+) with high de novo lipogenesis (DNL) tone inhibited DNL and enhanced glucose oxidation, which in turn led to net reduction of liver triglycerides (TG), reduced ambient glucose, and improved insulin sensitivity. The present study was conducted to explore translatability and the therapeutic potential of FAS inhibition for the treatment of diabetes in humans. Methods We tested PTM in animal models with different DNL tones, i.e. intrinsic synthesis rates, which vary among species and are regulated by nutritional and disease states, and confirmed glucose-lowering efficacy of PTM in lean NHPs with quantitation of liver lipid by MRS imaging. To understand the direct effect of PTM on liver metabolism, we performed ex vivo liver perfusion study to compare FAS inhibitor and carnitine palmitoyltransferase 1 (CPT1) inhibitor. Results The efficacy of PTM is generally reproduced in preclinical models with DNL tones comparable to humans, including lean and established diet-induced obese (eDIO) mice as well as non-human primates (NHPs). Similar effects of PTM on DNL reduction were observed in lean and type 2 diabetic rhesus and lean cynomolgus monkeys after acute and chronic treatment of PTM. Mechanistically, PTM lowers plasma glucose in part by enhancing hepatic glucose uptake and glycolysis. Teglicar, a CPT1 inhibitor, has similar effects on glucose uptake and glycolysis. In sharp contrast, Teglicar but not PTM significantly increased hepatic TG production, thus caused liver steatosis in eDIO mice. Conclusions These findings demonstrate unique properties of PTM and provide proof-of-concept of FAS inhibition having potential utility for the treatment of diabetes and related metabolic

  7. Effects of mechanistically distinct NF-kappaB inhibitors on glial inducible nitric-oxide synthase expression.

    PubMed

    Davis, Randall L; Sanchez, Alma C; Lindley, Daniel J; Williams, Simon C; Syapin, Peter J

    2005-06-01

    Nuclear factor (NF)-kappaB is an important regulator of inflammatory gene expression. Transcriptional regulation of Nos2, the inducible nitric-oxide synthase (iNOS) gene, is complex and not fully understood, but appears to be regulated in part by NF-kappaB. To further understand the role of NF-kappaB in Nos2 expression, we compared three functionally distinct NF-kappaB inhibitors on NF-kappaB transactivation and iNOS induction by rat C6 glial cells. Cytokine-induced activation of a consensus NF-kappaB-reporter gene was concentration-dependently inhibited by BAY 11-7082, MG-132, and helenalin. The rank order of potency was MG-132>helenalin>BAY 11-7082, with low concentrations of helenalin stimulating reporter gene activity. Cytokine-stimulated iNOS expression, measured by nitrite accumulation and in vitro l-citrulline production, was similarly reduced by exposing C6 cells to the NF-kappaB inhibitors. Surprisingly, activation of Nos2-reporter gene constructs containing the proximal 188 bp (containing one kappaB site) or proximal 94 bp (no kappaB site) of the rat promoter also was inhibited with the same rank order of potency. Interestingly, low concentrations of helenalin increased activity of both promoter constructs, while BAY 11-7082 poorly inhibited the 94-bp activity. This is the first report describing BAY 11-7082 and helenalin effects on iNOS expression in astroglia. Given the reported mechanism of actions for these inhibitors, cytokine-induced glial iNOS expression appears more sensitive to disruption of proteasome degradation and p65 function than modulation of IkappaB phosphorylation. These findings may foster the design of therapeutic agents aimed at NF-kappaB-associated pathways involved in neuroinflammation, especially iNOS expression.

  8. The fatty acid synthase inhibitor triclosan: repurposing an anti-microbial agent for targeting prostate cancer

    PubMed Central

    Sadowski, Martin C.; Pouwer, Rebecca H.; Gunter, Jennifer H.; Lubik, Amy A.; Quinn, Ronald J.; Nelson, Colleen C.

    2014-01-01

    Inhibition of FASN has emerged as a promising therapeutic target in cancer, and numerous inhibitors have been investigated. However, severe pharmacological limitations have challenged their clinical testing. The synthetic FASN inhibitor triclosan, which was initially developed as a topical antibacterial agent, is merely affected by these pharmacological limitations. Yet, little is known about its mechanism in inhibiting the growth of cancer cells. Here we compared the cellular and molecular effects of triclosan in a panel of eight malignant and non-malignant prostate cell lines to the well-known FASN inhibitors C75 and orlistat, which target different partial catalytic activities of FASN. Triclosan displayed a superior cytotoxic profile with a several-fold lower IC50 than C75 or orlistat. Structure-function analysis revealed that alcohol functionality of the parent phenol is critical for inhibitory action. Rescue experiments confirmed that end product starvation was a major cause of cytotoxicity. Importantly, triclosan, C75 and orlistat induced distinct changes to morphology, cell cycle, lipid content and the expression of key enzymes of lipid metabolism, demonstrating that inhibition of different partial catalytic activities of FASN activates different metabolic pathways. These finding combined with its well-documented pharmacological safety profile make triclosan a promising drug candidate for the treatment of prostate cancer. PMID:25313139

  9. Discovery of Bacterial Fatty Acid Synthase Type II Inhibitors Using a Novel Cellular Bioluminescent Reporter Assay

    PubMed Central

    Wallace, Joselynn; Bowlin, Nicholas O.; Mills, Debra M.; Saenkham, Panatda; Kwasny, Steven M.; Opperman, Timothy J.; Williams, John D.; Rock, Charles O.; Bowlin, Terry L.

    2015-01-01

    Novel, cellular, gain-of-signal, bioluminescent reporter assays for fatty acid synthesis type II (FASII) inhibitors were constructed in an efflux-deficient strain of Pseudomonas aeruginosa and based on the discovery that FASII genes in P. aeruginosa are coordinately upregulated in response to pathway disruption. A screen of 115,000 compounds identified a series of sulfonamidobenzamide (SABA) analogs, which generated strong luminescent signals in two FASII reporter strains but not in four control reporter strains designed to respond to inhibitors of pathways other than FASII. The SABA analogs selectively inhibited lipid biosynthesis in P. aeruginosa and exhibited minimal cytotoxicity to mammalian cells (50% cytotoxic concentration [CC50] ≥ 80 μM). The most potent SABA analogs had MICs of 0.5 to 7.0 μM (0.2 to 3.0 μg/ml) against an efflux-deficient Escherichia coli (ΔtolC) strain but had no detectable MIC against efflux-proficient E. coli or against P. aeruginosa (efflux deficient or proficient). Genetic, molecular genetic, and biochemical studies revealed that SABA analogs target the enzyme (AccC) catalyzing the biotin carboxylase half-reaction of the acetyl coenzyme A (acetyl-CoA) carboxylase step in the initiation phase of FASII in E. coli and P. aeruginosa. These results validate the capability and the sensitivity of this novel bioluminescent reporter screen to identify inhibitors of E. coli and P. aeruginosa FASII. PMID:26169404

  10. The fatty acid synthase inhibitor triclosan: repurposing an anti-microbial agent for targeting prostate cancer.

    PubMed

    Sadowski, Martin C; Pouwer, Rebecca H; Gunter, Jennifer H; Lubik, Amy A; Quinn, Ronald J; Nelson, Colleen C

    2014-10-15

    Inhibition of FASN has emerged as a promising therapeutic target in cancer, and numerous inhibitors have been investigated. However, severe pharmacological limitations have challenged their clinical testing. The synthetic FASN inhibitor triclosan, which was initially developed as a topical antibacterial agent, is merely affected by these pharmacological limitations. Yet, little is known about its mechanism in inhibiting the growth of cancer cells. Here we compared the cellular and molecular effects of triclosan in a panel of eight malignant and non-malignant prostate cell lines to the well-known FASN inhibitors C75 and orlistat, which target different partial catalytic activities of FASN. Triclosan displayed a superior cytotoxic profile with a several-fold lower IC50 than C75 or orlistat. Structure-function analysis revealed that alcohol functionality of the parent phenol is critical for inhibitory action. Rescue experiments confirmed that end product starvation was a major cause of cytotoxicity. Importantly, triclosan, C75 and orlistat induced distinct changes to morphology, cell cycle, lipid content and the expression of key enzymes of lipid metabolism, demonstrating that inhibition of different partial catalytic activities of FASN activates different metabolic pathways. These finding combined with its well-documented pharmacological safety profile make triclosan a promising drug candidate for the treatment of prostate cancer.

  11. Ozagrel hydrochloride, a selective thromboxane A2 synthase inhibitor, alleviates liver injury induced by acetaminophen overdose in mice

    PubMed Central

    2013-01-01

    Background Overdosed acetaminophen (paracetamol, N-acetyl-p-aminophenol; APAP) causes severe liver injury. We examined the effects of ozagrel, a selective thromboxane A2 (TXA2) synthase inhibitor, on liver injury induced by APAP overdose in mice. Methods Hepatotoxicity was induced to ICR male mice by an intraperitoneal injection with APAP (330 mg/kg). The effects of ozagrel (200 mg/kg) treatment 30 min after the APAP injection were evaluated with mortality, serum alanine aminotransferase (ALT) levels and hepatic changes, including histopathology, DNA fragmentation, mRNA expression and total glutathione contents. The impact of ozagrel (0.001-1 mg/mL) on cytochrome P450 2E1 (CYP2E1) activity in mouse hepatic microsome was examined. RLC-16 cells, a rat hepatocytes cell line, were exposed to 0.25 mM N-acetyl-p-benzoquinone imine (NAPQI), a hepatotoxic metabolite of APAP. In this model, the cytoprotective effects of ozagrel (1–100 muM) were evaluated by the WST-1 cell viability assay. Results Ozagel treatment significantly attenuated higher mortality, elevated serum alanine aminotransferase levels, excessive hepatic centrilobular necrosis, hemorrhaging and DNA fragmentation, as well as increase in plasma 2,3-dinor thromboxane B2 levels induced by APAP injection. Ozagrel also inhibited the hepatic expression of cell death-related mRNAs induced by APAP, such as jun oncogene, FBJ osteosarcoma oncogene (fos) and C/EBP homologous protein (chop), but did not suppress B-cell lymphoma 2-like protein11 (bim) expression and hepatic total glutathione depletion. These results show ozagrel can inhibit not all hepatic changes but can reduce the hepatic necrosis. Ozagrel had little impact on CYP2E1 activity involving the NAPQI production. In addition, ozagrel significantly attenuated cell injury induced by NAPQI in RLC-16. Conclusions We demonstrate that the TXA2 synthase inhibitor, ozagrel, dramatically alleviates liver injury induced by APAP in mice, and suggest that it is a

  12. Allosteric Inhibitors at the Heterodimer Interface of Imidazole Glycerol Phosphate Synthase

    NASA Astrophysics Data System (ADS)

    Snoeberger, Ning-Shiuan Nicole

    Imidazole glycerol phosphate synthase (IGPS) from Thermotoga maritima is a heterodimeric enzyme composed of the HisH and HisF proteins. It is attractive as a pathological target since it is absent in mammals but found in plant and opportunistic human pathogens. IGPS was experimentally determined to be a V-type allosteric enzyme that is involved in an essential biosynthetic pathway of microorganisms. The enzyme catalyzes the hydrolysis of glutamine to form NH3 in the HisH protein, followed by cyclization of NH3 with N'-[(5'-phosphoribulosyl)imino]-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR) in the HisF subunit, forming imidazole glycerol phosphate (IGP) and 5-aminoimidazole-4-carboxamide ribotide (AICAR) that enter the histidine and purine biosynthetic pathways. Allosteric motions induced upon the binding of the effector PRFAR to HisF propagate through the non-covalent HisH/HisF interface and synchronize catalytic activity at the two distant active sites. However, the nature of the allosteric pathway and the feasibility of manipulating signal transduction by using allosteric drug-like molecules remain to be established. Molecular docking studies of commercial drugs at the HisH/HisF interface were used to identify stable candidates with a potential allosteric effect on the reaction mechanism. Molecular dynamic simulations and calculations of NMR chemical shifts were combined to elucidate the allosteric pathway of IGPS.

  13. The crystal structure of spermidine synthase with a multisubstrate adduct inhibitor

    PubMed Central

    Korolev, Sergey; Ikeguchi, Yoshihiko; Skarina, Tatiana; Beasley, Steven; Arrowsmith, Cheryl; Edwards, Aled; Joachimiak, Andrzej; Pegg, Anthony E.; Savchenko, Alexei

    2009-01-01

    Polyamines are essential in all branches of life. Spermidine synthase (putrescine aminopropyltransferase, PAPT) catalyzes the biosynthesis of spermidine, a ubiquitous polyamine. The crystal structure of the PAPT from Thermotoga maritima (TmPAPT) has been solved to 1.5 Å resolution in the presence and absence of AdoDATO (S-adenosyl-1,8-diamino-3-thiooctane), a compound containing both substrate and product moieties. This, the first structure of an aminopropyltransferase, reveals deep cavities for binding substrate and cofactor, and a loop that envelops the active site. The AdoDATO binding site is lined with residues conserved in PAPT enzymes from bacteria to humans, suggesting a universal catalytic mechanism. Other conserved residues act sterically to provide a structural basis for polyamine specificity. The enzyme is tetrameric; each monomer consists of a C-terminal domain with a Rossmann-like fold and an N-terminal β-stranded domain. The tetramer is assembled using a novel barrel-type oligomerization motif. PMID:11731804

  14. Dynamics based pharmacophore models for screening potential inhibitors of mycobacterial cyclopropane synthase.

    PubMed

    Choudhury, Chinmayee; Priyakumar, U Deva; Sastry, G Narahari

    2015-04-27

    The therapeutic challenges in the treatment of tuberculosis demand multidisciplinary approaches for the identification of potential drug targets as well as fast and accurate techniques to screen huge chemical libraries. Mycobacterial cyclopropane synthase (CmaA1) has been shown to be essential for the survival of the bacteria due to its critical role in the synthesis of mycolic acids. The present study proposes pharmacophore models based on the structure of CmaA1 taking into account its various states in the cyclopropanation process, and their dynamic nature as assessed using molecular dynamics (MD) simulations. The qualities of these pharmacophore models were validated by mapping 23 molecules that have been previously reported to exhibit inhibitory activities on CmaA1. Additionally, 1398 compounds that have been shown to be inactive for tuberculosis were collected from the ChEMBL database and were screened against the models for validation. The models were further validated by comparing the results from pharmacophore mapping with the results obtained from docking these molecules with the respective protein structures. The best models are suggested by validating all the models based on their screening abilities and by comparing with docking results. The models generated from the MD trajectories were found to perform better than the one generated based on the crystal structure demonstrating the importance of incorporating receptor flexibility in drug design.

  15. Isoeugenin, a Novel Nitric Oxide Synthase Inhibitor Isolated from the Rhizomes of Imperata cylindrica.

    PubMed

    An, Hyo-Jin; Nugroho, Agung; Song, Byong-Min; Park, Hee-Juhn

    2015-01-01

    Phytochemical studies on the constituents of the rhizomes of Imperata cylindrica (Gramineae) were performed using high-performance liquid chromatography (HPLC). We also aimed to search for any biologically active substance capable of inhibiting nitric oxide (NO) formation in lipopolysaccharide (LPS)-activated macrophage 264.7 cells, by testing four compounds isolated from this plant. Four compounds, including a new chromone, isoeugenin, along with ferulic acid, p-coumaric acid, and caffeic acid were isolated and identified by NMR spectroscopy. The structure of isoeugenin was determined as 7-hydroxy-5-methoxy-2-methylchromone by the 2D-NMR technique. Among the four compounds, isoeugenin has the lowest IC50 value on the inhibition of NO production in LPS-activated macrophage RAW264.7 cells (IC50, 9.33 μg/mL). In addition, isoeugenin significantly suppressed the LPS-induced expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and proinflammatory cytokines mRNA levels. Taken together, these results suggest that the anti-inflammatory activity of isoeugenin is associated with the down-regulation of iNOS, COX-2, and pro-inflammatory cytokines in RAW264.7 cells. Accordingly, our results suggest that the new chromone isoegenin should be considered a potential treatment for inflammatory disease. PMID:26633331

  16. Pterin-sulfa conjugates as dihydropteroate synthase inhibitors and antibacterial agents.

    PubMed

    Zhao, Ying; Shadrick, William R; Wallace, Miranda J; Wu, Yinan; Griffith, Elizabeth C; Qi, Jianjun; Yun, Mi-Kyung; White, Stephen W; Lee, Richard E

    2016-08-15

    The sulfonamide class of antibiotics has been in continuous use for over 70years. They are thought to act by directly inhibiting dihydropteroate synthase (DHPS), and also acting as prodrugs that sequester pterin pools by forming dead end pterin-sulfonamide conjugates. In this study, eight pterin-sulfonamide conjugates were synthesized using a novel synthetic strategy and their biochemical and microbiological properties were investigated. The conjugates were shown to competitively inhibit DHPS, and inhibition was enhanced by the presence of pyrophosphate that is crucial to catalysis and is known to promote an ordering of the DHPS active site. The co-crystal structure of Yersinia pestis DHPS bound to one of the more potent conjugates revealed a mode of binding that is similar to that of the enzymatic product analog pteroic acid. The antimicrobial activities of the pterin-sulfonamide conjugates were measured against Escherichia coli in the presence and absence of folate precursors and dependent metabolites. These results show that the conjugates have appreciable antibacterial activity and act by an on target, anti-folate pathway mechanism rather than as simple dead end products. PMID:27423480

  17. Novel contraceptive targets to inhibit ovulation: the prostaglandin E2 pathway

    PubMed Central

    Duffy, Diane M.

    2015-01-01

    BACKGROUND Prostaglandin E2 (PGE2) is an essential intrafollicular regulator of ovulation. In contrast with the one-gene, one-protein concept for synthesis of peptide signaling molecules, production and metabolism of bioactive PGE2 requires controlled expression of many proteins, correct subcellular localization of enzymes, coordinated PGE2 synthesis and metabolism, and prostaglandin transport in and out of cells to facilitate PGE2 action and degradation. Elevated intrafollicular PGE2 is required for successful ovulation, so disruption of PGE2 synthesis, metabolism or transport may yield effective contraceptive strategies. METHODS This review summarizes case reports and studies on ovulation inhibition in women and macaques treated with cyclooxygenase inhibitors published from 1987 to 2014. These findings are discussed in the context of studies describing levels of mRNA, protein, and activity of prostaglandin synthesis and metabolic enzymes as well as prostaglandin transporters in ovarian cells. RESULTS The ovulatory surge of LH regulates the expression of each component of the PGE2 synthesis-metabolism-transport pathway within the ovulatory follicle. Data from primary ovarian cells and cancer cell lines suggest that enzymes and transporters can cooperate to optimize bioactive PGE2 levels. Elevated intrafollicular PGE2 mediates key ovulatory events including cumulus expansion, follicle rupture and oocyte release. Inhibitors of the prostaglandin-endoperoxide synthase 2 (PTGS2) enzyme (also known as cyclooxygenase-2 or COX2) reduce ovulation rates in women. Studies in macaques show that PTGS2 inhibitors can reduce the rates of cumulus expansion, oocyte release, follicle rupture, oocyte nuclear maturation and fertilization. A PTGS2 inhibitor reduced pregnancy rates in breeding macaques when administered to simulate emergency contraception. However, PTGS2 inhibition did not prevent pregnancy in monkeys when administered to simulate monthly contraceptive use. CONCLUSION

  18. Impaired learning in rats in a 14-unit T-maze by 7-nitroindazole, a neuronal nitric oxide synthase inhibitor, is attenuated by the nitric oxide donor, molsidomine.

    PubMed

    Meyer, R C; Spangler, E L; Patel, N; London, E D; Ingram, D K

    1998-01-01

    In previous experiments, it was demonstrated that systemic or central administration of the nitric oxide synthase (NO synthase) inhibitor, NG-nitro-L-arginine (N-Arg), produced dose-dependent learning impairments in rats in a 14-unit T-maze; and that sodium nitroprusside, a NO donor, could attenuate the impairment. Since N-Arg is not specific for neuronal NO synthase and produces hypertension, it is possible that effects on the cardiovasculature may have contributed to the impaired maze performance. In the present experiment, we have investigated the maze performance of 3-4 months old male Fischer-344 rats following treatment with 7-nitroindazole, a NO synthase inhibitor that is selective for neuronal NO synthase and does not produce hypertension. In addition, we examined the effects of the NO donor, molsidomine, which is much longer acting than sodium nitroprusside. Rats were pretrained to avoid footshock in a straight runway and received training in a 14-unit T-maze 24 h later. In an initial dose-response study, rats received intraperitoneal (i.p.) injections of either 7-nitroindazole (25, 50, or 65 mg/kg) or peanut oil 30 min prior to maze training. 7-nitroindazole produced significant, dose-dependent maze acquisition deficits, with 65 mg/kg producing the greatest learning impairment. This dose of 7-nitroindazole had no significant effect on systolic blood pressure. Following the dose-response study, rats were given i.p. injections of either 7-nitroindazole (70 mg/kg) plus saline, 7-nitroindazole (70 mg/kg) plus the NO donor, molsidomine (2 or 4 mg/kg), or peanut oil plus saline as controls. Both doses of molsidomine significantly attenuated the learning deficit induced by 7-nitroindazole relative to controls. These findings represent the first evidence that impaired learning produced by inhibition of neuronal NO synthase can be overcome by systemic administration of a NO donor. PMID:9489851

  19. Generation of human endometrial knockout cell lines with the CRISPR/Cas9 system confirms the prostaglandin F2α synthase activity of aldo-ketoreductase 1B1.

    PubMed

    Lacroix Pépin, Nicolas; Chapdelaine, Pierre; Rodriguez, Yoima; Tremblay, Jacques-P; Fortier, Michel A

    2014-07-01

    Prostaglandins (PGs) are important regulators of female reproductive function. The primary PGs produced in the endometrium are PGE2 and PGF2α. Relatively little is known about the biosynthetic pathways leading to the formation of PGF2α. We have described the role of aldo-ketoreductase (AKR)1B1 in increased PGF2α production by human endometrial cells following stimulation with interleukin-1β (IL-1β). However, alternate PGF synthases are expressed concurrently in endometrial cells. A definite proof of the role of AKR1B1 would require gene knockout; unfortunately, this gene has no direct equivalent in the mouse. Recently, an efficient genome-editing technology using RNA-guided DNase Cas9 and the clustered regularly interspaced short palindromic repeats (CRISPR) system has been developed. We have adapted this approach to knockout AKR1B1 gene expression in human endometrial cell lines. One clone (16-2) of stromal origin generated by the CRISPR/Cas9 system exhibited a complete loss of AKR1B1 protein and mRNA expression, whereas other clones presented with partial edition. The present report focuses on the characterization of clone 16-2 exhibiting deletion of 68 and 2 nucleotides, respectively, on each of the alleles. Cells from this clone lost their ability to produce PGF2α but maintained their original stromal cell (human endometrial stromal cells-2) phenotype including the capacity to decidualize in the presence of progesterone (medroxyprogesterone acetate) and 8-bromo-cAMP. Knockout cells also maintained their ability to increase PGE2 production in response to IL-1β. In summary, we demonstrate that the new genome editing CRISPR/Cas9 system can be used in human cells to generate stable knockout cell line models. Our results suggest that genome editing of human cell lines can be used to complement mouse KO models to validate the function of genes in differentiated tissues and cells. Our results also confirm that AKR1B1 is involved in the synthesis of PGF2α.

  20. Selective Inducible Nitric Oxide Synthase Inhibitor Reversed Zinc Chloride-Induced Spatial Memory Impairment via Increasing Cholinergic Marker Expression.

    PubMed

    Tabrizian, Kaveh; Azami, Kian; Belaran, Maryam; Soodi, Maliheh; Abdi, Khosrou; Fanoudi, Sahar; Sanati, Mehdi; Mottaghi Dastjerdi, Negar; Soltany Rezaee-Rad, Mohammad; Sharifzadeh, Mohammad

    2016-10-01

    Zinc, an essential micronutrient and biochemical element of the human body, plays structural, catalytic, and regulatory roles in numerous physiological functions. In the current study, the effects of a pretraining oral administration of zinc chloride (10, 25, and 50 mg/kg) for 14 consecutive days and post-training bilateral intra-hippocampal infusion of 1400W as a selective inducible nitric oxide synthase (iNOS) inhibitor (10, 50, and 100 μM/side), alone and in combination, on the spatial memory retention in Morris water maze (MWM) were investigated. Animals were trained for 4 days and tested 48 h after completion of training. Also, the molecular effects of these compounds on the expression of choline acetyltransferase (ChAT), as a cholinergic marker in the CA1 region of the hippocampus and medial septal area (MSA), were evaluated. Behavioral and molecular findings of this study showed that a 2-week oral administration of zinc chloride (50 mg/kg) impaired spatial memory retention in MWM and decreased ChAT expression. Immunohistochemical analysis of post-training bilateral intra-hippocampal infusion of 1400W revealed a significant increase in ChAT immunoreactivity. Furthermore, post-training bilateral intra-hippocampal infusion of 1400W into the CA1 region of the hippocampus reversed zinc chloride-induced spatial memory impairment in MWM and significantly increased ChAT expression in comparison with zinc chloride-treated animals. Taken together, these results emphasize the role of selective iNOS inhibitors in reversing zinc chloride-induced spatial memory deficits via modulation of cholinergic marker expression.

  1. 5-imino-1,2,4-thiadiazoles: first small molecules as substrate competitive inhibitors of glycogen synthase kinase 3.

    PubMed

    Palomo, Valle; Perez, Daniel I; Perez, Concepcion; Morales-Garcia, Jose A; Soteras, Ignacio; Alonso-Gil, Sandra; Encinas, Arantxa; Castro, Ana; Campillo, Nuria E; Perez-Castillo, Ana; Gil, Carmen; Martinez, Ana

    2012-02-23

    Cumulative evidence strongly supports that glycogen synthase kinase-3 (GSK-3) is a pathogenic molecule when it is up-dysregulated, emerging as an important therapeutic target in severe unmet human diseases. GSK-3 specific inhibitors might be promising effective drugs for the treatment of devastating pathologies such as neurodegenerative diseases, stroke, and mood disorders. As GSK-3 has the ability to phosphorylate primed substrates, small molecules able to bind to this site should be perfect drug candidates, able to partially block the activity of the enzyme over some specific substrates. Here, we report substituted 5-imino-1,2,4-thiadiazoles as the first small molecules able to inhibit GSK-3 in a substrate competitive manner. These compounds are cell permeable, able to decrease inflammatory activation and to selectively differentiate neural stem cells. Overall, 5-imino-1,2,4-thiadiazoles are presented here as new molecules able to decrease neuronal cell death and to increase endogenous neurogenesis blocking the GSK-3 substrate site.

  2. Selective Nitric Oxide Synthase Inhibitor 7-Nitroindazole Protects against Cocaine-Induced Oxidative Stress in Rat Brain.

    PubMed

    Vitcheva, Vessela; Simeonova, Rumyana; Kondeva-Burdina, Magdalena; Mitcheva, Mitka

    2015-01-01

    One of the mechanisms involved in the development of addiction, as well as in brain toxicity, is the oxidative stress. The aim of the current study was to investigate the effects of 7-nitroindazole (7-NI), a selective inhibitor of neuronal nitric oxide synthase (nNOS), on cocaine withdrawal and neurotoxicity in male Wistar rats. The animals were divided into four groups: control; group treated with cocaine (15 mg/kg(-1), i.p., 7 days); group treated with 7-NI (25 mg/kg(-1), i.p., 7 days); and a combination group (7-NI + cocaine). Cocaine repeated treatment resulted in development of physical dependence, judged by withdrawal symptoms (decreased locomotion, increased salivation and breathing rate), accompanied by an increased nNOS activity and oxidative stress. The latter was discerned by an increased formation of malondialdehyde (MDA), depletion of reduced glutathione (GSH) levels, and impairment of the enzymatic antioxidant defense system measured in whole brain. In synaptosomes, isolated from cocaine-treated rats, mitochondrial activity and GSH levels were also decreased. 7-NI administered along with cocaine not only attenuated the withdrawal, due to its nNOS inhibition, but also reversed both the GSH levels and antioxidant enzyme activities near control levels. PMID:26576217

  3. Selective Nitric Oxide Synthase Inhibitor 7-Nitroindazole Protects against Cocaine-Induced Oxidative Stress in Rat Brain

    PubMed Central

    Vitcheva, Vessela; Simeonova, Rumyana; Kondeva-Burdina, Magdalena; Mitcheva, Mitka

    2015-01-01

    One of the mechanisms involved in the development of addiction, as well as in brain toxicity, is the oxidative stress. The aim of the current study was to investigate the effects of 7-nitroindazole (7-NI), a selective inhibitor of neuronal nitric oxide synthase (nNOS), on cocaine withdrawal and neurotoxicity in male Wistar rats. The animals were divided into four groups: control; group treated with cocaine (15 mg/kg−1, i.p., 7 days); group treated with 7-NI (25 mg/kg−1, i.p., 7 days); and a combination group (7-NI + cocaine). Cocaine repeated treatment resulted in development of physical dependence, judged by withdrawal symptoms (decreased locomotion, increased salivation and breathing rate), accompanied by an increased nNOS activity and oxidative stress. The latter was discerned by an increased formation of malondialdehyde (MDA), depletion of reduced glutathione (GSH) levels, and impairment of the enzymatic antioxidant defense system measured in whole brain. In synaptosomes, isolated from cocaine-treated rats, mitochondrial activity and GSH levels were also decreased. 7-NI administered along with cocaine not only attenuated the withdrawal, due to its nNOS inhibition, but also reversed both the GSH levels and antioxidant enzyme activities near control levels. PMID:26576217

  4. NMDA receptor antagonist AP5 and nitric oxide synthase inhibitor 7-NI affect different phases of learning and memory in goldfish.

    PubMed

    Xu, X; Russell, T; Bazner, J; Hamilton, J

    2001-01-19

    The present study investigated the amnestic effects of N-methyl-D-aspartate (NMDA) receptor antagonist AP5 and nitric oxide (NO) synthase inhibitor 7-NI in avoidance conditioning in goldfish. The results showed that both AP5 and 7-NI, without impairing performance processes, produced anterograde amnesia when given before training. Furthermore, 7-NI produced retrograde amnesia when given immediately following training while AP5 did not. Thus, AP5 and 7-NI affected different phases of learning and memory.

  5. Discovery of Isonicotinamides as Highly Selective, Brain Penetrable, and Orally Active Glycogen Synthase Kinase-3 Inhibitors.

    PubMed

    Luo, Guanglin; Chen, Ling; Burton, Catherine R; Xiao, Hong; Sivaprakasam, Prasanna; Krause, Carol M; Cao, Yang; Liu, Nengyin; Lippy, Jonathan; Clarke, Wendy J; Snow, Kimberly; Raybon, Joseph; Arora, Vinod; Pokross, Matt; Kish, Kevin; Lewis, Hal A; Langley, David R; Macor, John E; Dubowchik, Gene M

    2016-02-11

    GSK-3 is a serine/threonine kinase that has numerous substrates. Many of these proteins are involved in the regulation of diverse cellular functions, including metabolism, differentiation, proliferation, and apoptosis. Inhibition of GSK-3 may be useful in treating a number of diseases including Alzheimer's disease (AD), type II diabetes, mood disorders, and some cancers, but the approach poses significant challenges. Here, we present a class of isonicotinamides that are potent, highly kinase-selective GSK-3 inhibitors, the members of which demonstrated oral activity in a triple-transgenic mouse model of AD. The remarkably high kinase selectivity and straightforward synthesis of these compounds bode well for their further exploration as tool compounds and therapeutics.

  6. Discovery of Isonicotinamides as Highly Selective, Brain Penetrable, and Orally Active Glycogen Synthase Kinase-3 Inhibitors.

    PubMed

    Luo, Guanglin; Chen, Ling; Burton, Catherine R; Xiao, Hong; Sivaprakasam, Prasanna; Krause, Carol M; Cao, Yang; Liu, Nengyin; Lippy, Jonathan; Clarke, Wendy J; Snow, Kimberly; Raybon, Joseph; Arora, Vinod; Pokross, Matt; Kish, Kevin; Lewis, Hal A; Langley, David R; Macor, John E; Dubowchik, Gene M

    2016-02-11

    GSK-3 is a serine/threonine kinase that has numerous substrates. Many of these proteins are involved in the regulation of diverse cellular functions, including metabolism, differentiation, proliferation, and apoptosis. Inhibition of GSK-3 may be useful in treating a number of diseases including Alzheimer's disease (AD), type II diabetes, mood disorders, and some cancers, but the approach poses significant challenges. Here, we present a class of isonicotinamides that are potent, highly kinase-selective GSK-3 inhibitors, the members of which demonstrated oral activity in a triple-transgenic mouse model of AD. The remarkably high kinase selectivity and straightforward synthesis of these compounds bode well for their further exploration as tool compounds and therapeutics. PMID:26751161

  7. Inhibition of invasion by glycogen synthase kinase-3 beta inhibitors through dysregulation of actin re-organisation via down-regulation of WAVE2.

    PubMed

    Yoshino, Yuki; Suzuki, Manami; Takahashi, Hidekazu; Ishioka, Chikashi

    2015-08-14

    Cancer cell invasion is a critical phenomenon in cancer pathogenesis. Glycogen synthase kinase-3β (GSK-3β) has been reported to regulate cancer cell invasion both negatively and positively. Thus, the net effect of GSK-3β on invasion is unclear. In this report, we showed that GSK-3β inhibitors induced dysregulation of the actin cytoskeleton and functional insufficiency of focal adhesion, which resulted in suppressed invasion. In addition, WAVE2, an essential molecule for actin fibre branching, was down-regulated after GSK-3β inhibition. Collectively, we propose that the WAVE2-actin cytoskeleton axis is an important target of GSK-3β inhibitors in cancer cell invasion.

  8. Modulation of IL-1-induced cartilage injury by NO synthase inhibitors: a comparative study with rat chondrocytes and cartilage entities

    PubMed Central

    Cipolletta, Christine; Jouzeau, Jean-Yves; Gegout-Pottie, Pascale; Presle, Nathalie; Bordji, Karim; Netter, Patrick; Terlain, Bernard

    1998-01-01

    Nitric oxide (NO) is produced in diseased joints and may be a key mediator of IL-1 effects on cartilage. Therefore, we compared the potency of new [aminoguanidine (AG), S-methylisothiourea (SMT), S-aminoethylisothiourea (AETU)] and classical [Nω-monomethyl-L-arginine (L-NMMA), Nω-nitro-L-arginine methyl ester (L-NAME)] NO synthase (NOS) inhibitors on the inhibitory effect of recombinant human interleukin-1β (rhIL-1β) on rat cartilage anabolism. Three different culture systems were used: (1) isolated chondrocytes encapsulated in alginate beads; (2) patellae and (3) femoral head caps. Chondrocyte beads and cartilage entities were incubated in vitro for 48 h in the presence of rhIL-1β with a daily change of incubation medium to obtain optimal responses on proteoglycan synthesis and NO production. Proteoglycan synthesis was assessed by incorporation of radiolabelled sodium sulphate [Na235SO4] and NO production by cumulated nitrite release during the period of study. Chondrocytes and patellae, as well as femoral head caps, responded concentration-dependently to IL-1β challenge (0 to 250 U ml−1 and 0 to 15 U ml−1 respectively) by a large increase in nitrite level and a marked suppression of proteoglycan synthesis. Above these concentrations of IL-1β (2500 U ml−1 and 30 U ml−1 respectively), proteoglycan synthesis plateaued whereas nitrite release still increased thus suggesting different concentration-response curves. When studying the effect of NOS inhibitors (1 to 1000 μM) on NO production by cartilage cells stimulated with IL-1β (25 U ml−1 or 5 U ml−1), we observed that: (i) their ability to reduce nitrite level decreased from chondrocytes to cartilage samples, except for L-NMMA and AETU; (ii) they could be roughly classified in the following rank order of potency: AETU>L-NMMA⩾SMT>AG⩾L-NAME and (iii) AETU was cytotoxic when used in the millimolar range. When studying the effect of NOS inhibitors on proteoglycan

  9. Prostaglandin D2 Inhibits Hair Growth and Is Elevated in Bald Scalp of Men with Androgenetic Alopecia

    PubMed Central

    Garza, Luis A.; Liu, Yaping; Yang, Zaixin; Alagesan, Brinda; Lawson, John A.; Norberg, Scott M.; Loy, Dorothy E.; Zhao, Tailun; Blatt, Hanz B.; Stanton, David C.; Carrasco, Lee; Ahluwalia, Gurpreet; Fischer, Susan M.; FitzGerald, Garret A.; Cotsarelis, George

    2012-01-01

    Testosterone is necessary for the development of male pattern baldness, known as androgenetic alopecia (AGA); yet, the mechanisms for decreased hair growth in this disorder are unclear. We show that prostaglandin D2 synthase (PTGDS) is elevated at the mRNA and protein levels in bald scalp compared to haired scalp of men with AGA. The product of PTGDS enzyme activity, prostaglandin D2 (PGD2), is similarly elevated in bald scalp. During normal follicle cycling in mice, Ptgds and PGD2 levels increase immediately preceding the regression phase, suggesting an inhibitory effect on hair growth. We show that PGD2 inhibits hair growth in explanted human hair follicles and when applied topically to mice. Hair growth inhibition requires the PGD2 receptor G protein (heterotrimeric guanine nucleotide)–coupled receptor 44 (GPR44), but not the PGD2 receptor 1 (PTGDR). Furthermore, we find that a transgenic mouse, K14-Ptgs2, which targets prostaglandin-endoperoxide synthase 2 expression to the skin, demonstrates elevated levels of PGD2 in the skin and develops alopecia, follicular miniaturization, and sebaceous gland hyperplasia, which are all hallmarks of human AGA. These results define PGD2 as an inhibitor of hair growth in AGA and suggest the PGD2-GPR44 pathway as a potential target for treatment. PMID:22440736

  10. Molecular Mechanism of Silver Nanoparticles-Induced Human Osteoblast Cell Death: Protective Effect of Inducible Nitric Oxide Synthase Inhibitor

    PubMed Central

    Zielinska, Ewelina; Tukaj, Cecylia; Radomski, Marek Witold; Inkielewicz-Stepniak, Iwona

    2016-01-01

    Background Silver nanoparticles (AgNPs) show strong antibacterial properties, making them excellent candidates to be used in orthopaedic repair and regeneration. However, there are concerns regarding the cytotoxicity of AgNPs and molecular mechanisms underlying AgNPs-induced bone cells toxicity have not been elucidated. Therefore, the aim of our study was to explore mechanisms of AgNPs-induced osteoblast cell death with particular emphasis on the role of nitric oxide (NO) generated by inducible nitric oxide synthase (iNOS). Methods and Result Silver nanoparticles used in this study were 18.3±2.6 nm in size, uncoated, spherical, regular shape and their zeta potential was -29.1±2.4 mV as measured by transmission electron microscopy (TEM) and zetasizer. The release of silver (Ag) from AgNPs was measured in cell culture medium by atomic absorption spectroscopy (AAS). The exposure of human osteoblast cells (hFOB 1.19) to AgNPs at concentration of 30 or 60 μg/mL for 24 or 48 hours, respectively resulted in cellular uptake of AgNPs and changes in cell ultrastructure. These changes were associated with apoptosis and necrosis as shown by flow cytometry and lactate dehydrogenase (LDH) assay as well as increased levels of pro-apoptotic Bax and decreased levels of anti-apoptotic Bcl-2 mRNA and protein. Importantly, we have found that AgNPs elevated the levels of nitric oxide (NO) with concomitant upregulation of inducible nitric oxide synthase (iNOS) mRNA and protein. A significant positive correlation was observed between the concentration of AgNPs and iNOS at protein and mRNA level (r = 0.837, r = 0.721, respectively; p<0.001). Finally, preincubation of osteoblast cells with N-iminoethyl-l-lysine (L-NIL), a selective iNOS inhibitor, as well as treating cells with iNOS small interfering RNAs (siRNA) significantly attenuated AgNPs-induced apoptosis and necrosis. Moreover, we have found that AgNPs-induced cells death is not related to Ag dissolution is cell culture medium

  11. Novel benzothiazinones (BTOs) as allosteric modulator or substrate competitive inhibitor of glycogen synthase kinase 3β (GSK-3β) with cellular activity of promoting glucose uptake.

    PubMed

    Zhang, Peng; Li, Shufen; Gao, Yang; Lu, Wenbo; Huang, Ke; Ye, Deyong; Li, Xi; Chu, Yong

    2014-12-15

    Glycogen synthase kinase 3β (GSK-3β) plays a key role in insulin metabolizing pathway and therefore inhibition of the enzyme might provide an important therapeutic approach for treatment of insulin resistance and type 2 diabetes. Recently, discovery of ATP noncompetitive inhibitors is gaining importance not only due to their generally increased selectivity but also for the potentially subtle modulation of the target. These kinds of compounds include allosteric modulators and substrate competitive inhibitors. Here we reported two benzothiazinone compounds (BTO), named BTO-5h (IC50=8 μM) and BTO-5s (IC50=10 μM) as novel allosteric modulator and substrate competitive inhibitor of GSK-3β, respectively. Their different action modes were proved by kinetic experiments. Furthermore, BTO-5s was selected to check the kinases profile and showed little or even no activity to a panel of ten protein kinases at 100 μM, indicating it has good selectivity. Docking studies were performed to give suggesting binding modes which can well explain their impacts on the enzyme. Moreover, cell experiments displayed both compounds reduced the phosphorylation level of glycogen synthase in an intact cell, and greatly enhanced the glucose uptake in both HpG2 and 3T3-L1 cells. All of these results suggested BTO-5s and BTO-5h maybe have potentially therapeutic value for anti-diabetes. The results also offer a new scaffold for designing and developing selective inhibitors with novel mechanisms of action.

  12. Inhibitor-κB kinase attenuates Hsp90-dependent endothelial nitric oxide synthase function in vascular endothelial cells

    PubMed Central

    Konopinski, Ryszard; Krishnan, Manickam; Roman, Linda; Bera, Alakesh; Hongying, Zheng; Habib, Samy L.; Mohan, Sumathy

    2015-01-01

    Endothelial nitric oxide (NO) synthase (eNOS) is the predominant isoform that generates NO in the blood vessels. Many different regulators, including heat shock protein 90 (Hsp90), govern eNOS function. Hsp90-dependent phosphorylation of eNOS is a critical event that determines eNOS activity. In our earlier study we demonstrated an inhibitor-κB kinase-β (IKKβ)-Hsp90 interaction in a high-glucose environment. In the present study we further define the putative binding domain of IKKβ on Hsp90. Interestingly, IKKβ binds to the middle domain of Hsp90, which has been shown to interact with eNOS to stimulate its activity. This new finding suggests a tighter regulation of eNOS activity than was previously assumed. Furthermore, addition of purified recombinant IKKβ to the eNOS-Hsp90 complex reduces the eNOS-Hsp90 interaction and eNOS activity, indicating a competition for Hsp90 between eNOS and IKKβ. The pathophysiological relevance of the IKKβ-Hsp90 interaction has also been demonstrated using in vitro vascular endothelial growth factor-mediated signaling and an Ins2Akita in vivo model. Our study further defines the preferential involvement of α- vs. β-isoforms of Hsp90 in the IKKβ-eNOS-Hsp90 interaction, even though both Hsp90α and Hsp90β stimulate NO production. These studies not only reinforce the significance of maintaining a homeostatic balance of eNOS and IKKβ within the cell system that regulates NO production, but they also confirm that the IKKβ-Hsp90 interaction is favored in a high-glucose environment, leading to impairment of the eNOS-Hsp90 interaction, which contributes to endothelial dysfunction and vascular complications in diabetes. PMID:25652452

  13. Selective enhancement of the uptake and bioactivity of a TAT-conjugated peptide inhibitor of glycogen synthase kinase-3.

    PubMed

    Manceur, Aziza P; Driscoll, Brandon D; Sun, Wei; Audet, Julie

    2009-03-01

    The use of cell-penetrating peptides as transduction vectors is a promising approach to deliver peptides and proteins into cells. However, the uptake and bioavailability of trans-activating transcriptor (TAT)-conjugated molecules vary depending on the cell type and the cargo. This study aimed to determine whether a low-voltage electrical pulse can enhance the TAT-mediated delivery of peptide cargoes in different cell types. In TF-1 and mouse embryonic stem cells, the uptake of a novel detachable TAT-conjugated glycogen synthase kinase-3 (GSK-3) peptide inhibitor was enhanced by an order of magnitude without affecting the cell viability. A similar increase in uptake was achieved in primary mouse bone marrow cells while maintaining >80% of their viability. Interestingly, under these low-voltage conditions, the uptake of a control peptide not conjugated to TAT was not significantly increased. A T-cell factor/lymphoid enhancer factor (TCF/LEF) luciferase reporter assay was also used to assess the bioactivity of the TAT construct. The results indicated that cells loaded with a low-voltage electrical pulse had a twofold increase in TCF/LEF activity, which was equivalent to a level of GSK-3 inhibition similar to that of cells treated with 20 mmol/l lithium or 500 nmol/l (2'Z,3'E)-6-bromoindirubin-3'-oxime. These results demonstrate the usefulness of low-voltage electrical pulses to enhance the uptake and bioactivity of TAT-conjugated molecules in different cell types. PMID:19107119

  14. Reactivated memory of an inhibitory avoidance response in mice is sensitive to a nitric oxide synthase inhibitor.

    PubMed

    Baratti, Carlos M; Boccia, Mariano M; Blake, Mariano G; Acosta, Gabriela B

    2008-05-01

    It is accepted that once consolidation is completed memory becomes permanent. However, it has also been suggested that reactivation (retrieval) of the original memory, again, makes it sensitive to the same treatments that affect memory consolidation when given after training. Previous results demonstrated that the immediate post-training intraperitoneal administration of N(omega)-nitro-l-arginine methyl ester (L-NAME), a non-specific inhibitor of nitric oxide synthase (NOS), impairs retention test performance of a one-trial step-through inhibitory avoidance response in adult mice. The effect of L-NAME on retention was attributed to an action on memory consolidation of the original learning. For the first time, we report that the administration of L-NAME after the first retention test (memory reactivation) of the inhibitory avoidance response impairs retention performance over six consecutive days. This impairment effect is dose-and-time dependent and could not be attributed to a retrieval deficit since a mild footshock did not reinstate the original avoidance response and no spontaneous recovery was observed at least 21 days after training. Further support for a storage deficit interpretation as opposed to a retrieval deficit was obtained from the fact that L-NAME's effects after retrieval were not due to state-dependency. The impairment effect of L-NAME was dependent on the age of the original memory. That is, there was an inverse correlation between the susceptibility of the memory trace when reactivated and the time elapsed between training and the first retrieval session. We suggest an action of L-NAME on memory reactivation-induced processes that are different from memory extinction of the original learning extending the biological significance of nitric oxide on memory.

  15. The effect of a selective neuronal nitric oxide synthase inhibitor 3-bromo 7-nitroindazole on spatial learning and memory in rats.

    PubMed

    Gocmez, Semil Selcen; Yazir, Yusufhan; Sahin, Deniz; Karadenizli, Sabriye; Utkan, Tijen

    2015-04-01

    Since the discovery of nitric oxide (NO) as a neuronal messenger, its way to modulate learning and memory functions is subject of intense research. NO is an intercellular messenger in the central nervous system and is formed on demand through the conversion of L-arginine to L-citrulline via the enzyme nitric oxide synthase (NOS). Neuronal form of nitric oxide synthase may play an important role in a wide range of physiological and pathological conditions. Therefore the aim of this study was to investigate the effects of chronic 3-bromo 7-nitroindazole (3-Br 7-NI), specific neuronal nitric oxide synthase (nNOS) inhibitor, administration on spatial learning and memory performance in rats using the Morris water maze (MWM) paradigm. Male rats received either 3-Br 7-NI (20mg/kg/day) or saline via intraperitoneal injection for 5days. Daily administration of the specific neuronal nitric oxide synthase (nNOS) inhibitor, 3-Br 7-NI impaired the acquisition of the MWM task. 3-Br 7-NI also impaired the probe trial. The MWM training was associated with a significant increase in the brain-derived neurotrophic factor (BDNF) mRNA expression in the hippocampus. BDNF mRNA expression in the hippocampus did not change after 3-Br 7-NI treatment. L-arginine significantly reversed behavioural parameters, and the effect of 3-Br 7-NI was found to be NO-dependent. There were no differences in locomotor activity and blood pressure in 3-Br 7-NI treated rats. Our results may suggest that nNOS plays a key role in spatial memory formation in rats.

  16. Use of bacterial surrogates as a tool to explore antimalarial drug interaction: Synergism between inhibitors of malarial dihydrofolate reductase and dihydropteroate synthase.

    PubMed

    Talawanich, Yuwadee; Kamchonwongpaisan, Sumalee; Sirawaraporn, Worachart; Yuthavong, Yongyuth

    2015-09-01

    Interaction between antimalarial drugs is important in determining the outcome of chemotherapy using drug combinations. Inhibitors of dihydrofolate reductase (DHFR) such as pyrimethamine and of dihydropteroate synthase (DHPS) such as sulfa drugs are known to have synergistic interactions. However, studies of the synergism are complicated by the fact that the malaria parasite can also salvage exogenous folates, and the salvage may also be affected by the drugs. It is desirable to have a convenient system to study interaction of DHFR and DHPS inhibitors without such complications. Here, we describe the use of Escherichia coli transformed with malarial DHFR and DHPS, while its own corresponding genes have been inactivated by optimal concentration of trimethoprim and genetic knockout, respectively, to study the interaction of the inhibitors. Marked synergistic effects are observed for all combinations of pyrimethamine and sulfa inhibitors in the presence of trimethoprim. At 0.05μM trimethoprim, sum of fractional inhibitory concentrations, ΣFIC of pyrimethamine with sulfadoxine, pyrimethamine with sulfathiazole, pyrimethamine with sulfamethoxazole, and pyrimethamine with dapsone are in the range of 0.24-0.41. These results show synergism between inhibitors of the two enzymes even in the absence of folate transport and uptake. This bacterial surrogate system should be useful as a tool for assessing the interactions of drug combinations between the DHFR and DHPS inhibitors.

  17. S-2-amino-5-(2-nitroimidazol-1-yl)pentanoic acid: a model for potential bioreductively activated prodrugs for inhibitors of nitric oxide synthase (NOS) activity.

    PubMed

    Ulhaq, S; Naylor, M A; Chinje, E C; Threadgill, M D; Stratford, I J

    1997-01-01

    Treatment of 1,1-dimethylethyl S-(2-1,1-dimethylethoxycarbonylamino)-5-bromopentanoate with 1-potassio-2-nitroimidazole, followed by deprotection, afforded S-2-amino-5-(2-nitroimidazol-1-yl)pentanoic acid, which was reduced to S-2-amino-5-(2-aminoimidazol-1-yl)pentanoic acid. This aminoimadazole inhibited rat brain nitric oxide synthase (NOS) activity 3.2 times more potently than did the nitro analogue. Thus S-2-amino-5-(2-nitroimidazol-1-yl)pentanoic acid is a potent prodrug which may be bioreductively activated to a NOS inhibitor in hypoxic solid tumours.

  18. Discovery of inducible nitric oxide synthase (iNOS) inhibitor development candidate KD7332, part 1: Identification of a novel, potent, and selective series of quinolinone iNOS dimerization inhibitors that are orally active in rodent pain models.

    PubMed

    Bonnefous, Céline; Payne, Joseph E; Roppe, Jeffrey; Zhuang, Hui; Chen, Xiaohong; Symons, Kent T; Nguyen, Phan M; Sablad, Marciano; Rozenkrants, Natasha; Zhang, Yan; Wang, Li; Severance, Daniel; Walsh, John P; Yazdani, Nahid; Shiau, Andrew K; Noble, Stewart A; Rix, Peter; Rao, Tadimeti S; Hassig, Christian A; Smith, Nicholas D

    2009-05-14

    There are three isoforms of dimeric nitric oxide synthases (NOS) that convert arginine to citrulline and nitric oxide. Inducible NOS is implicated in numerous inflammatory diseases and, more recently, in neuropathic pain states. The majority of existing NOS inhibitors are either based on the structure of arginine or are substrate competitive. We describe the identification from an ultra high-throughput screen of a novel series of quinolinone small molecule, nonarginine iNOS dimerization inhibitors. SAR studies on the screening hit, coupled with an in vivo lipopolysaccharide (LPS) challenge assay measuring plasma nitrates and drug levels, rapidly led to the identification of compounds 12 and 42--potent inhibitors of the human and mouse iNOS enzyme that were highly selective over endothelial NOS (eNOS). Following oral dosing, compounds 12 and 42 gave a statistical reduction in pain behaviors in the mouse formalin model, while 12 also statistically reduced neuropathic pain behaviors in the chronic constriction injury (Bennett) model.

  19. Mechanical stimulation of skeletal muscle mitigates glucocorticoid induced decreases in prostaglandin synthesis

    NASA Technical Reports Server (NTRS)

    Chromiak, Joseph A.; Vandenburgh, Herman H.

    1993-01-01

    The glucocorticoid dexamethasone (Dex) induces a decline in protein synthesis and protein content of tissue cultured, avian skeletal muscle cells, and this atrophy is attenuated by repetitive mechanical stretch. Since the prostaglandin synthesis inhibitor indomethacin mitigated this stretch attenuation of muscle atrophy, the role of prostaglandins as growth modulators in these processes was examined. Dex at 10(exp -8) M reduced PGF(sub 2(alpha)) production 55 percent - 65 percent and PGE(sub 2) production 84 - 90 percent after 24 - 72 h of incubation in static cultures. Repetitive 10 percent stretch-relaxations of the non-Dex treated cultures increased PGF(sub 2(alpha)) efflux 41 percent at 24 h and 276 percent at 72 h and increased PGE(sub 2) production 51 percent at 24 h and 236 percent at 72 h. Mechanical stimulation of Dex treated cultures increased PGF(sub 2(alpha)) production 162 percent after 24 h, thus returning PGF(sub 2(alpha)) efflux to the level of non-Dex treated cultures. At 72 h, stretch increased PGF(sub 2(alpha)) efflux 65 percent in Dex treated cultures, but PGF(sub 2(alpha)) production was 45-84 percent less than non-Dex treated cultures. Mechanical stimulation of Dex treated cultures increased PGE(sub 2) production at 24 h, but not at 72 h. Dex reduced prostaglandin H synthase (PGHS) activity in the muscle cultures by 70 percent after 8 - 24 h of incubation, and mechanical stimulation increased PGHS activity of the Dex treated cultures by 98 percent. It is concluded that repetitive mechanical stimulation attenuates the catabolic effects of Dex on cultured skeletal muscle cells in part by reversing the Dex-induced declines in PGHS activity and prostaglandin production.

  20. Design, synthesis and biological evaluation of benzothiazepinones (BTZs) as novel non-ATP competitive inhibitors of glycogen synthase kinase-3β (GSK-3β).

    PubMed

    Zhang, Peng; Hu, Hai-Rong; Bian, Shi-Hui; Huang, Zhao-Hui; Chu, Yong; Ye, De-Yong

    2013-03-01

    Glycogen synthase kinase-3β (GSK-3β) plays a key role in type II diabetes and Alzheimer's diseases, to which non-ATP competitive inhibitors represent an effectively therapeutical approach due to their good specificity. Herein, a series of small molecules benzothiazepinones (BTZs) as novel non-ATP competitive inhibitors of GSK-3β have been designed and synthesized. The in vitro evaluation performed by luminescent assay showed most BTZ derivatives have inhibitory effects in micromolar scale. Among them compounds 6l, 6t and 6v have the IC50 values of 25.0 μM, 27.8 μM and 23.0 μM, respectively. Moreover 6v is devoid of any inhibitory activity in the assays to other thirteen protein kinases. Besides, SAR is analyzed and a hypothetical enzymatic binding mode is proposed by molecular docking study, which would be useful for new candidates design.

  1. Differential Expression of 1-Aminocyclopropane-1-Carboxylate Synthase Genes during Orchid Flower Senescence Induced by the Protein Phosphatase Inhibitor Okadaic Acid1

    PubMed Central

    Wang, Ning Ning; Yang, Shang Fa; Charng, Yee-yung

    2001-01-01

    Applying 10 pmol of okadaic acid (OA), a specific inhibitor of type 1 or type 2A serine/threonine protein phosphatases, to the orchid (Phalaenopsis species) stigma induced a dramatic increase in ethylene production and an accelerated senescence of the whole flower. Aminoethoxyvinylglycine or silver thiosulfate, inhibitors of ethylene biosynthesis or action, respectively, effectively inhibited the OA-induced ethylene production and retarded flower senescence, suggesting that the protein phosphatase inhibitor induced orchid flower senescence through an ethylene-mediated signaling pathway. OA treatment induced a differential expression pattern for the 1-aminocyclopropane-1-carboxylic acid synthase multigene family. Accumulation of Phal-ACS1 transcript in the stigma, labelum, and ovary induced by OA were higher than those induced by pollination as determined by “semiquantitative” reverse transcriptase-polymerase chain reaction. In contrast, the transcript levels of Phal-ACS2 and Phal-ACS3 induced by OA were much lower than those induced by pollination. Staurosporine, a protein kinase inhibitor, on the other hand, inhibited the OA-induced Phal-ACS1 expression in the stigma and delayed flower senescence. Our results suggest that a hyper-phosphorylation status of an unidentified protein(s) is involved in up-regulating the expression of Phal-ACS1 gene resulting in increased ethylene production and accelerated the senescence process of orchid flower. PMID:11351088

  2. Effects of terahertz radiation at atmospheric oxygen frequency of 129 GHz on blood nitrite concentrations under conditions of different types of stress against the background of administration of nonselective inhibitor of constitutive NO-synthases.

    PubMed

    Kirichuk, V F; Tsymbal, A A

    2012-02-01

    We studied the effect of terahertz radiation at atmospheric oxygen frequency 129 GHz on blood nitrite concentration in different types of experimental stress against the background of administration of nonselective inhibitor of constitutive NO-synthases. Normalizing effects of radiation on blood nitrite dynamics in animals with acute stress was shown after 15-min exposure and in animals with chronic stress after 30-min exposure. No positive effect of terahertz radiation was observed on altered blood nitrite concentration in male rats after preliminary administration of nonselective constitutive NO-synthase isoform inhibitor L-NAME.

  3. Effects of terahertz radiation at atmospheric oxygen frequency of 129 GHz on blood nitrite concentrations under conditions of different types of stress against the background of administration of nonselective inhibitor of constitutive NO-synthases.

    PubMed

    Kirichuk, V F; Tsymbal, A A

    2012-02-01

    We studied the effect of terahertz radiation at atmospheric oxygen frequency 129 GHz on blood nitrite concentration in different types of experimental stress against the background of administration of nonselective inhibitor of constitutive NO-synthases. Normalizing effects of radiation on blood nitrite dynamics in animals with acute stress was shown after 15-min exposure and in animals with chronic stress after 30-min exposure. No positive effect of terahertz radiation was observed on altered blood nitrite concentration in male rats after preliminary administration of nonselective constitutive NO-synthase isoform inhibitor L-NAME. PMID:22803105

  4. An Innovative Strategy for Dual Inhibitor Design and Its Application in Dual Inhibition of Human Thymidylate Synthase and Dihydrofolate Reductase Enzymes

    PubMed Central

    Arooj, Mahreen; Sakkiah, Sugunadevi; Cao, Guang ping; Lee, Keun Woo

    2013-01-01

    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. PMID:23577115

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

    PubMed

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

    2001-12-25

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

  6. The Enteropathy of Prostaglandin Deficiency

    PubMed Central

    Adler, David H.; Phillips, John A.; Cogan, Joy D.; Iverson, Tina M.; Stein, Jeffrey A.; Brenner, David A.; Morrow, Jason D.; Boutaud, Olivier; Oates, John A.

    2009-01-01

    Purpose Small intestinal ulcers are frequent complications of therapy with non-steroidal anti-inflammatory drugs (NSAIDs). We present here a genetic deficiency of eicosanoid biosynthesis that illuminates the mechanism of NSAID-induced ulcers of the small intestine. Methods Eicosanoids and metabolites were measured by isotope-dilution with mass spectrometry. cDNA was obtained by reverse transcription and sequenced following amplification with RT-PCR. Results We investigated the cause of chronic recurrent small intestinal ulcers, small bowel perforations, and gastrointestinal blood loss in a 45 year old male who was not taking any cyclooxygenase inhibitor. Prostaglandin metabolites in urine were significantly depressed. Serum thromboxane B2 (TxB2) production was 4.6% of normal controls (p<0.006) and serum 12-HETE was 1.3% of controls (p<0.005). Optical platelet aggregation with simultaneous monitoring of ATP release demonstrated absent granule secretion in response to ADP and a blunted aggregation response to ADP and collagen, but normal response to arachidonic acid (AA). LTB4 biosynthesis by ionophore activated leukocytes was only 3% of controls and urinary LTE4 was undetectable. These findings suggested deficient AA release from membrane phospholipids by cytosolic phospholipase A2-α (cPLA2-α) which regulates cyclooxygenase and lipoxygenase mediated eicosanoid production by catalyzing the release of their substrate, AA. Sequencing of cPLA2-α cDNA demonstrated 2 heterozygous non-synonymous single base pair mutations: Ser111Pro (S111P) and Arg485His (R485H), as well as a known SNP: Lys651Arg (K651R). Conclusion Characterization of this cPLA2-α deficiency provides support for the importance of prostaglandins in protecting small intestinal integrity, and indicates that loss of prostaglandin biosynthesis is sufficient to produce small intestinal ulcers. PMID:19148786

  7. Synergistic neuroprotective effects of lithium and valproic acid or other histone deacetylase inhibitors in neurons: roles of glycogen synthase kinase-3 inhibition.

    PubMed

    Leng, Yan; Liang, Min-Huei; Ren, Ming; Marinova, Zoya; Leeds, Peter; Chuang, De-Maw

    2008-03-01

    Lithium and valproic acid (VPA) are two primary drugs used to treat bipolar mood disorder and have frequently been used in combination to treat bipolar patients resistant to monotherapy with either drug. Lithium, a glycogen synthase kinase-3 (GSK-3) inhibitor, and VPA, a histone deacetylase (HDAC) inhibitor, have neuroprotective effects. The present study was undertaken to demonstrate synergistic neuroprotective effects when both drugs were coadministered. Pretreatment of aging cerebellar granule cells with lithium or VPA alone provided little or no neuroprotection against glutamate-induced cell death. However, copresence of both drugs resulted in complete blockade of glutamate excitotoxicity. Combined treatment with lithium and VPA potentiated serine phosphorylation of GSK-3 alpha and beta isoforms and inhibition of GSK-3 enzyme activity. Transfection with GSK-3alpha small interfering RNA (siRNA) and/or GSK-3beta siRNA mimicked the ability of lithium to induce synergistic protection with VPA. HDAC1 siRNA or other HDAC inhibitors (phenylbutyrate, sodium butyrate or trichostatin A) also caused synergistic neuroprotection together with lithium. Moreover, combination of lithium and HDAC inhibitors potentiated beta-catenin-dependent, Lef/Tcf-mediated transcriptional activity. An additive increase in GSK-3 serine phosphorylation was also observed in mice chronically treated with lithium and VPA. Together, for the first time, our results demonstrate synergistic neuroprotective effects of lithium and HDAC inhibitors and suggest that GSK-3 inhibition is a likely molecular target for the synergistic neuroprotection. Our results may have implications for the combined use of lithium and VPA in treating bipolar disorder. Additionally, combined use of both drugs may be warranted for clinical trials to treat glutamate-related neurodegenerative diseases.

  8. Screening of inhibitors of glycogen synthase kinase-3β from traditional Chinese medicines using enzyme-immobilized magnetic beads combined with high-performance liquid chromatography.

    PubMed

    Li, Yunfang; Xu, Jia; Chen, Yu; Mei, Zhinan; Xiao, Yuxiu

    2015-12-18

    Glycogen synthase kinase-3β (GSK-3β) was immobilized on magnetic beads (MBs) by affinity method for the first time. The enzyme-immobilized MBs were coupled with high-performance liquid chromatography-ultraviolet (HPLC-UV) technique to establish a cost-effective and reliable method for screening of inhibitors of GSK-3β. A peptide substrate of GSK-3β containing a tyrosine residue was employed since it can be sensitively detected by UV detector at 214nm. The substrate and its phosphorylated product were separated by baseline within 10min. The enzyme activity was determined by the quantification of peak area of the product. Parameters including enzyme immobilization, enzyme reaction and the performance of immobilized-enzyme were investigated. The immobilized enzyme can be reused for 10 times and remain stable for 4 days at 4°C. The inhibitory activities of extracts of 15 traditional Chinese medicines (TCMs) were screened. As a result, three of them including Euonymus fortunei, Amygdalus communis and Garcinia xanthochymus were found possessing high inhibitory activities (inhibition rate >90%). From G. xanthochymus, a new inhibitor of GSK-3β, fukugetin, was discovered with an IC50 value of 3.18±0.07μM. Enzyme kinetics and molecular docking experiments further revealed the inhibitory mechanism, indicating fukugetin was a non-ATP competitive inhibitor interacting with the phosphate recognizing substrate binding site of GSK-3β.

  9. In Vitro and In Vivo Activities of E5700 and ER-119884, Two Novel Orally Active Squalene Synthase Inhibitors, against Trypanosoma cruzi

    PubMed Central

    Urbina, Julio A.; Concepcion, Juan Luis; Caldera, Aura; Payares, Gilberto; Sanoja, Cristina; Otomo, Takeshi; Hiyoshi, Hironobu

    2004-01-01

    Chagas' disease is a serious public health problem in Latin America, and no treatment is available for the prevalent chronic stage. Its causative agent, Trypanosoma cruzi, requires specific endogenous sterols for survival, and we have recently demonstrated that squalene synthase (SQS) is a promising target for antiparasitic chemotherapy. E5700 and ER-119884 are quinuclidine-based inhibitors of mammalian SQS that are currently in development as cholesterol- and triglyceride-lowering agents in humans. These compounds were found to be potent noncompetitive or mixed-type inhibitors of T. cruzi SQS with Ki values in the low nanomolar to subnanomolar range in the absence or presence of 20 μM inorganic pyrophosphate. The antiproliferative 50% inhibitory concentrations of the compounds against extracellular epimastigotes and intracellular amastigotes were ca. 10 nM and 0.4 to 1.6 nM, respectively, with no effects on host cells. When treated with these compounds at the MIC, all of the parasite's sterols disappeared from the parasite cells. In vivo studies indicated that E5700 was able to provide full protection against death and completely arrested the development of parasitemia when given at a concentration of 50 mg/kg of body weight/day for 30 days, while ER-119884 provided only partial protection. This is the first report of an orally active SQS inhibitor that is capable of providing complete protection against fulminant, acute Chagas' disease. PMID:15215084

  10. Structure of N-acetyl-L-glutamate synthase/kinase from Maricaulis maris with the allosteric inhibitor L-arginine bound.

    PubMed

    Zhao, Gengxiang; Haskins, Nantaporn; Jin, Zhongmin; M Allewell, Norma; Tuchman, Mendel; Shi, Dashuang

    2013-08-01

    Maricaulis maris N-acetylglutamate synthase/kinase (mmNAGS/K) catalyzes the first two steps in L-arginine biosynthesis and has a high degree of sequence and structural homology to human N-acetylglutamate synthase, a regulator of the urea cycle. The synthase activity of both mmNAGS/K and human NAGS are regulated by L-arginine, although L-arginine is an allosteric inhibitor of mmNAGS/K, but an activator of human NAGS. To investigate the mechanism of allosteric inhibition of mmNAGS/K by L-arginine, we have determined the structure of the mmNAGS/K complexed with L-arginine at 2.8 Å resolution. In contrast to the structure of mmNAGS/K in the absence of L-arginine where there are conformational differences between the four subunits in the asymmetric unit, all four subunits in the L-arginine liganded structure have very similar conformations. In this conformation, the AcCoA binding site in the N-acetyltransferase (NAT) domain is blocked by a loop from the amino acid kinase (AAK) domain, as a result of a domain rotation that occurs when L-arginine binds. This structural change provides an explanation for the allosteric inhibition of mmNAGS/K and related enzymes by L-arginine. The allosterically regulated mechanism for mmNAGS/K differs significantly from that for Neisseria gonorrhoeae NAGS (ngNAGS). To define the active site, several residues near the putative active site were mutated and their activities determined. These experiments identify roles for Lys356, Arg386, Asn391 and Tyr397 in the catalytic mechanism. PMID:23850694

  11. Effects of the dual TP receptor antagonist and thromboxane synthase inhibitor EV-077 on human endothelial and vascular smooth muscle cells

    SciTech Connect

    Petri, Marcelo H.; Tellier, Céline; Michiels, Carine; Ellertsen, Ingvill; Dogné, Jean-Michel; Bäck, Magnus

    2013-11-15

    Highlights: •EV-077 reduced TNF-α induced inflammation in endothelial cells. •The thromboxane mimetic U69915 enhanced vascular smooth muscle cell proliferation. •EV-077 inhibited smooth muscle cell proliferation. -- Abstract: The prothrombotic mediator thromboxane A{sub 2} is derived from arachidonic acid metabolism through the cyclooxygenase and thromboxane synthase pathways, and transduces its effect through the thromboxane prostanoid (TP) receptor. The aim of this study was to determine the effect of the TP receptor antagonist and thromboxane synthase inhibitor EV-077 on inflammatory markers in human umbilical vein endothelial cells and on human coronary artery smooth muscle cell proliferation. To this end, mRNA levels of different proinflammatory mediators were studied by real time quantitative PCR, supernatants were analyzed by enzyme immune assay, and cell proliferation was assessed using WST-1. EV-077 significantly decreased mRNA levels of ICAM-1 and PTX3 after TNFα incubation, whereas concentrations of 6-keto PGF1α in supernatants of endothelial cells incubated with TNFα were significantly increased after EV-077 treatment. Although U46619 did not alter coronary artery smooth muscle cell proliferation, this thromboxane mimetic enhanced the proliferation induced by serum, insulin and growth factors, which was significantly inhibited by EV-077. In conclusion, EV-077 inhibited TNFα-induced endothelial inflammation and reduced the enhancement of smooth muscle cell proliferation induced by a thromboxane mimetic, supporting that the thromboxane pathway may be associated with early atherosclerosis in terms of endothelial dysfunction and vascular hypertrophy.

  12. In silico screening for identification of novel β-1,3-glucan synthase inhibitors using pharmacophore and 3D-QSAR methodologies.

    PubMed

    Meetei, Potshangbam Angamba; Rathore, R S; Prabhu, N Prakash; Vindal, Vaibhav

    2016-01-01

    The enzyme β-1,3-glucan synthase, which catalyzes the synthesis of β-1,3-glucan, an essential and unique structural component of the fungal cell wall, has been considered as a promising target for the development of less toxic anti-fungal agents. In this study, a robust pharmacophore model was developed and structure activity relationship analysis of 42 pyridazinone derivatives as β-1,3-glucan synthase inhibitors were carried out. A five-point pharmacophore model, consisting of two aromatic rings (R) and three hydrogen bond acceptors (A) was generated. Pharmacophore based 3D-QSAR model was developed for the same reported data sets. The generated 3D-QSAR model yielded a significant correlation coefficient value (R (2) = 0.954) along with good predictive power confirmed by the high value of cross-validated correlation coefficient (Q (2) = 0.827). Further, the pharmacophore model was employed as a 3D search query to screen small molecules database retrieved from ZINC to select new scaffolds. Finally, ADME studies revealed the pharmacokinetic efficiency of these compounds. PMID:27429875

  13. 1-(Fluoroalkylidene)-1,1-bisphosphonic Acids are Potent and Selective Inhibitors of the Enzymatic Activity of Toxoplasma gondii Farnesyl Pyrophosphate Synthase

    PubMed Central

    Szajnman, Sergio H.; Rosso, Valeria S.; Malayil, Leena; Smith, Alyssa; Moreno, Silvia N. J.; Docampo, Roberto

    2012-01-01

    α-Fluorinated-1,1-bisphosphonic acids derived from fatty acids were designed, synthesized and biologically evaluated against Trypanosoma cruzi, the etiologic agent of Chagas disease and against Toxoplasma gondii, the responsible agent of toxoplasmosis and also towards the target parasitic enzymes farnesyl pyrophosphate synthase of T. cruzi (TcFPPS) and T gondii (TgFPPS), respectively. Interestingly, 1-fluorononylidene-1,1-bisphosphonic acid (compound 43) has proven to be an extremely potent inhibitor of the enzymatic activity of TgFPPS at the low nanomolar range exhibiting an IC50 of 30 nM. This compound was two-fold more potent than risedronate (IC50 = 74 nM) taken as a positive control. This enzymatic activity was associated to a strong cell growth inhibition against tachyzoites of T. gondii having an IC50 value of 2.7 μM. PMID:22215028

  14. Structural basis for the design of potent and species-specific inhibitors of 3-hydroxy-3-methylglutaryl CoA synthases.

    PubMed

    Pojer, Florence; Ferrer, Jean-Luc; Richard, Stéphane B; Nagegowda, Dinesh A; Chye, Mee-Len; Bach, Thomas J; Noel, Joseph P

    2006-08-01

    3-Hydroxy-3-methylglutaryl CoA synthase (HMGS) catalyzes the first committed step in the mevalonate metabolic pathway for isoprenoid biosynthesis and serves as an alternative target for cholesterol-lowering and antibiotic drugs. We have determined a previously undescribed crystal structure of a eukaryotic HMGS bound covalently to a potent and specific inhibitor F-244 [(E,E)-11-[3-(hydroxymethyl)-4-oxo-2-oxytanyl]-3,5,7-trimethyl-2,4-undecadienenoic acid]. Given the accessibility of synthetic analogs of the F-244 natural product, this inhibited eukaryotic HMGS structure serves as a necessary starting point for structure-based methods that may improve the potency and species-specific selectivity of the next generation of F-244 analogs designed to target particular eukaryotic and prokaryotic HMGS.

  15. Structural Basis for the Design of Potent and Species-specific Inhibitors of 3-hydroxy-3-methylglutaryl CoA Synthases

    SciTech Connect

    Pojer,F.; Ferrer, J.; Richard, S.; Nagegowda, D.; Chye, M.; Bach, T.; Noel, J.

    2006-01-01

    3-Hydroxy-3-methylglutaryl CoA synthase (HMGS) catalyzes the first committed step in the mevalonate metabolic pathway for isoprenoid biosynthesis and serves as an alternative target for cholesterol-lowering and antibiotic drugs. We have determined a previously undescribed crystal structure of a eukaryotic HMGS bound covalently to a potent and specific inhibitor F-244 [(E,E)-11-[3-(hydroxymethyl)-4-oxo-2-oxytanyl]-3,5,7-trimethyl-2,4-undecadienenoic acid]. Given the accessibility of synthetic analogs of the F-244 natural product, this inhibited eukaryotic HMGS structure serves as a necessary starting point for structure-based methods that may improve the potency and species-specific selectivity of the next generation of F-244 analogs designed to target particular eukaryotic and prokaryotic HMGS.

  16. Prostaglandins as abortifacients.

    PubMed

    Karim, S M

    1971-12-30

    Clinical trials have demonstrated the use of prostaglandins as effective abortifacients. Continuous intravenous infusion of the drugs however has been associated with certain side effects at therapeutically effective doses, such as nausea, vomiting, diarrhea and a local erythematous reaction at the site of venepuncture. Higher doses result in more serious side effects such as vasovagal symptoms, pyrexia and tachycardia. Direct application of prostaglandins E2 or F2a into the uterine cavity has been shown to minimize the side effects. Appropriate doses of prostaglandins every one or 2 hours administered at the site of action between the fetal membrane and uterine wall (via the cervix) produce the strong and frequent uterine contractions necessary for the expulsion of the products of conception. A drawback of this method is the need for the uterine cavity to be continuously monitored as dosage is determined by the uterine response. Another effective method of terminating 1st and 2nd trimester pregnacy with minimal side effects is vaginal administration (into the posterior fornix) of 50 mg PGF2a or 20 mg PGE2 every 2 or 3 hours. Single injection of prostaglandins into the amniotic sac usually results in complete abortion. The method is simple but should be used only in pregnancies of over 12 weeks' gestation as the amniotic sac is inaccessible in the 1st trimester. The prostaglandin method, compared with other methods of abortion in the 1st trimester of pregnancy (e.g., suction or dilatation and curettage) is inferior in terms of time, expense and convenience. Incomplete abortion is quite common in the 1st trimester when prostaglandins are used. With respect to 2nd trimester methods (hypertonic saline and hysterotomy) however, prostaglandins given by intravaginal, intrauterine, or intraamniotic routes offer clear advantages.

  17. The effect of nitric oxide synthase inhibitors nitro-L-arginine and 7-nitroindazole on spatial learning and motor functions in Lurcher mutant and wild type mice.

    PubMed

    Markvartová, V; Vozeh, F

    2008-01-01

    Nitric oxide (NO) is an intercellular messenger that, among other things, plays an important role in the nervous system as a gaseous neurotransmitter, modulating long-term potentiation (LTP) induction of synaptic transmission. LTP has been suggested to be the basis of memory formation. On the other hand NO also participates in excitotoxic processes which play an important role in many neuropathological states. The aim of this work was to observe the effect of two NO synthase (NOS) inhibitors (N omega-Nitro-L-arginine, NA; 7-nitroindazole, NI) on spontaneous behaviour, spatial learning and motor functions in Lurcher (+/Lc) and wild type (+/+) mice, derived from the B6CBA strain. Heterozygous Lurcher mutant mice represent a natural model of the olivocerebellar degeneration. They suffer from postnatal, practically total, extinction of cerebellar Purkinje cells (due to the excitotoxic apoptosis) and a partial decrease of granule cells and inferior olive neurons (ION) because of the lost target of their axons. +/+ animals are healthy littermates of +/Lc. NA is a nonselective NOS inhibitor which influences, except neuronal (n), also endothelial (e) NOS with an impact on blood pressure, NI is a selective nNOS inhibitor without any circulatory effect. The adult animals of both types (+/Lc; +/+) were influenced by acute administration of both inhibitors (25 mg/kg i.p. 30 min. before experiments) and newborns only by both acute and long-term administration of NI (1 month, starting from postnatal day 2, P2). Control solutions - saline or solvents of both NA and NI inhibitors--diluted 1M HCl and dimethyl sulfoxide (DMSO) respectively, were given at a relevant volume in the same way. The effect of both inhibitors and control solutions on motor functions was tested using four standard procedures (horizontal wire, slanting ladder, rotating cylinder, foot-bridge); in newborns at the age of 14 days. Spatial learning ability was examined in five-day long procedure in the Morris

  18. Structure-Based Design and Synthesis of Nω-Nitro-L-Arginine-Containing Peptidomimetics as Selective Inhibitors of Neuronal Nitric Oxide Synthase. Displacement of the Heme Structural Water

    PubMed Central

    Seo, Jiwon; Igarashi, Jotato; Li, Huiying; Martásek, Pavel; Roman, Linda J.; Poulos, Thomas L.; Silverman, Richard B.

    2008-01-01

    The neuronal isoform of nitric oxide synthase (nNOS), the enzyme responsible for the production of nitric oxide in the central nervous system, represents an attractive target for the treatment of various neurodegenerative disorders. X-ray crystal structures of complexes of nNOS with two nNOS-selective inhibitors, (4S)-N-{4-amino-5-[(2-aminoethylamino]pentyl}-N′-nitroguanidine (1) and 4-N-(Nω-nitro-L-argininyl)-trans-4-amino-L-proline amide (2), led to the discovery of a conserved structural water molecule that was hydrogen bonded between the two heme propionates and the inhibitors (Figure 2). Based on this observation, we hypothesized that by attaching a hydrogen bond donor group to the amide nitrogen of 2 or to the secondary amine nitrogen of 1, the inhibitor molecules could displace the structural water molecule and obtain a direct interaction with the heme cofactor. To test this hypothesis, peptidomimetic analogues 3–5, which have either an N-hydroxyl (3 and 5) or N-amino (4) donor group, were designed and synthesized. X-ray crystal structures of nNOS with inhibitors 3 and 5 bound verified that the N-hydroxyl group had, indeed, displaced the structural water molecule and provided a direct interaction with the heme propionate moiety (Figures 4 and 5). Surprisingly, in vitro activity assay results indicated that the addition of a hydroxyl group (3) only increased the potency slightly against the neuronal isoform over the parent compound (1). Rationalizations for the small increase in potency are consistent with other changes in the crystal structures. PMID:17425297

  19. Enhancement of the post-training cholinergic tone antagonizes the impairment of retention induced by a nitric oxide synthase inhibitor in mice.

    PubMed

    Kopf, S R; Baratti, C M

    1996-05-01

    The present experiments examined the role of the central cholinergic system in the memory impairment induced by post-training administration of a nitric oxide synthase (NOS) inhibitor in mice. Male Swiss mice received a one-trial inhibitory avoidance training (0.8 mA, 50 Hz, 1-s footshock) followed immediately by an ip injection of the NOS inhibitor L-NG-nitroarginine methyl ester (L-NAME; 100 mg/kg). Retention (cut-off time, 300 s) was tested 48 h after training. The administration of L-NAME results in memory impairment for the inhibitory avoidance task. The effects of L-NAME (100 mg/kg, ip) on retention were reversed in a dose-related manner by the centrally acting anticholinesterase physostigmine (35, 70, or 150 microg/kg, sc) administered 30 min after the NOS inhibitor. Further, L-NAME (100 mg/kg, ip)-induced memory impairment was completely antagonized by the centrally acting muscarinic cholinergic agonist oxotremorine (OTM; 25, 50, or 100 microg/kg, sc) when given 30 min after L-NAME. The peripherally acting anticholinesterase neostigmine (150 microg/kg, sc) did not modify the memory-impairing effects of L-NAME. These findings suggest that the memory impairment following post-training administration of a NOS inhibitor is mediated, at least in part, by a reduction of the activity of central muscarinic cholinergic mechanisms and are consistent with our previous view that nitric oxide may be involved in post-training neural processes underlying the storage of newly acquired information. PMID:8616584

  20. Effect of nitric oxide synthase inhibitors on preventing ethanol-induced suppression of the hypothalamic-pituitary-gonadal axis in the male rat.

    PubMed

    Shi, Q; Emanuele, N V; Emanuele, M A

    1998-11-01

    Ethanol (EtOH) suppression of the hypothalamic-pituitary-gonadal (HPG) axis results in broad reproductive malfunction. In the HPG axis, the suppressive effects of EtOH are manifested by decreased serum testosterone, reduced testicular luteinizing hormone (LH) receptor numbers, lowered serum LH and pituitary beta-LH mRNA levels (in castrated animals), and impaired luteinizing hormone releasing hormone (LHRH) release from the hypothalamus. Increasing evidence has suggested that nitric oxide (NO) plays a role in regulation of the HPG axis. NO was shown to stimulate LHRH secretion from the hypothalamus and to have variable effects on LH release from the pituitary. At the gonadal level, NO is inhibitory to testosterone production. NO may directly inhibit some testicular steroidogenic enzymes. To investigate the effect of EtOH, NO, and their interaction on the male HPG axis, three NO synthase (NOS) inhibitors, N(G)-nitro-L-arginine methyl ester, N(G)-nitro-L-arginine, and 7-nitro indazole were used to study overall HPG function in the presence and absence of EtOH. Animals were given intraperitoneal injections of saline, EtOH, various NOS inhibitors, or EtOH, along with NOS inhibitors 2 hr before sacrifice. Serum testosterone and LH concentrations, pituitary beta-LH mRNA levels, hypothalamic LHRH mRNA levels, and LHRH content were determined. It was found that blocking NOS by these NOS inhibitors prevented EtOH-induced suppression of testosterone and, in some cases, serum LH. However, this was not accompanied by concurrent changes with NOS blockade on LHRH mRNA, hypothalamic pro-LHRH or LHRH content or pituitary LH beta mRNA levels. It appears that the protective effect of NOS blockade was largely, although not completely, due to a direct effect at the gonadal level.

  1. Minimal pharmacophoric elements and fragment hopping, an approach directed at molecular diversity and isozyme selectivity. Design of selective neuronal nitric oxide synthase inhibitors.

    PubMed

    Ji, Haitao; Stanton, Benjamin Z; Igarashi, Jotaro; Li, Huiying; Martásek, Pavel; Roman, Linda J; Poulos, Thomas L; Silverman, Richard B

    2008-03-26

    Fragment hopping, a new fragment-based approach for de novo inhibitor design focusing on ligand diversity and isozyme selectivity, is described. The core of this approach is the derivation of the minimal pharmacophoric element for each pharmacophore. Sites for both ligand binding and isozyme selectivity are considered in deriving the minimal pharmacophoric elements. Five general-purpose libraries are established: the basic fragment library, the bioisostere library, the rules for metabolic stability, the toxicophore library, and the side chain library. These libraries are employed to generate focused fragment libraries to match the minimal pharmacophoric elements for each pharmacophore and then to link the fragment to the desired molecule. This method was successfully applied to neuronal nitric oxide synthase (nNOS), which is implicated in stroke and neurodegenerative diseases. Starting with the nitroarginine-containing dipeptide inhibitors we developed previously, a small organic molecule with a totally different chemical structure was designed, which showed nanomolar nNOS inhibitory potency and more than 1000-fold nNOS selectivity. The crystallographic analysis confirms that the small organic molecule with a constrained conformation can exactly mimic the mode of action of the dipeptide nNOS inhibitors. Therefore, a new peptidomimetic strategy, referred to as fragment hopping, which creates small organic molecules that mimic the biological function of peptides by a pharmacophore-driven strategy for fragment-based de novo design, has been established as a new type of fragment-based inhibitor design. As an open system, the newly established approach efficiently incorporates the concept of early "ADME/Tox" considerations and provides a basic platform for medicinal chemistry-driven efforts. PMID:18321097

  2. Minimal Pharmacophoric Elements and Fragment Hopping, an Approach Directed at Molecular Diversity and Isozyme Selectivity. Design of Selective Neuronal Nitric Oxide Synthase Inhibitors

    PubMed Central

    Ji, Haitao; Stanton, Benjamin Z.; Igarashi, Jotaro; Li, Huiying; Martásek, Pavel; Roman, Linda J.; Poulos, Thomas L.; Silverman, Richard B.

    2010-01-01

    Fragment hopping, a new fragment-based approach for de novo inhibitor design focusing on ligand diversity and isozyme selectivity, is described. The core of this approach is the derivation of the minimal pharmacophoric element for each pharmacophore. Sites for both ligand binding and isozyme selectivity are considered in deriving the minimal pharmacophoric elements. Five general-purpose libraries are established: the basic fragment library, the bioisostere library, the rules for metabolic stability, the toxicophore library, and the side chain library. These libraries are employed to generate focused fragment libraries to match the minimal pharmacophoric elements for each pharmacophore and then to link the fragment to the desired molecule. This method was successfully applied to neuronal nitric oxide synthase (nNOS), which is implicated in stroke and neurodegenerative diseases. Starting with the nitroarginine-containing dipeptide inhibitors we developed previously, a small organic molecule with a totally different chemical structure was designed, which showed nanomolar nNOS inhibitory potency and more than 1000-fold nNOS selectivity. The crystallographic analysis confirms that the small organic molecule with a constrained conformation can exactly mimic the mode of action of the dipeptide nNOS inhibitors. Therefore, a new peptidomimetic strategy, referred to as fragment hopping, which creates small organic molecules that mimic the biological function of peptides by a pharmacophore-driven strategy for fragment-based de novo design, has been established as a new type of fragment-based inhibitor design. As an open system, the newly established approach efficiently incorporates the concept of early “ADME/Tox” considerations and provides a basic platform for medicinal chemistry-driven efforts. PMID:18321097

  3. Enhancement of the post-training cholinergic tone antagonizes the impairment of retention induced by a nitric oxide synthase inhibitor in mice.

    PubMed

    Kopf, S R; Baratti, C M

    1996-05-01

    The present experiments examined the role of the central cholinergic system in the memory impairment induced by post-training administration of a nitric oxide synthase (NOS) inhibitor in mice. Male Swiss mice received a one-trial inhibitory avoidance training (0.8 mA, 50 Hz, 1-s footshock) followed immediately by an ip injection of the NOS inhibitor L-NG-nitroarginine methyl ester (L-NAME; 100 mg/kg). Retention (cut-off time, 300 s) was tested 48 h after training. The administration of L-NAME results in memory impairment for the inhibitory avoidance task. The effects of L-NAME (100 mg/kg, ip) on retention were reversed in a dose-related manner by the centrally acting anticholinesterase physostigmine (35, 70, or 150 microg/kg, sc) administered 30 min after the NOS inhibitor. Further, L-NAME (100 mg/kg, ip)-induced memory impairment was completely antagonized by the centrally acting muscarinic cholinergic agonist oxotremorine (OTM; 25, 50, or 100 microg/kg, sc) when given 30 min after L-NAME. The peripherally acting anticholinesterase neostigmine (150 microg/kg, sc) did not modify the memory-impairing effects of L-NAME. These findings suggest that the memory impairment following post-training administration of a NOS inhibitor is mediated, at least in part, by a reduction of the activity of central muscarinic cholinergic mechanisms and are consistent with our previous view that nitric oxide may be involved in post-training neural processes underlying the storage of newly acquired information.

  4. L-Citrulline dilates rat retinal arterioles via nitric oxide- and prostaglandin-dependent pathways in vivo.

    PubMed

    Mori, Asami; Morita, Masahiko; Morishita, Koji; Sakamoto, Kenji; Nakahara, Tsutomu; Ishii, Kunio

    2015-04-01

    L-Citrulline is an effective precursor of L-arginine produced by the L-citrulline/L-arginine cycle, and it exerts beneficial effects on the cardiovascular system by supporting enhanced nitric oxide (NO) production. NO dilates retinal blood vessels via the cyclooxygenase-mediated pathway. The purpose of this study was to examine the effects of L-citrulline on retinal circulation and to investigate the potential involvement of NO and prostaglandins in L-citrulline-induced responses in rats. L-Citrulline (10-300 μg kg(-1) min(-1), i.v.) increased the diameter of retinal arterioles without significantly changing mean blood pressure, heart rate, and fundus blood flow. The vasodilator response of retinal arterioles to l-citrulline was significantly diminished following treatment with N(G)-nitro-L-arginine methyl ester (30 mg/kg, i.v.), an NO synthase inhibitor, or indomethacin (5 mg/kg, i.v.), a cyclooxygenase inhibitor. In addition, α-methyl-dl-aspartic acid (147 mg/kg, i.v.), an inhibitor of argininosuccinate synthase, the rate-limiting enzyme for the recycling of l-citrulline to l-arginine, diminished the L-citrulline-induced retinal vasodilation. These results suggest that both NO- and prostaglandin-dependent pathways contribute to the L-citrulline-induced vasodilation of rat retinal arterioles. The L-citrulline/L-arginine recycling pathway may have more importance in regulating vascular tone in retinal blood vessels than in peripheral resistance vessels. PMID:25953269

  5. The roles of prostaglandin E2 and D2 in lipopolysaccharide-mediated changes in sleep

    PubMed Central

    Oishi, Yo; Yoshida, Kyoko; Scammell, Thomas E.; Urade, Yoshihiro; Lazarus, Michael; Saper, Clifford B.

    2014-01-01

    When living organisms become sick as a result of a bacterial infection, a suite of brain-mediated responses occur, including fever, anorexia and sleepiness. Systemic administration of lipopolysaccharide (LPS), a common constituent of bacterial cell walls, increases body temperature and non-rapid eye movement (NREM) sleep in animals and induces the production of pro-inflammatory prostaglandins (PGs). Prostaglandin E2 (PGE2) is the principal mediator of fever, and both PGE2 and PGD2 regulate sleep–wake behavior. The extent to which PGE2 and PGD2 are involved in the effect of LPS on NREM sleep remains to be clarified. Therefore, we examined LPS-induced changes in body temperature and NREM sleep in mice with nervous system-specific knockouts (KO) for the PGE2 receptors, EP3 or EP4; in mice with total body KO of microsomal PGE synthase-1, or the PGD2 receptor DP; and in mice treated with the cyclooxygenase (COX) inhibitor meloxicam. We observed that LPS-induced NREM sleep was slightly attenuated in mice lacking EP4 receptors in the nervous system, but was not affected in any of the other KO mice or in mice pretreated with the COX inhibitor. These results suggest that the effect of LPS on NREM sleep is partially dependent on PGs and is likely mediated mainly by other pro-inflammatory substances. In addition, our data show that the main effect of LPS on body temperature is hypothermia in the absence of nervous system EP3 receptors or in the presence of a COX inhibitor. PMID:25532785

  6. Structure-Based Design of Novel Pyrimido[4,5-c]pyridazine Derivatives as Dihydropteroate Synthase Inhibitors with Increased Affinity

    SciTech Connect

    Zhao, Ying; Hammoudeh, Dalia; Yun, Mi-Kyung; Qi, Jianjun; White, Stephen W.; Lee, Richard E.

    2012-05-29

    Dihydropteroate synthase (DHPS) is the validated drug target for sulfonamide antimicrobial therapy. However, due to widespread drug resistance and poor tolerance, the use of sulfonamide antibiotics is now limited. The pterin binding pocket in DHPS is highly conserved and is distinct from the sulfonamide binding site. It therefore represents an attractive alternative target for the design of novel antibacterial agents. We previously carried out the structural characterization of a known pyridazine inhibitor in the Bacillus anthracis DHPS pterin site and identified a number of unfavorable interactions that appear to compromise binding. With this structural information, a series of 4,5-dioxo-1,4,5,6-tetrahydropyrimido[4,5-c]pyridazines were designed to improve binding affinity. Most importantly, the N-methyl ring substitution was removed to improve binding within the pterin pocket, and the length of the side chain carboxylic acid was optimized to fully engage the pyrophosphate binding site. These inhibitors were synthesized and evaluated by an enzyme activity assay, X-ray crystallography, isothermal calorimetry, and surface plasmon resonance to obtain a comprehensive understanding of the binding interactions from structural, kinetic, and thermodynamic perspectives. This study clearly demonstrates that compounds lacking the N-methyl substitution exhibit increased inhibition of DHPS, but the beneficial effects of optimizing the side chain length are less apparent.

  7. High resolution genetic mapping uncovers chitin synthase-1 as the target-site of the structurally diverse mite growth inhibitors clofentezine, hexythiazox and etoxazole in Tetranychus urticae

    PubMed Central

    Demaeght, Peter; Osborne, Edward J.; Odman-Naresh, Jothini; Grbić, Miodrag; Nauen, Ralf; Merzendorfer, Hans

    2014-01-01

    The acaricides clofentezine, hexythiazox and etoxazole are commonly referred to as ‘mite growth inhibitors’, and clofentezine and hexythiazox have been used successfully for the integrated control of plant mite pests for decades. Although they are still important today, their mode of action has remained elusive. Recently, a mutation in chitin synthase 1 (CHS1) was linked to etoxazole resistance. In this study, we identified and investigated a T. urticae strain (HexR) harboring recessive, monogenic resistance to each of hexythiazox, clofentezine, and etoxazole. To elucidate if there is a common genetic basis for the observed cross-resistance, we adapted a previously developed bulk segregant analysis method to map with high resolution a single, shared resistance locus for all three compounds. This finding indicates that the underlying molecular basis for resistance to all three compounds is identical. This locus is centered on the CHS1 gene, and as supported by additional genetic and biochemical studies, a non-synonymous variant (I1017F) in CHS1 associates with resistance to each of the tested acaricides in HexR. Our findings thus demonstrate a shared molecular mode of action for the chemically diverse mite growth inhibitors clofentezine, hexythiazox and etoxazole as inhibitors of an essential, non-catalytic activity of CHS1. Given the previously documented cross-resistance between clofentezine, hexythiazox and the benzyolphenylurea compounds flufenoxuron and cycloxuron, CHS1 should be also considered as a potential target-site of insecticidal BPUs. PMID:24859419

  8. 7-Nitroindazole and methylene blue, inhibitors of neuronal nitric oxide synthase and NO-stimulated guanylate cyclase, block MK-801-elicited behaviors in mice.

    PubMed

    Deutsch, S I; Rosse, R B; Paul, S M; Tomasino, V; Koetzner, L; Morn, C B; Mastropaolo, J

    1996-07-01

    We examined the abilities of 7-nitroindazole and methylene blue, inhibitors of the neuronal isoform of nitric oxide synthase (NOS) and nitric oxide-stimulated guanylate cyclase activity respectively, to attenuate explosive episodic jumping behavior(s) ("popping") elicited by MK-801 in mice. MK-801, like phencyclidine (PCP), is a high-affinity, noncompetitive antagonist of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor. We have postulated that MK-801-elicited popping behavior in mice represents an animal model of schizophrenia, because popping behavior is markedly inhibited/antagonized by both typical and atypical antipsychotic drugs. In the present study, popping behavior induced by MK-801 was measured using an automated detection system that quantifies vertical displacements on the testing platform. 7-Nitroindazole (100 mg/kg) and methylene blue (32 and 100 mg/kg) significantly reduced the number and force of MK-801-elicited popping behavior. Mouse rotorod performance did not differ between animals receiving 7-nitroindazole, methylene blue, or their respective vehicles, suggesting that attenuation of MK-801-elicited popping behavior was not due to either sedation or ataxia caused by 7-nitroindazole or methylene blue. Our findings suggest that nitric oxide may, in part, mediate behaviors induced by NMDA receptor antagonists, like MK-801, and that inhibitors of NOS may have antipsychotic actions.

  9. The role of S-methylisothiourea hemisulfate as inducible nitric oxide synthase inhibitor against kidney iron deposition in iron overload rats

    PubMed Central

    Maleki, Maryam; Samadi, Melika; Khanmoradi, Mehrangiz; Nematbakhsh, Mehdi; Talebi, Ardeshir; Nasri, Hamid

    2016-01-01

    Background: Iron dextran is in common use to maintain iron stores. However, it is potentially toxic and may lead to iron deposition (ID) and impair functions of organs. Iron overload can regulate the expression of inducible nitric oxide synthase (iNOS) in some cells that has an important role in tissue destruction. S-methylisothiourea hemisulfate (SMT) is a direct inhibitor of iNOS, and this study was designed to investigate the effect of SMT against kidney ID in iron overload rats. Materials and Methods: 24 Wistar rats (male and female) were randomly assigned to two groups. Iron overloading was performed by iron dextran 100 mg/kg/day every other day for 2 weeks. In addition, during the study, groups 1 and 2 received vehicle and SMT (10 mg/kg, ip), respectively. Finally, blood samples were obtained, and the kidneys were prepared for histopathological procedures. Results: SMT significantly reduced the serum levels of creatinine and blood urea nitrogen. However, SMT did not alter the serum levels of iron and nitrite, and the kidney tissue level of nitrite. Co-administration of SMT with iron dextran did not attenuate the ID in the kidney. Conclusion: SMT, as a specific iNOS inhibitor, could not protect the kidney from ID while it attenuated the serum levels of kidney function biomarkers. PMID:27308268

  10. Glycogen synthase kinase 3β inhibitors protect hippocampal neurons from radiation-induced apoptosis by regulating MDM2-p53 pathway

    PubMed Central

    Thotala, D K; Hallahan, D E; Yazlovitskaya, E M

    2012-01-01

    Exposure of the brain to ionizing radiation can cause neurocognitive deficiencies. The pathophysiology of these neurological changes is complex and includes radiation-induced apoptosis in the subgranular zone of the hippocampus. We have recently found that inhibition of glycogen synthase kinase 3β (GSK-3β) resulted in significant protection from radiation-induced apoptosis in hippocampal neurons. The molecular mechanisms of this cytoprotection include abrogation of radiation-induced accumulation of p53. Here we show that pretreatment of irradiated HT-22 hippocampal-derived neurons with small molecule inhibitors of GSK-3β SB216763 or SB415286, or with GSK-3β-specific shRNA resulted in accumulation of the p53-specific E3 ubiquitin ligase MDM2. Knockdown of MDM2 using specific shRNA or chemical inhibition of MDM2-p53 interaction prevented the protective changes triggered by GSK-3β inhibition in irradiated HT-22 neurons and restored radiation cytotoxicity. We found that this could be due to regulation of apoptosis by subcellular localization and interaction of GSK-3β, p53 and MDM2. These data suggest that the mechanisms of radioprotection by GSK-3β inhibitors in hippocampal neurons involve regulation of MDM2-dependent p53 accumulation and interactions between GSK-3β, MDM2 and p53. PMID:21738215

  11. Thiolactomycin-based β-ketoacyl-AcpM synthase A (KasA) inhibitors: fragment-based inhibitor discovery using transient one-dimensional nuclear overhauser effect NMR spectroscopy.

    PubMed

    Kapilashrami, Kanishk; Bommineni, Gopal R; Machutta, Carl A; Kim, Pilho; Lai, Cheng-Tsung; Simmerling, Carlos; Picart, Francis; Tonge, Peter J

    2013-03-01

    Thiolactomycin (TLM) is a natural product inhibitor of KasA, the β-ketoacyl synthase A from Mycobacterium tuberculosis. To improve the affinity of TLM for KasA, a series of TLM analogs have been synthesized based on interligand NOEs between TLM and a pantetheine analog when both are bound simultaneously to the enzyme. Kinetic binding data reveal that position 3 of the thiolactone ring is a suitable position for elaboration of the TLM scaffold, and the structure-activity relationship studies provide information on the molecular features that govern time-dependent inhibition in this enzyme system. These experiments also exemplify the utility of transient one-dimensional NOE spectroscopy for obtaining interligand NOEs compared with traditional steady state two-dimensional NOESY spectroscopy. PMID:23306195

  12. HMG-CoA Reductase Inhibitor Improves Endothelial Dysfunction in Spontaneous Hypertensive Rats Via Down-regulation of Caveolin-1 and Activation of Endothelial Nitric Oxide Synthase

    PubMed Central

    Suh, Jung-Won; Chang, Hyuk-Jae; Cho, Young-Seok; Youn, Tae-Jin; Chae, In-Ho; Kim, Kwang-Il; Kim, Cheol-Ho; Kim, Hyo-soo; Oh, Buyng-Hee; Park, Young-Bae

    2010-01-01

    Hypertension is associated with endothelial dysfunction and increased cardiovascular risk. Caveolin-1 regulates nitric oxide (NO) signaling by modulating endothelial nitric oxide synthase (eNOS). The purpose of this study was to examine whether HMG-CoA reductase inhibitor improves impaired endothelial function of the aorta in spontaneous hypertensive rat (SHR) and to determine the underlying mechanisms involved. Eight-week-old male SHR were assigned to either a control group (CON, n=11) or a rosuvastatin group (ROS, n=12), rosuvastatin (10 mg/kg/day) administered for eight weeks. Abdominal aortic rings were prepared and responses to acetylcholine (10-9-10-4 M) were determined in vitro. To evaluate the potential role of NO and caveolin-1, we examined the plasma activity of NOx, eNOS, phosphorylated-eNOS and expression of caveolin-1. The relaxation in response to acetylcholine was significantly enhanced in ROS compared to CON. Expression of eNOS RNA was unchanged, whereas NOx level and phosphorylated-eNOS at serine-1177 was increased accompanied with depressed level of caveolin-1 in ROS. We conclude that 3-Hydroxy-3-methylglutaryl Coenzyme-A (HMG-CoA) reductase inhibitor can improve impaired endothelial dysfunction in SHR, and its underlying mechanisms are associated with increased NO production. Furthermore, HMG-CoA reductase inhibitor can activate the eNOS by phosphorylation related to decreased caveolin-1 abundance. These results imply the therapeutic strategies for the high blood pressure-associated endothelial dysfunction through modifying caveolin status. PMID:20052342

  13. Structure determination of glycogen synthase kinase-3 from Leishmania major and comparative inhibitor structure-activity relationships with Trypanosoma brucei GSK-3

    SciTech Connect

    Ojo, Kayode K; Arakaki, Tracy L; Napuli, Alberto J; Inampudi, Krishna K; Keyloun, Katelyn R; Zhang, Li; Hol, Wim G.J.; Verlind, Christophe L.M.J.; Merritt, Ethan A; Van Voorhis, Wesley C

    2012-04-24

    Glycogen synthase kinase-3 (GSK-3) is a drug target under intense investigation in pharmaceutical companies and constitutes an attractive piggyback target for eukaryotic pathogens. Two different GSKs are found in trypanosomatids, one about 150 residues shorter than the other. GSK-3 short (GeneDB: Tb927.10.13780) has previously been validated genetically as a drug target in Trypanosoma brucei by RNAi induced growth retardation; and chemically by correlation between enzyme and in vitro growth inhibition. Here, we report investigation of the equivalent GSK-3 short enzymes of L. major (LmjF18.0270) and L. infantum (LinJ18_V3.0270, identical in amino acid sequences to LdonGSK-3 short) and a crystal structure of LmajGSK-3 short at 2 Å resolution. The inhibitor structure-activity relationships (SARs) of L. major and L. infantum are virtually identical, suggesting that inhibitors could be useful for both cutaneous and visceral leishmaniasis. Leishmania spp. GSK-3 short has different inhibitor SARs than TbruGSK-3 short, which can be explained mostly by two variant residues in the ATP-binding pocket. Indeed, mutating these residues in the ATP-binding site of LmajGSK-3 short to the TbruGSK-3 short equivalents results in a mutant LmajGSK-3 short enzyme with SAR more similar to that of TbruGSK-3 short. The differences between human GSK-3β (HsGSK-3β) and LmajGSK-3 short SAR suggest that compounds which selectively inhibit LmajGSK-3 short may be found.

  14. The role of prostaglandines in peristalsis of the human colon.

    PubMed

    Bruch, H P; Schmidt, E; Laven, R; Kehrer, G; Wasner, K H

    1978-08-01

    Prostaglandines (PG) of the E and F series cause peristaltic activity in isolated longitudinal muscle strips of the human colon. As this phasic motor reaction can be varied by acetyl choline and adrenaline it was supposed, that prostaglandines contribute to peristalsis. The role of PG E and F in the human colon was studied by inhibiting the prostaglandine synthesis and by antagonizing the prostaglandine-effects. Indomethacin proved to be a suitable inhibitor. HR 546 was found a powerful antagonist. The effect of Pentagastrin and Cholecystokinin (CCK) on peristaltic activity were suppressed by Indomethacin and HR 546. The inhibition of peristalsis by Indomethacin and HR 546 was removed by high doses of PG E and F. On the basis of these results the role of PG for the motility of the gut is discussed.

  15. Cytochrome P450 CYP81A12 and CYP81A21 Are Associated with Resistance to Two Acetolactate Synthase Inhibitors in Echinochloa phyllopogon1[W

    PubMed Central

    Iwakami, Satoshi; Endo, Masaki; Saika, Hiroaki; Okuno, Junichi; Nakamura, Naoki; Yokoyama, Masao; Watanabe, Hiroaki; Toki, Seiichi; Uchino, Akira; Inamura, Tatsuya

    2014-01-01

    Previous studies have demonstrated multiple herbicide resistance in California populations of Echinochloa phyllopogon, a noxious weed in rice (Oryza sativa) fields. It was suggested that the resistance to two classes of acetolactate synthase-inhibiting herbicides, bensulfuron-methyl (BSM) and penoxsulam (PX), may be caused by enhanced activities of herbicide-metabolizing cytochrome P450. We investigated BSM metabolism in the resistant (R) and susceptible (S) lines of E. phyllopogon, which were originally collected from different areas in California. R plants metabolized BSM through O-demethylation more rapidly than S plants. Based on available information about BSM tolerance in rice, we isolated and analyzed P450 genes of the CYP81A subfamily in E. phyllopogon. Two genes, CYP81A12 and CYP81A21, were more actively transcribed in R plants compared with S plants. Transgenic Arabidopsis (Arabidopsis thaliana) expressing either of the two genes survived in media containing BSM or PX at levels at which the wild type stopped growing. Segregation of resistances in the F2 generation from crosses of R and S plants suggested that the resistance to BSM and PX were each under the control of a single regulatory element. In F6 recombinant inbred lines, BSM and PX resistances cosegregated with increased transcript levels of CYP81A12 and CYP81A21. Heterologously produced CYP81A12 and CYP81A21 proteins in yeast (Saccharomyces cerevisiae) metabolized BSM through O-demethylation. Our results suggest that overexpression of the two P450 genes confers resistance to two classes of acetolactate synthase inhibitors to E. phyllopogon. The overexpression of the two genes could be regulated simultaneously by a single trans-acting element in the R line of E. phyllopogon. PMID:24760819

  16. Alkaloids as Inhibitors of Malate Synthase from Paracoccidioides spp.: Receptor-Ligand Interaction-Based Virtual Screening and Molecular Docking Studies, Antifungal Activity, and the Adhesion Process

    PubMed Central

    Costa, Fausto Guimaraes; Neto, Benedito Rodrigues da Silva; Gonçalves, Ricardo Lemes; da Silva, Roosevelt Alves; de Oliveira, Cecília Maria Alves; Kato, Lucília; Freitas, Carla dos Santos; Giannini, Maria José Soares Mendes; da Silva, Julhiany de Fátima; Soares, Célia Maria de Almeida

    2015-01-01

    Paracoccidioides is the agent of paracoccidioidomycosis. Malate synthase plays a crucial role in the pathogenicity and virulence of various fungi, such as those that are human pathogens. Thus, an inhibitor of this enzyme may be used as a powerful antifungal without side effects in patients once these enzymes are absent in humans. Here, we searched for compounds with inhibitory capacity against the malate synthase of Paracoccidioides species (PbMLS). The three-dimensional (3D) structure of PbMLS was determined using the I-TASSER server. Compounds were selected from the ZINC database. Based on the mechanism underlying the interaction of the compounds with PbMLS, it was possible to identify β-carboline moiety as a standard key structure. The compounds with β-carboline moiety that are available in our laboratories were investigated. A total of nine alkaloid compounds were selected. The primary mechanisms of interaction of the alkaloid compounds in the binding pocket of PbMLS were identified and compared with the mechanism of interaction of acetyl coenzyme A (acetyl-CoA). We discovered that the amphipathic nature of the compounds, concomitant with the presence of β-carboline moiety, was crucial for their stability in the binding pocket of PbMLS. In addition, the importance of a critical balance of the polar and nonpolar contacts of the compounds in this region was observed. Four β-carboline alkaloid compounds showed the ability to inhibit recombinant PbMLS (PbMLSr) activity, Paracoccidioides species growth, and adhesion of the fungus and PbMLSr to the extracellular matrix components. The cytotoxicity of the alkaloids was also evaluated. PMID:26124176

  17. Alkaloids as inhibitors of malate synthase from Paracoccidioides spp.: receptor-ligand interaction-based virtual screening and molecular docking studies, antifungal activity, and the adhesion process.

    PubMed

    Costa, Fausto Guimaraes; Neto, Benedito Rodrigues da Silva; Gonçalves, Ricardo Lemes; da Silva, Roosevelt Alves; de Oliveira, Cecília Maria Alves; Kato, Lucília; Freitas, Carla Dos Santos; Giannini, Maria José Soares Mendes; da Silva, Julhiany de Fátima; Soares, Célia Maria de Almeida; Pereira, Maristela

    2015-09-01

    Paracoccidioides is the agent of paracoccidioidomycosis. Malate synthase plays a crucial role in the pathogenicity and virulence of various fungi, such as those that are human pathogens. Thus, an inhibitor of this enzyme may be used as a powerful antifungal without side effects in patients once these enzymes are absent in humans. Here, we searched for compounds with inhibitory capacity against the malate synthase of Paracoccidioides species (PbMLS). The three-dimensional (3D) structure of PbMLS was determined using the I-TASSER server. Compounds were selected from the ZINC database. Based on the mechanism underlying the interaction of the compounds with PbMLS, it was possible to identify β-carboline moiety as a standard key structure. The compounds with β-carboline moiety that are available in our laboratories were investigated. A total of nine alkaloid compounds were selected. The primary mechanisms of interaction of the alkaloid compounds in the binding pocket of PbMLS were identified and compared with the mechanism of interaction of acetyl coenzyme A (acetyl-CoA). We discovered that the amphipathic nature of the compounds, concomitant with the presence of β-carboline moiety, was crucial for their stability in the binding pocket of PbMLS. In addition, the importance of a critical balance of the polar and nonpolar contacts of the compounds in this region was observed. Four β-carboline alkaloid compounds showed the ability to inhibit recombinant PbMLS (PbMLSr) activity, Paracoccidioides species growth, and adhesion of the fungus and PbMLSr to the extracellular matrix components. The cytotoxicity of the alkaloids was also evaluated.

  18. Effect of 16.16 dimethyl prostaglandin E2, N-acetyl-cysteine and the proton pump inhibitor BY 831-78 on hydrogen peroxide-induced mucosal damage in the rat stomach.

    PubMed

    Schürer-Maly, C C; Haussner, V; Halter, F

    1990-01-01

    Reactive oxygen species are noxious to gastrointestinal mucosa and contribute to a variety of gastrointestinal diseases. We examined whether 16.16 dimethyl prostaglandin E2 (PG) is protective against the oxidizing action of 6% H2O2 causing gross hemorrhagic lesions in rat gastric mucosa. Male Wistar rats were treated with PG, 0.005-5 micrograms/kg, either intragastrically (i.g.) or subcutaneously, 30 min prior to i.g. administration of 6% H2O2, 0.5 ml/100 g. Further animals received 25 mg of the mucus dissolvent N-acetyl-cystein (NAC) following oral PG treatment or 30 mumol/kg of the H+K(+)-ATPase inhibitor BY 831-78 (BY), 4 h before onset of the experiments. Volume, pH and beta-N-acetyl-glucosaminidase and lactate dehydrogenase as parameters of cell damage were determined in the gastric juice. i.g. PG treatment achieved 60 and 55% reduction of the mucosal lesions in doses between 5 and 0.05 micrograms/kg, respectively. i.p. PG administration was effective in all doses tested. Gastric juice volume was only slightly and enzymes were not significantly affected by PG treatment. NAC did not diminish PG efficacy or aggravate mucosal lesions. Gastric acid suppression did not increase PG-induced protection but was strongly protective by itself, reducing damage by 75%. Low-dose PG treatment achieves an effective protection against oxidative damage in gastric mucosa, which is not the result of dilution or enhanced mucus production. PMID:2147665

  19. Effect of 16.16 dimethyl prostaglandin E2, N-acetyl-cysteine and the proton pump inhibitor BY 831-78 on hydrogen peroxide-induced mucosal damage in the rat stomach.

    PubMed

    Schürer-Maly, C C; Haussner, V; Halter, F

    1990-01-01

    Reactive oxygen species are noxious to gastrointestinal mucosa and contribute to a variety of gastrointestinal diseases. We examined whether 16.16 dimethyl prostaglandin E2 (PG) is protective against the oxidizing action of 6% H2O2 causing gross hemorrhagic lesions in rat gastric mucosa. Male Wistar rats were treated with PG, 0.005-5 micrograms/kg, either intragastrically (i.g.) or subcutaneously, 30 min prior to i.g. administration of 6% H2O2, 0.5 ml/100 g. Further animals received 25 mg of the mucus dissolvent N-acetyl-cystein (NAC) following oral PG treatment or 30 mumol/kg of the H+K(+)-ATPase inhibitor BY 831-78 (BY), 4 h before onset of the experiments. Volume, pH and beta-N-acetyl-glucosaminidase and lactate dehydrogenase as parameters of cell damage were determined in the gastric juice. i.g. PG treatment achieved 60 and 55% reduction of the mucosal lesions in doses between 5 and 0.05 micrograms/kg, respectively. i.p. PG administration was effective in all doses tested. Gastric juice volume was only slightly and enzymes were not significantly affected by PG treatment. NAC did not diminish PG efficacy or aggravate mucosal lesions. Gastric acid suppression did not increase PG-induced protection but was strongly protective by itself, reducing damage by 75%. Low-dose PG treatment achieves an effective protection against oxidative damage in gastric mucosa, which is not the result of dilution or enhanced mucus production.

  20. A novel Pro197Glu substitution in acetolactate synthase (ALS) confers broad-spectrum resistance across ALS inhibitors.

    PubMed

    Liu, Weitang; Yuan, Guohui; Du, Long; Guo, Wenlei; Li, Lingxu; Bi, Yaling; Wang, Jinxin

    2015-01-01

    Water chickweed (Myosoton aquaticum L.), a competitive broadleaf weed, is widespread in wheat fields in China. Tribenuron and pyroxsulam failed to control water chickweed in the same field in Qiaotian Village in 2011 and 2012, respectively. An initial tribenuron resistance confirmation test identified a resistant population (AH02). ALS gene sequencing revealed a previously unreported substitution of Glu for Pro at amino acid position 197 in resistant individuals. A purified subpopulation (WRR04) that was individually homozygous for the Pro197Glu substitution was generated and characterized in terms of its response to different classes of ALS inhibitors. A whole-plant experiment showed that the WRR04 population exhibited broad-spectrum resistance to tribenuron (SU, 318-fold), pyrithiobac sodium (PTB, > 197-fold), pyroxsulam (TP, 81-fold), florasulam (TP, > 36-fold) and imazethapyr (IMI, 11-fold). An in vitro ALS assay confirmed that the ALS from WRR04 showed high resistance to all the tested ALS inhibitors. These results established that the Pro197Glu substitution endows broad-spectrum resistance across ALS inhibitors in water chickweed. In addition, molecular markers were developed to rapidly identify the Pro197Glu mutation.

  1. Molecular Docking Studies of Catechin and Its Derivatives as Anti-bacterial Inhibitor for Glucosamine-6-Phosphate Synthase

    NASA Astrophysics Data System (ADS)

    Fikrika, H.; Ambarsari, L.; Sumaryada, T.

    2016-01-01

    Molecular docking simulation of catechin and its derivatives on Glucosamine-6- Phosphate Synthase (GlmS) has been performed in this research. GlmS inhibition by a particular ligand will suppress the production of bacterial cell wall and significantly reduce the population of invading bacteria. In this study, catechin derivatives i.e epicatechin, galloatechin and epigalloatechin were found to have stronger binding affinities as compared to natural ligand of GlmS, Fructose-6-Phosphate (F6P). Those three ligands were docked on the same pocket in GlmS target as F6P, with 70% binding sites similarity. Based on the docking results, gallocatechin turns out to be the most potent ligand for anti-bacterial agent with ΔG= -8.00 kcal/mol. The docking between GlmS and catechin derivatives are characterized by a constant present of a strong hydrogen bond between functional group O3 and Ser-349. This hydrogen bond most likely plays a significant role in the docking mechanism and binding modes selection. The surprising result is catechin itself exhibited a quite strong binding with GlmS (ΔG= -7.80 kcal.mol), but docked on a completely different pocket compared to other ligands. This results suggest that catechin might still have a curing effect but with a completely different pathway and mechanism as compared to its derivatives.

  2. Human stem cell osteoblastogenesis mediated by novel glycogen synthase kinase 3 inhibitors induces bone formation and a unique bone turnover biomarker profile in rats

    SciTech Connect

    Gilmour, Peter S.; O'Shea, Patrick J.; Fagura, Malbinder; Pilling, James E.; Sanganee, Hitesh; Wada, Hiroki; Courtney, Paul F.; Kavanagh, Stefan; Hall, Peter A.; Escott, K. Jane

    2013-10-15

    Wnt activation by inhibiting glycogen synthase kinase 3 (GSK-3) causes bone anabolism in rodents making GSK-3 a potential therapeutic target for osteoporotic and osteolytic metastatic bone disease. To understand the wnt pathway related to human disease translation, the ability of 3 potent inhibitors of GSK-3 (AZD2858, AR79, AZ13282107) to 1) drive osteoblast differentiation and mineralisation using human adipose-derived stem cells (hADSC) in vitro; and 2) stimulate rat bone formation in vivo was investigated. Bone anabolism/resorption was determined using clinically relevant serum biomarkers as indicators of bone turnover and bone formation assessed in femurs by histopathology and pQCT/μCT imaging. GSK-3 inhibitors caused β-catenin stabilisation in human and rat mesenchymal stem cells, stimulated hADSC commitment towards osteoblasts and osteogenic mineralisation in vitro. AZD2858 produced time-dependent changes in serum bone turnover biomarkers and increased bone mass over 28 days exposure in rats. After 7 days, AZD2858, AR79 or AZ13282107 exposure increased the bone formation biomarker P1NP, and reduced the resorption biomarker TRAcP-5b, indicating increased bone anabolism and reduced resorption in rats. This biomarker profile was differentiated from anabolic agent PTH{sub 1–34} or the anti-resorptive Alendronate-induced changes. Increased bone formation in cortical and cancellous bone as assessed by femur histopathology supported biomarker changes. 14 day AR79 treatment increased bone mineral density and trabecular thickness, and decreased trabecular number and connectivity assessed by pQCT/μCT. GSK-3 inhibition caused hADSC osteoblastogenesis and mineralisation in vitro. Increased femur bone mass associated with changes in bone turnover biomarkers confirmed in vivo bone formation and indicated uncoupling of bone formation and resorption. - Highlights: • Wnt modulation with 3 novel GSK-3 inhibitors alters bone growth. • Human stem cell osteoblastogenesis

  3. Inhibition of GSK-3 reduces prostaglandin E2 production by decreasing the expression levels of COX-2 and mPGES-1 in monocyte/macrophage lineage cells.

    PubMed

    Noma, Toshihiro; Takahashi-Yanaga, Fumi; Arioka, Masaki; Mori, Yoshihide; Sasaguri, Toshiyuki

    2016-09-15

    Inflammatory stimuli induce prostaglandin E2 (PGE2) synthesis by upregulating cycloxgenase-2 (COX-2) and microsomal PGE synthase-1 (mPGES-1). Glycogen synthase kinase-3 (GSK-3) reportedly plays an important role in inflammatory reactions, whereas the role of this enzyme in inflammatory PGE2 production remains unclear. In the present study, therefore, we examined whether inhibition of GSK-3 can reduce inflammatory PGE2 production in vitro and in vivo. When macrophage-like cells differentiated from THP-1 were stimulated with lipopolysaccharide (LPS), PGE2 production and the expression levels of COX-2 and mPGES-1 were markedly elevated. GSK-3 inhibitors LiCl and SB216763 strongly suppressed their protein levels through inhibition of mRNA expressions. Subsequently, we examined the effect of GSK-3 inhibitors on nuclear factor κB (NF-κB) and early growth response-1 (Egr-1). The GSK-3 inhibitors had no significant effect on the NF-κB pathway, whereas they significantly decreased the expression level of Egr-1. Pharmacological and genetic inhibitions of GSK-3 also strongly suppressed PGE2 production in cultured peritoneal macrophages and in inflammatory air pouches made under the skin of living mice. These results suggested that GSK-3 plays a key role in PGE2 production by increasing COX-2 and mPGES-1 probably through Egr-1-mediated transcription and GSK-3 inhibitors may be potential as novel anti-inflammatory drugs. PMID:27453433

  4. Prostaglandin receptor EP4 in abdominal aortic aneurysms.

    PubMed

    Cao, Richard Y; St Amand, Tim; Li, XinZhi; Yoon, Sung-Hee; Wang, Carol P; Song, Hui; Maruyama, Takayuki; Brown, Peter M; Zelt, David T; Funk, Colin D

    2012-07-01

    Abdominal aortic aneurysm (AAA) pathogenesis is distinguished by vessel wall inflammation. Cyclooxygenase (COX)-2 and microsomal prostaglandin E synthase-1, key components of the most well-characterized inflammatory prostaglandin pathway, contribute to AAA development in the 28-day angiotensin II infusion model in mice. In this study, we used this model to examine the role of the prostaglandin E receptor subtype 4 (EP4) and genetic knockdown of COX-2 expression (70% to 90%) in AAA pathogenesis. The administration of the prostaglandin receptor EP4 antagonist AE3-208 (10 mg/kg per day) to apolipoprotein E (apoE)-deficient mice led to active drug plasma concentrations and reduced AAA incidence and severity compared with control apoE-deficient mice (P < 0.01), whereas COX-2 genetic knockdown/apoE-deficient mice displayed only a minor, nonsignificant decrease in incidence of AAA. EP4 receptor protein was present in human and mouse AAA, as observed by using Western blot analysis. Aortas from AE3-208-treated mice displayed evidence of a reduced inflammatory phenotype compared with controls. Atherosclerotic lesion size at the aortic root was similar between all groups. In conclusion, the prostaglandin E(2)-EP4 signaling pathway plays a role in the AAA inflammatory process. Blocking the EP4 receptor pharmacologically reduces both the incidence and severity of AAA in the angiotensin II mouse model, potentially via attenuation of cytokine/chemokine synthesis and the reduction of matrix metalloproteinase activities.

  5. Attenuation of acute nitrogen mustard-induced lung injury, inflammation and fibrogenesis by a nitric oxide synthase inhibitor

    SciTech Connect

    Malaviya, Rama; Venosa, Alessandro; Hall, LeRoy; Gow, Andrew J.; Sinko, Patrick J.; Laskin, Jeffrey D.; Laskin, Debra L.

    2012-12-15

    Nitrogen mustard (NM) is a toxic vesicant known to cause damage to the respiratory tract. Injury is associated with increased expression of inducible nitric oxide synthase (iNOS). In these studies we analyzed the effects of transient inhibition of iNOS using aminoguanidine (AG) on NM-induced pulmonary toxicity. Rats were treated intratracheally with 0.125 mg/kg NM or control. Bronchoalveolar lavage fluid (BAL) and lung tissue were collected 1 d–28 d later and lung injury, oxidative stress and fibrosis assessed. NM exposure resulted in progressive histopathological changes in the lung including multifocal lesions, perivascular and peribronchial edema, inflammatory cell accumulation, alveolar fibrin deposition, bronchiolization of alveolar septal walls, and fibrosis. This was correlated with trichrome staining and expression of proliferating cell nuclear antigen (PCNA). Expression of heme oxygenase (HO)-1 and manganese superoxide dismutase (Mn-SOD) was also increased in the lung following NM exposure, along with levels of protein and inflammatory cells in BAL, consistent with oxidative stress and alveolar-epithelial injury. Both classically activated proinflammatory (iNOS{sup +} and cyclooxygenase-2{sup +}) and alternatively activated profibrotic (YM-1{sup +} and galectin-3{sup +}) macrophages appeared in the lung following NM administration; this was evident within 1 d, and persisted for 28 d. AG administration (50 mg/kg, 2 ×/day, 1 d–3 d) abrogated NM-induced injury, oxidative stress and inflammation at 1 d and 3 d post exposure, with no effects at 7 d or 28 d. These findings indicate that nitric oxide generated via iNOS contributes to acute NM-induced lung toxicity, however, transient inhibition of iNOS is not sufficient to protect against pulmonary fibrosis. -- Highlights: ► Nitrogen mustard (NM) induces acute lung injury and fibrosis. ► Pulmonary toxicity is associated with increased expression of iNOS. ► Transient inhibition of iNOS attenuates acute

  6. Promotion of purine nucleotide binding to thymidylate synthase by a potent folate analogue inhibitor, 1843U89.

    PubMed Central

    Weichsel, A; Montfort, W R; Cieśla, J; Maley, F

    1995-01-01

    A folate analogue, 1843U89 (U89), with potential as a chemotherapeutic agent due to its potent and specific inhibition of thymidylate synthase (TS; EC 2.1.1.45), greatly enhances not only the binding of 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) and dUMP to Escherichia coli TS but also that of dGMP, GMP, dIMP, and IMP. Guanine nucleotide binding was first detected by CD analysis, which revealed a unique spectrum for the TS-dGMP-U89 ternary complex. The quantitative binding of dGMP relative to GMP, FdUMP, and dUMP was determined in the presence and absence of U89 by ultrafiltration analysis, which revealed that although the binding of GMP and dGMP could not be detected in the absence of U89 both were bound in its presence. The Kd for dGMP was about the same as that for dUMP and FdUMP, with binding of the latter two nucleotides being increased by two orders of magnitude by U89. An explanation for the binding of dGMP was provided by x-ray diffraction studies that revealed an extensive stacking interaction between the guanine of dGMP and the benzoquinazoline ring of U89 and hydrogen bonds similar to those involved in dUMP binding. In addition, binding energy was provided through a water molecule that formed hydrogen bonds to both N7 of dGMP and the hydroxyl of Tyr-94. Accommodation of the larger dGMP molecule was accomplished through a distortion of the active site and a shift of the deoxyribose moiety to a new position. These rearrangements also enabled the binding of GMP to occur by creating a pocket for the ribose 2' hydroxyl group, overcoming the normal TS discrimination against nucleotides containing the 2' hydroxyl. Images Fig. 3 Fig. 4 Fig. 5 PMID:7724588

  7. Identification and in vitro evaluation of new leads as selective and competitive glycogen synthase kinase-3β inhibitors through ligand and structure based drug design.

    PubMed

    Darshit, B S; Balaji, B; Rani, P; Ramanathan, M

    2014-09-01

    Glycogen synthase kinase-3β elicits multi-functional effects on intracellular signaling pathways, thereby making the kinase a therapeutic target in multiple pathologies. Hence, it is important to selectively inhibit GSK-3β over structurally and biologically similar targets, such as CDK5. The current study was designed to identify and evaluate novel ATP-competitive GSK-3β inhibitors. The study was designed to identify new leads by ligand based drug design, structure based drug design and in vitro evaluation. The best validated pharmacophore model (AADRRR) identified using LBDD was derived from a dataset of 135 molecules. There were 357 primary hits within the SPECS database using this pharmacophore model. A SBDD approach to the GSK-3β and CDK5 proteins was applied to all primary hits, and 5 selective inhibitors were identified for GSK-3β. GSK-3β and CDK5 in vitro kinase inhibition assays were performed with these molecules to confirm their selectivity for GSK-3β. The molecules showed IC50 values ranging from 0.825μM to 1.116μM and were 23- to 57-fold selective for GSK-3β. Of all the molecules, molecule 3 had the lowest IC50 value of 0.825μM. Our research identified molecules possessing benzothiophene, isoquinoline, thiazolidinedione imidazo-isoquinoline and quinazolinone scaffolds. Potency of these molecules may be due to H-bond interaction with backbone residues of Val135, Asp133 and side chain interaction with Tyr134. Selectivity over CDK5 may be due to side chain interactions with Asp200, backbone of Val61, ionic interaction with Lys60 and π-cationic interaction with Arg141. These selective molecules were also exhibited small atom hydrophobicity and H-bond interaction with water molecule.

  8. Increased anticancer activity of the thymidylate synthase inhibitor BGC9331 combined with the topoisomerase I inhibitor SN-38 in human colorectal and breast cancer cells: induction of apoptosis and ROCK cleavage through caspase-3-dependent and -independent mechanisms.

    PubMed

    Coudray, Anne-Marie; Louvet, Christophe; Kornprobst, Michel; Raymond, Eric; André, Thierry; Tournigand, Christophe; Faivre, Sandrine; De Gramont, Aimery; Larsen, Annette K; Gespach, Christian

    2005-08-01

    The folate analogue BGC9331 is a new thymidylate synthase (TS) inhibitor showing a broad spectrum of cyto-toxic activity against several human solid tumors, including colorectal cancer. In this study, we investigated the anticancer activity of BGC9331 either alone or combined with 5-fluorouracil (5-FU), MTA (multi-target antifolate), oxali-platin and SN-38, the active metabolite of the topoisomerase I inhibitor CPT-11. The antiproliferative activity of each drug and BGC9331-based combinations was investigated in the HT-29 human colorectal cancer cell line and its HT-29/5-FU counterparts selected for resistance to 5-FU. BGC9331 combined with MTA or SN-38 induced synergistic responses in HT-29 cells. Treatment of HT-29 cells with either BGC9331 or SN-38 increased caspase-3 activity and the percentage of apoptotic cells from 3 to 13%. Both drugs also augmented the proteolytic cleavage of the Rho-kinase ROCK-1 that was attenuated by the caspase-3 pathway inhibitor z-DEVD-fmk. BGC9331 combined with SN-38 further increased the percentage of apoptotic cells to 25%, and inhibited cell cycle progression and cell proliferation by 65%. This was accompanied by proteolytic activation of ROCK-1, through both caspase-3-dependent and -independent mechanisms, as shown in caspase-3-deficient MCF-7 breast cancer cells. These encouraging results warrant further preclinical investigations and clinical trials on the use of BGC9331 combined with SN-38/CPT-11 in treatment of patients with advanced colorectal or gastric cancers.

  9. Lapatinib, a Dual EGFR and HER2 Tyrosine Kinase Inhibitor, Downregulates Thymidylate Synthase by Inhibiting the Nuclear Translocation of EGFR and HER2

    PubMed Central

    Kim, Hwang-Phill; Yoon, Young-Kwang; Kim, Jin-Won; Han, Sae-Won; Hur, Hyung-Seok; Park, Jinah; Lee, Ju-Hee; Oh, Do-Youn; Im, Seock-Ah; Bang, Yung-Jue; Kim, Tae-You

    2009-01-01

    Background Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) has been shown to exert a synergistic antitumor effect when combined with fluoropyrimidine. This synergy may be attributable to the downregulation of thymidylate synthase (TS), which is frequently overexpressed in fluoropyrimidine-resistant cancer cells. However, the molecular mechanism underlying the downregulation of TS has yet to be clearly elucidated. Methodology and Principal Findings In this study, we demonstrate that lapatinib, a dual TKI of EGFR and HER2 downregulates TS via inhibition of the nuclear translocation of EGFR and HER2. From our cDNA microarray experiments, we determined that a variety of nucleotide synthesis-related genes, including TS, were downregulated with lapatinib, and this was apparent in HER2-amplified cells. Targeted and pharmacologic inhibition assays confirmed that the dual inhibition of EGFR and HER2 is required for the more effective reduction of TS as compared to what was observed with gefitinib or trasutuzumab alone. Additionally, we determined that co-transfected EGFR and HER2 activate the TS gene promoter more profoundly than do either EGFR or HER2 alone. The translocation of EGFR and HER2 into the nucleus and the subsequent activation of the TS promoter were inhibited by lapatinib. Conclusions and Significance These results demonstrate that lapatinib inhibits the nuclear translocation of EGFR and HER2 and downregulates TS, thus sensitizing cancer cells to fluoropyrimidine. PMID:19529774

  10. A quantitative structure-activity relationship (QSAR) study on a few series of potent, highly selective inhibitors of nitric oxide synthase.

    PubMed

    Bharti, Vishwa Deepak; Gupta, Satya P; Kumar, Harish

    2014-02-01

    QSAR study was performed on a series of 1,2-dihydro-4-quinazolinamines, 4,5-dialkylsubstituted-2-imino-1,3-thiazolidine derivatives and 4,5-disubstituted-1,3-oxazolidin-2-imine derivatives studied by Tinker et al. [J Med Chem (2003), 46, 913-916], Ueda et al. [Bioorg Med Chem (2004) 12, 4101-4116] and Ueda et al. [Bioorg Med Chem Lett (2004) 14, 313-316], respectively, as potent, highly selective inhibitors of inducible nitric oxide synthase (iNOS). The iNOS inhibition activity of the whole series of compounds was analyzed in relation to the physicochemical and molecular properties of the compounds. The QSAR analysis revealed that the inhibition potency of the compounds was controlled by a topological parameter 1chi(v) (Kier's first order valence molecular connectivity index), density (D), surface tension (St) and length (steric parameter) of a substituent. This suggested that the drug-receptor interaction predominantly involved the dispersion interaction, but the bulky molecule would face steric problem because of which the molecule may not completely fit in active sites of the receptor and thus may not have the optimum interaction.

  11. Characterization of 5-aminolevulinate synthase from Agrobacterium radiobacter, screening new inhibitors for 5-aminolevulinate dehydratase from Escherichia coli and their potential use for high 5-aminolevulinate production.

    PubMed

    Lin, Jianping; Fu, Weiqi; Cen, Peilin

    2009-04-01

    The hemA gene encoding 5-aminolevulinate synthase (ALAS) from Agrobacterium radiobacter zju-0121 showed 92.6% homology with that from A. radiobacter ATCC4718 and contained several rare codons. To enhance the expression of this gene, Escherichia coli Rosetta(DE3), which is a rare codon optimizer strain, was used as the host to construct an efficient recombinant strain. And the encoded protein was over-expressed as fusion protein and was purified by affinity purification on Ni-NTA agarose and by gel filtration chromatography on Sephadex G-25 Medium resin. The recombinant protein was partly characterized, and D-glucose, D-fructose, D-xylose, D-mannose, L-arabinose, D-galactose, lactose, sucrose and maltose were detected to have no distinct inhibition on this recombinant ALAS. Meanwhile, 20mM D-glucose or D-xylose inhibited about 20% activity of ALA dehydratase (ALAD) from Escherichia coli Rosetta(DE3). Combining D-xylose as a new inhibitor for ALAD with D-glucose in fed-batch culture and based on the optimal culture system using Rosetta(DE3)/pET28a-hemA, the yield of ALA achieved was 7.3g/l (56 mM) under the appropriate conditions in the fermenter.

  12. The neuronal nitric oxide synthase inhibitor NANT blocks acetaminophen toxicity and protein nitration in freshly isolated hepatocytes.

    PubMed

    Banerjee, Sudip; Melnyk, Stepan B; Krager, Kimberly J; Aykin-Burns, Nukhet; Letzig, Lynda G; James, Laura P; Hinson, Jack A

    2015-12-01

    3-Nitrotyrosine (3NT) in liver proteins of mice treated with hepatotoxic doses of acetaminophen (APAP) has been postulated to be causative in toxicity. Nitration is by a reactive nitrogen species formed from nitric oxide (NO). The source of the NO is unclear. iNOS knockout mice were previously found to be equally susceptible to APAP toxicity as wildtype mice and iNOS inhibitors did not decrease toxicity in mice or in hepatocytes. In this work we examined the potential role of nNOS in APAP toxicity in hepatocytes using the specific nNOS inhibitor NANT (10 µM)(N-[(4S)-4-amino-5-[(2-aminoethyl)amino]pentyl]-N'-nitroguanidinetris (trifluoroacetate)). Primary hepatocytes (1 million/ml) from male B6C3F1 mice were incubated with APAP (1mM). Cells were removed and assayed spectrofluorometrically for reactive nitrogen and oxygen species using diaminofluorescein (DAF) and Mitosox red, respectively. Cytotoxicity was determined by LDH release into media. Glutathione (GSH, GSSG), 3NT, GSNO, acetaminophen-cysteine adducts, NAD, and NADH were measured by HPLC. APAP significantly increased cytotoxicity at 1.5-3.0 h. The increase was blocked by NANT. NANT did not alter APAP mediated GSH depletion or acetaminophen-cysteine adducts in proteins which indicated that NANT did not inhibit metabolism. APAP significantly increased spectroflurometric evidence of reactive nitrogen and oxygen formation at 0.5 and 1.0 h, respectively, and increased 3NT and GSNO at 1.5-3.0 h. These increases were blocked by NANT. APAP dramatically increased NADH from 0.5-3.0 h and this increase was blocked by NANT. Also, APAP decreased the Oxygen Consumption Rate (OCR), decreased ATP production, and caused a loss of mitochondrial membrane potential, which were all blocked by NANT. PMID:26454079

  13. The neuronal nitric oxide synthase inhibitor NANT blocks acetaminophen toxicity and protein nitration in freshly isolated hepatocytes.

    PubMed

    Banerjee, Sudip; Melnyk, Stepan B; Krager, Kimberly J; Aykin-Burns, Nukhet; Letzig, Lynda G; James, Laura P; Hinson, Jack A

    2015-12-01

    3-Nitrotyrosine (3NT) in liver proteins of mice treated with hepatotoxic doses of acetaminophen (APAP) has been postulated to be causative in toxicity. Nitration is by a reactive nitrogen species formed from nitric oxide (NO). The source of the NO is unclear. iNOS knockout mice were previously found to be equally susceptible to APAP toxicity as wildtype mice and iNOS inhibitors did not decrease toxicity in mice or in hepatocytes. In this work we examined the potential role of nNOS in APAP toxicity in hepatocytes using the specific nNOS inhibitor NANT (10 µM)(N-[(4S)-4-amino-5-[(2-aminoethyl)amino]pentyl]-N'-nitroguanidinetris (trifluoroacetate)). Primary hepatocytes (1 million/ml) from male B6C3F1 mice were incubated with APAP (1mM). Cells were removed and assayed spectrofluorometrically for reactive nitrogen and oxygen species using diaminofluorescein (DAF) and Mitosox red, respectively. Cytotoxicity was determined by LDH release into media. Glutathione (GSH, GSSG), 3NT, GSNO, acetaminophen-cysteine adducts, NAD, and NADH were measured by HPLC. APAP significantly increased cytotoxicity at 1.5-3.0 h. The increase was blocked by NANT. NANT did not alter APAP mediated GSH depletion or acetaminophen-cysteine adducts in proteins which indicated that NANT did not inhibit metabolism. APAP significantly increased spectroflurometric evidence of reactive nitrogen and oxygen formation at 0.5 and 1.0 h, respectively, and increased 3NT and GSNO at 1.5-3.0 h. These increases were blocked by NANT. APAP dramatically increased NADH from 0.5-3.0 h and this increase was blocked by NANT. Also, APAP decreased the Oxygen Consumption Rate (OCR), decreased ATP production, and caused a loss of mitochondrial membrane potential, which were all blocked by NANT.

  14. Reducing prostaglandin E2 production to raise cancer immunogenicity.

    PubMed

    Zelenay, Santiago; Reis E Sousa, Caetano

    2016-05-01

    Cyclooxygenases (COX), commonly upregulated in numerous cancers, generate prostaglandin E2 (PGE2), which has been implicated in key aspects of malignant growth including proliferation, invasion and angiogenesis. Recently, we showed that production of PGE2 by cancer cells dominantly enables progressive tumor growth via immune escape and that cyclooxygenase inhibitors synergize with immunotherapy to enhance tumor eradication. PMID:27467936

  15. Inhibitor of neuronal nitric oxide synthase improves gas exchange in ventilator-induced lung injury after pneumonectomy

    PubMed Central

    2012-01-01

    Background Mechanical ventilation with high tidal volumes may cause ventilator-induced lung injury (VILI) and enhanced generation of nitric oxide (NO). We demonstrated in sheep that pneumonectomy followed by injurious ventilation promotes pulmonary edema. We wished both to test the hypothesis that neuronal NOS (nNOS), which is distributed in airway epithelial and neuronal tissues, could be involved in the pathogenesis of VILI and we also aimed at investigating the influence of an inhibitor of nNOS on the course of VILI after pneumonectomy. Methods Anesthetized sheep underwent right pneumonectomy, mechanical ventilation with tidal volumes (VT) of 6 mL/kg and FiO2 0.5, and were subsequently randomized to a protectively ventilated group (PROTV; n = 8) keeping VT and FiO2 unchanged, respiratory rate (RR) 25 inflations/min and PEEP 4 cm H2O for the following 8 hrs; an injuriously ventilated group with VT of 12 mL/kg, zero end-expiratory pressure, and FiO2 and RR unchanged (INJV; n = 8) and a group, which additionally received the inhibitor of nNOS, 7-nitroindazole (NI) 1.0 mg/kg/h intravenously from 2 hours after the commencement of injurious ventilation (INJV + NI; n = 8). We assessed respiratory, hemodynamic and volumetric variables, including both the extravascular lung water index (EVLWI) and the pulmonary vascular permeability index (PVPI). We measured plasma nitrite/nitrate (NOx) levels and examined lung biopsies for lung injury score (LIS). Results Both the injuriously ventilated groups demonstrated a 2–3-fold rise in EVLWI and PVPI, with no significant effects of NI. In the INJV group, gas exchange deteriorated in parallel with emerging respiratory acidosis, but administration of NI antagonized the derangement of oxygenation and the respiratory acidosis significantly. NOx displayed no significant changes and NI exerted no significant effect on LIS in the INJV group. Conclusion Inhibition of nNOS improved gas exchange, but did not

  16. Development of potent and selective indomethacin analogues for the inhibition of AKR1C3 (Type 5 17β-hydroxysteroid dehydrogenase/prostaglandin F synthase) in castrate-resistant prostate cancer.

    PubMed

    Liedtke, Andy J; Adeniji, Adegoke O; Chen, Mo; Byrns, Michael C; Jin, Yi; Christianson, David W; Marnett, Lawrence J; Penning, Trevor M

    2013-03-28

    Castrate-resistant prostate cancer (CRPC) is a fatal, metastatic form of prostate cancer. CRPC is characterized by reactivation of the androgen axis due to changes in androgen receptor signaling and/or adaptive intratumoral androgen biosynthesis. AKR1C3 is upregulated in CRPC where it catalyzes the formation of potent androgens. This makes AKR1C3 a target for the treatment of CRPC. AKR1C3 inhibitors should not inhibit AKR1C1/AKR1C2, which inactivate 5α-dihydrotestosterone. Indomethacin, used to inhibit cyclooxygenase, also inhibits AKR1C3 and displays selectivity over AKR1C1/AKR1C2. Parallel synthetic strategies were used to generate libraries of indomethacin analogues, which exhibit reduced cyclooxygenase inhibitory activity but retain AKR1C3 inhibitory potency and selectivity. The lead compounds inhibited AKR1C3 with nanomolar potency, displayed >100-fold selectivity over AKR1C1/AKR1C2, and blocked testosterone formation in LNCaP-AKR1C3 cells. The AKR1C3·NADP(+)·2'-des-methyl-indomethacin crystal structure was determined, and it revealed a unique inhibitor binding mode. The compounds reported are promising agents for the development of therapeutics for CRPC.

  17. Development of potent and selective indomethacin analogues for the inhibition of AKR1C3 (Type 5 17β-hydroxysteroid dehydrogenase/prostaglandin F synthase) in castrate-resistant prostate cancer.

    PubMed

    Liedtke, Andy J; Adeniji, Adegoke O; Chen, Mo; Byrns, Michael C; Jin, Yi; Christianson, David W; Marnett, Lawrence J; Penning, Trevor M

    2013-03-28

    Castrate-resistant prostate cancer (CRPC) is a fatal, metastatic form of prostate cancer. CRPC is characterized by reactivation of the androgen axis due to changes in androgen receptor signaling and/or adaptive intratumoral androgen biosynthesis. AKR1C3 is upregulated in CRPC where it catalyzes the formation of potent androgens. This makes AKR1C3 a target for the treatment of CRPC. AKR1C3 inhibitors should not inhibit AKR1C1/AKR1C2, which inactivate 5α-dihydrotestosterone. Indomethacin, used to inhibit cyclooxygenase, also inhibits AKR1C3 and displays selectivity over AKR1C1/AKR1C2. Parallel synthetic strategies were used to generate libraries of indomethacin analogues, which exhibit reduced cyclooxygenase inhibitory activity but retain AKR1C3 inhibitory potency and selectivity. The lead compounds inhibited AKR1C3 with nanomolar potency, displayed >100-fold selectivity over AKR1C1/AKR1C2, and blocked testosterone formation in LNCaP-AKR1C3 cells. The AKR1C3·NADP(+)·2'-des-methyl-indomethacin crystal structure was determined, and it revealed a unique inhibitor binding mode. The compounds reported are promising agents for the development of therapeutics for CRPC. PMID:23432095

  18. Inhibitors

    MedlinePlus

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

  19. Effects of S-ethylisothiourea, a potent inhibitor of nitric oxide synthase, alone or in combination with a nitric oxide donor in splanchnic artery occlusion shock.

    PubMed Central

    Squadrito, F.; Altavilla, D.; Squadrito, G.; Campo, G. M.; Ioculano, M.; Canale, P.; Rossi, F.; Saitta, A.; Caputi, A. P.

    1996-01-01

    1. The aim of this study was to compare the effects of an intravenous infusion of a potent and non selective nitric oxide synthase inhibitor S-ethylisothiourea (Ethyl-TU) with that of a nitric oxide (NO) donor on the pathological sequelae associated with splanchnic artery occlusion (SAO) shock. In addition the effects of the combination of these two treatments were also investigated. 2. SAO shock was induced in anaesthetized rats by clamping splanchnic arteries for 45 min. Sham operated animals were used as controls. Survival time, white blood cell (WBC) count, mean arterial blood pressure, myeloperoxidase activity (MPO; studied as a quantitative means to evaluate neutrophil accumulation) and the responsiveness of aortic rings to acetylcholine (ACh, 10 nM-10 microM) and to phenylephrine (PE, 1 nM-10 microM) were studied. 3. SAO shocked rats had a decreased survival rate (0% survival 2 h after the release of occlusion) and survival time (76 +/- 10 min), increased MPO activity in the ileum (3.39 +/- 0.8 u x 10(-3) g-1 tissue), a marked leukopenia and a profound hypotension. In addition aortic rings from shocked rats showed a marked hyporeactivity to PE and reduced responsiveness to ACh. Endothelium denuded aortic rings had also a marked hyporeactivity to PE. 4. In vivo administration of Ethyl-TU (0.1 mg kg-1 h-1, beginning 1 min after the onset of reperfusion) significantly increased survival time and rate, improved mean arterial blood pressure, restored the responsiveness to PE, but did not change MPO activity, leukopenia or the impairment in the responsiveness of aortic rings to ACh. Addition of Ethyl-TU (2 microM) to endothelium denuded aortic rings in vitro, restored the marked hyporeactivity to PE. Administration of the NO donor C87-3754 (0.75 mg kg-1 h-1, beginning 1 min after the onset of reperfusion) slightly increased survival time and reduced MPO activity and leukopenia, but did not change survival rate and mean arterial blood pressure. In addition C87

  20. The Nitric Oxide Synthase Inhibitor NG-Nitro-L-Arginine Methyl Ester Diminishes the Immunomodulatory Effects of Parental Arginine in Rats with Subacute Peritonitis.

    PubMed

    Lo, Hui-Chen; Hung, Ching-Yi; Huang, Fu-Huan; Su, Tzu-Cheng; Lee, Chien-Hsing

    2016-01-01

    The combined treatment of parenteral arginine and the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) have been shown to improve liver function and systemic inflammation in subacute peritonitic rats. Here, we investigated the effects of single and combined parenteral arginine and L-NAME treatments on leukocyte and splenocyte immunity. Male Wistar rats were subjected to cecal punctures and were intravenously given total parenteral nutrition solutions with or without arginine and/or L-NAME supplementations for 7 days. Non-surgical and sham-operated rats with no cecal puncture were given a chow diet and parenteral nutrition, respectively. Parenteral feeding elevated the white blood cell numbers and subacute peritonitis augmented the parenteral nutrition-induced alterations in the loss of body weight gain, splenomegaly, and splenocyte decreases. Parenteral arginine significantly increased the B-leukocyte level, decreased the natural killer T (NKT)-leukocyte and splenocyte levels, alleviated the loss in body weight gain and total and cytotoxic T-splenocyte levels, and attenuated the increases in plasma nitrate/nitrite and interferon-gamma production by T-splenocytes. L-NAME infusion significantly decreased NKT-leukocyte level, tumor-necrosis factor (TNF)-alpha production by T-splenocytes and macrophages, and interferon-gamma production by T-leukocytes, monocytes, and T-splenocytes, as well as increased interleukin-6 production by T-leukocytes and monocytes and nitrate/nitrite production by T-leukocytes. Combined treatment significantly decreased plasma nitrate/nitrite, the NKT-leukocyte level, and TNF-alpha production by T-splenocytes. Parenteral arginine may attenuate immune impairment and L-NAME infusion may augment leukocyte proinflammatory response, eliminate splenocyte proinflammatory and T-helper 1 responses, and diminish arginine-induced immunomodulation in combined treatment in subacute peritonitic rats.

  1. Resistance of a Rodent Malaria Parasite to a Thymidylate Synthase Inhibitor Induces an Apoptotic Parasite Death and Imposes a Huge Cost of Fitness

    PubMed Central

    Muregi, Francis W.; Ohta, Isao; Masato, Uchijima; Kino, Hideto; Ishih, Akira

    2011-01-01

    Background The greatest impediment to effective malaria control is drug resistance in Plasmodium falciparum, and thus understanding how resistance impacts on the parasite's fitness and pathogenicity may aid in malaria control strategy. Methodology/Principal Findings To generate resistance, P. berghei NK65 was subjected to 5-fluoroorotate (FOA, an inhibitor of thymidylate synthase, TS) pressure in mice. After 15 generations of drug pressure, the 2% DT (the delay time for proliferation of parasites to 2% parasitaemia, relative to untreated wild-type controls) reduced from 8 days to 4, equalling the controls. Drug sensitivity studies confirmed that FOA-resistance was stable. During serial passaging in the absence of drug, resistant parasite maintained low growth rates (parasitaemia, 15.5%±2.9, 7 dpi) relative to the wild-type (45.6%±8.4), translating into resistance cost of fitness of 66.0%. The resistant parasite showed an apoptosis-like death, as confirmed by light and transmission electron microscopy and corroborated by oligonucleosomal DNA fragmentation. Conclusions/Significance The resistant parasite was less fit than the wild-type, which implies that in the absence of drug pressure in the field, the wild-type alleles may expand and allow drugs withdrawn due to resistance to be reintroduced. FOA resistance led to depleted dTTP pools, causing thymineless parasite death via apoptosis. This supports the tenet that unicellular eukaryotes, like metazoans, also undergo apoptosis. This is the first report where resistance to a chemical stimulus and not the stimulus itself is shown to induce apoptosis in a unicellular parasite. This finding is relevant in cancer therapy, since thymineless cell death induced by resistance to TS-inhibitors can further be optimized via inhibition of pyrimidine salvage enzymes, thus providing a synergistic impact. We conclude that since apoptosis is a process that can be pharmacologically modulated, the parasite's apoptotic machinery may

  2. Discovery of novel, non-acidic mPGES-1 inhibitors by virtual screening with a multistep protocol

    PubMed Central

    Noha, Stefan M.; Fischer, Katrin; Koeberle, Andreas; Garscha, Ulrike; Werz, Oliver; Schuster, Daniela

    2015-01-01

    Microsomal prostaglandin E2 synthase-1 (mPGES-1) inhibitors are considered as potential therapeutic agents for the treatment of inflammatory pain and certain types of cancer. So far, several series of acidic as well as non-acidic inhibitors of mPGES-1 have been discovered. Acidic inhibitors, however, may have issues, such as loss of potency in human whole blood and in vivo, stressing the importance of the design and identification of novel, non-acidic chemical scaffolds of mPGES-1 inhibitors. Using a multistep virtual screening protocol, the Vitas-M compound library (∼1.3 million entries) was filtered and 16 predicted compounds were experimentally evaluated in a biological assay in vitro. This approach yielded two molecules active in the low micromolar range (IC50 values: 4.5 and 3.8 μM, respectively). PMID:26088337

  3. “Zipped Synthesis” by Cross-Metathesis Provides a Cystathionine β-Synthase Inhibitor that Attenuates Cellular H2S Levels and Reduces Neuronal Infarction in a Rat Ischemic Stroke Model

    PubMed Central

    2016-01-01

    The gaseous neuromodulator H2S is associated with neuronal cell death pursuant to cerebral ischemia. As cystathionine β-synthase (CBS) is the primary mediator of H2S biogenesis in the brain, it has emerged as a potential target for the treatment of stroke. Herein, a “zipped” approach by alkene cross-metathesis into CBS inhibitor candidate synthesis is demonstrated. The inhibitors are modeled after the pseudo-C2-symmetric CBS product (l,l)-cystathionine. The “zipped” concept means only half of the inhibitor needs be constructed; the two halves are then fused by olefin cross-metathesis. Inhibitor design is also mechanism-based, exploiting the favorable kinetics associated with hydrazine-imine interchange as opposed to the usual imine–imine interchange. It is demonstrated that the most potent “zipped” inhibitor 6S reduces H2S production in SH-SY5Y cells overexpressing CBS, thereby reducing cell death. Most importantly, CBS inhibitor 6S dramatically reduces infarct volume (1 h post-stroke treatment; ∼70% reduction) in a rat transient middle cerebral artery occlusion model for ischemia. PMID:27163055

  4. "Zipped Synthesis" by Cross-Metathesis Provides a Cystathionine β-Synthase Inhibitor that Attenuates Cellular H2S Levels and Reduces Neuronal Infarction in a Rat Ischemic Stroke Model.

    PubMed

    McCune, Christopher D; Chan, Su Jing; Beio, Matthew L; Shen, Weijun; Chung, Woo Jin; Szczesniak, Laura M; Chai, Chou; Koh, Shu Qing; Wong, Peter T-H; Berkowitz, David B

    2016-04-27

    The gaseous neuromodulator H2S is associated with neuronal cell death pursuant to cerebral ischemia. As cystathionine β-synthase (CBS) is the primary mediator of H2S biogenesis in the brain, it has emerged as a potential target for the treatment of stroke. Herein, a "zipped" approach by alkene cross-metathesis into CBS inhibitor candidate synthesis is demonstrated. The inhibitors are modeled after the pseudo-C 2-symmetric CBS product (l,l)-cystathionine. The "zipped" concept means only half of the inhibitor needs be constructed; the two halves are then fused by olefin cross-metathesis. Inhibitor design is also mechanism-based, exploiting the favorable kinetics associated with hydrazine-imine interchange as opposed to the usual imine-imine interchange. It is demonstrated that the most potent "zipped" inhibitor 6S reduces H2S production in SH-SY5Y cells overexpressing CBS, thereby reducing cell death. Most importantly, CBS inhibitor 6S dramatically reduces infarct volume (1 h post-stroke treatment; ∼70% reduction) in a rat transient middle cerebral artery occlusion model for ischemia. PMID:27163055

  5. Misidentification of prostamides as prostaglandins.

    PubMed

    Glass, Michelle; Hong, Jiwon; Sato, Timothy A; Mitchell, Murray D

    2005-07-01

    Prostaglandins and endogenous cannabinoid metabolites share the same lipid backbone with differing polar head groups at exactly the position through which a large molecule is attached to provide antigenicity and thus raise antisera. Hence, we hypothesized that antisera raised against prostaglandins linked to a large molecule such as BSA at the carboxyl functional group would also recognize endogenous cannabinoid metabolites and lead to highly misleading interpretations of data. We found major cross-reactivity of commercial antisera raised to prostaglandins with endocannabinoid metabolites. Furthermore, in a well-characterized cell line (WISH) or primary amnion tissue explants, endocannabinoid treatment led to increased production of endocannabinoid metabolites as opposed to primary prostaglandins. This was apparent only after separation of products by thin-layer chromatography, because they measured as prostaglandins by radioimmunoassay. These findings have major implications for our interpretation of data in situations in which these prostaglandin-like molecules are formed, and they stress the need for chromatographic or spectrometric confirmation of prostaglandin production detected by antibody-based methods. PMID:15863842

  6. A bilateral antidiuresis to renal artery infusion of prostaglandin E1 in dogs treated with phenylbutazone

    PubMed Central

    Hall, W. J.; Hensey, O. J.; O'Neill, P.; Sheehan, J. D.

    1978-01-01

    1. In acute experiments, high levels of endogenous prostaglandins, provoked by operative stress, could obscure or alter the actions of infused prostaglandins on the kidney. For this reason we decided to compare the effects of infusing prostaglandin E1 into the renal artery of the dog before and after the administration of phenylbutazone, a prostaglandin synthetase inhibitor. 2. Infusion of prostaglandin E1 into the left renal artery of the pre-phenylbutazone treated dog undergoing a mannitol diuresis increased renal plasma flow, glomerular filtration rate and the excretion of salt and water. The findings are in general agreement with those reported by others. 3. Following phenylbutazone administration the vascular and saluretic actions of prostaglandin E1 were unchanged but a reduced diuretic effect was observed. The response to a low dose of prostaglandin E1 (0·05 μg/min) was reduced from 1·46 ± 0·15 to 0·96 ± 0·16 ml./min (P < 0·001) and the response to a high dose (0·5 μg/min) from 1·82 ± 0·19 to 0·99 ± 0·31 ml./min (P < 0·002). 4. A significantly less dilute urine was excreted during prostaglandin infusion in the dog after phenylbutazone treatment than before. The reduction in the diuretic response was of the same order as the decrease in the free water clearance response, while the increase in osmolar clearance was unchanged. 5. In water-loaded dogs treated with phenylbutazone, infusion of prostaglandin E1 into the left renal artery had a biphasic effect on urine output from the left kidney. An initial diuretic response to a low dose of prostaglandin E1 disappeared with the infusion of higher doses, and antidiuresis developed in the immediate post-infusion period. 6. As prostaglandin was infused into the left kidney progressive antidiuresis was seen in the non-infused right kidney. 7. It is concluded that endogenous prostaglandins do not obscure or alter the vascular and saluretic actions of intrarenal prostaglandin E1. The findings question

  7. Nitric oxide donors prevent while the nitric oxide synthase inhibitor L-NAME increases arachidonic acid plus CYP2E1-dependent toxicity

    SciTech Connect

    Wu Defeng; Cederbaum, Arthur . E-mail: arthur.cederbaum@mssm.edu

    2006-10-15

    Polyunsaturated fatty acids such as arachidonic acid (AA) play an important role in alcohol-induced liver injury. AA promotes toxicity in rat hepatocytes with high levels of cytochrome P4502E1 and in HepG2 E47 cells which express CYP2E1. Nitric oxide (NO) participates in the regulation of various cell activities as well as in cytotoxic events. NO may act as a protectant against cytotoxic stress or may enhance cytotoxicity when produced at elevated concentrations. The goal of the current study was to evaluate the effect of endogenously or exogenously produced NO on AA toxicity in liver cells with high expression of CYP2E1 and assess possible mechanisms for its actions. Pyrazole-induced rat hepatocytes or HepG2 cells expressing CYP2E1 were treated with AA in the presence or absence of an inhibitor of nitric oxide synthase L-N {sup G}-Nitroarginine Methylester (L-NAME) or the NO donors S-nitroso-N-acetylpenicillamine (SNAP), and (Z)-1-[-(2-aminoethyl)-N-(2-aminoethyl)]diazen-1-ium-1,2-diolate (DETA-NONO). AA decreased cell viability from 100% to 48 {+-} 6% after treatment for 48 h. In the presence of L-NAME, viability was further lowered to 23 {+-} 5%, while, SNAP or DETA-NONO increased viability to 66 {+-} 8 or 71 {+-} 6%. The L-NAME potentiated toxicity was primarily necrotic in nature. L-NAME did not affect CYP2E1 activity or CYP2E1 content. SNAP significantly lowered CYP2E1 activity but not protein. AA treatment increased lipid peroxidation and lowered GSH levels. L-NAME potentiated while SNAP prevented these changes. Thus, L-NAME increased, while NO donors decreased AA-induced oxidative stress. Antioxidants prevented the L-NAME potentiation of AA toxicity. Damage to mitochondria by AA was shown by a decline in the mitochondrial membrane potential (MMP). L-NAME potentiated this decline in MMP in association with its increase in AA-induced oxidative stress and toxicity. NO donors decreased this decline in MMP in association with their decrease in AA

  8. 1400W, a highly selective inducible nitric oxide synthase inhibitor is a potential disease modifier in the rat kainate model of temporal lobe epilepsy.

    PubMed

    Puttachary, Sreekanth; Sharma, Shaunik; Verma, Saurabh; Yang, Yang; Putra, Marson; Thippeswamy, Achala; Luo, Diou; Thippeswamy, Thimmasettappa

    2016-09-01

    Status epilepticus (SE) initiates epileptogenesis to transform normal brain to epileptic state which is characterized by spontaneous recurrent seizures (SRS). Prior to SRS, progressive changes occur in the brain soon after SE, for example, loss of blood-brain barrier (BBB) integrity, neuronal hyper-excitability (epileptiform spiking), neuroinflammation [reactive gliosis, high levels of reactive oxygen/nitrogen species (ROS/RNS)], neurodegeneration and synaptic re-organization. Our hypothesis was that modification of early epileptogenic events will alter the course of disease development and its progression. We tested the hypothesis in the rat kainate model of chronic epilepsy using a novel disease modifying drug, 1400W, a highly selective inhibitor of inducible nitric oxide synthase (iNOS/NOS-II). In an in vitro mouse brain slice model, using a multi-electrode array system, co-application of 1400W with kainate significantly suppressed kainate-induced epileptiform spiking. In the rats, in vivo, 4h after the induction of SE with kainate, 1400W (20mg/kg, i.p.) was administered twice daily for three days to target early events of epileptogenesis. The rats were subjected to continuous (24/7) video-EEG monitoring, remotely, for six months from epidurally implanted cortical electrodes. The 1400W treatment significantly reduced the epileptiform spike rate during the first 12-74h post-SE, which resulted in >90% reduction in SRS in long-term during the six month period when compared to the vehicle-treated control group (257±113 versus 19±10 episodes). Immunohistochemistry (IHC) of brain sections at seven days and six months revealed a significant reduction in; reactive astrogliosis and microgliosis (M1 type), extravascular serum albumin (a marker for BBB leakage) and neurodegeneration in the hippocampus, amygdala and entorhinal cortex in the 1400W-treated rats when compared to the vehicle control. In the seven day group, hippocampal Western blots revealed downregulation of

  9. Effects of the nitric oxide synthase inhibitor L-NMMA on cerebrovascular and cardiovascular responses to hypoxia and hypercapnia in humans

    PubMed Central

    Ide, Kojiro; Worthley, Matthew; Anderson, Todd; Poulin, Marc J

    2007-01-01

    Cerebral blood flow is highly sensitive to alterations in the partial pressures of O2 and CO2 (PO2 and PCO2, respectively) in the arterial blood. In humans, the extent to which nitric oxide (NO) is involved in this regulation is unclear. We hypothesized that the NO synthase (NOS) inhibitor NG-monomethyl-l-arginine (l-NMMA), attenuates the sensitivity of middle cerebral artery blood velocity () to isocapnic hypoxia (end-tidal PO2 = 50 Torr) and euoxic hypercapnia (end-tidal PCO2 =+9 Torr above resting values) in 10 volunteers (age, 28.7 ± 1.3 years; height, 179.2 ± 2.4 cm; weight, 78.0 ± 3.7 kg; mean ±s.e.m.). The techniques of transcranial Doppler ultrasound and dynamic end-tidal forcing were used to measure , and control end-tidal PO2 and end-tidal PCO2, respectively. At baseline (isocapnic euoxia), following intravenous administration of l-NMMA, mean arterial blood pressure (MAP) increased (76.3 ± 7.3 to 86.2 ± 9.4 mmHg) and heart rate (HR) decreased (59.5 ± 9.0 to 55.2 ± 9.5 beats min−1) but was unchanged. Hypoxia-induced increases in MAP, HR and were similar with and without l-NMMA (5.0 ± 0.7 versus 7.1 ± 1.0 mmHg, 11.5 ± 1.4 versus 12.4 ± 1.5 beats min−1, 6.5 ± 0.8 versus 6.6 ± 0.8 cm s−1 for ΔMAP, ΔHR and Δ, respectively). Hypercapnia-induced increases in MAP, HR and were similar with and without l-NMMA (7.4 ± 3.1 versus 8.1 ± 2.2 mmHg, 10.4 ± 4.6 versus 10.0 ± 4.2 beats min−1, 16.5 ± 1.5 versus 17.6 ± 1.5 cm s−1 for ΔMAP, ΔHR and Δ, respectively) but the sensitivity of the response at the removal of hypercapnia was attenuated with l-NMMA. In young healthy humans, pharmacological blockade of nitric oxide synthesis does not affect the increases in cerebral blood flow with hypoxia and hypercapnia, suggesting that nitric oxide is not required for the cerbrovascular responses to hypoxia and hypercapnia. PMID:17673507

  10. Characterization of Prostaglandin E2 Production by Candida albicans▿

    PubMed Central

    Erb-Downward, John R.; Noverr, Mairi C.

    2007-01-01

    Candida albicans produces lipid metabolites that are functionally similar to host prostaglandins. These studies, using mass spectrometry, demonstrate that C. albicans produces authentic prostaglandin E2 (PGE2) from arachidonic acid. Maximal PGE2 production was achieved at 37°C in stationary-phase culture supernatants and in cell-free lysates generated from stationary-phase cells. Interestingly, PGE2 production is inhibited by both nonspecific cyclooxygenase and lipoxygenase inhibitors but not by inhibitors specific for the cyclooxygenase 2 isoenzyme. The C. albicans genome does not possess a cyclooxygenase homolog; however, several genes that may play a role in prostaglandin production from C. albicans were investigated. It was found that a C. albicans fatty acid desaturase homolog (Ole2) and a multicopper oxidase homolog (Fet3) play roles in prostaglandin production, with ole2/ole2 and fet3/fet3 mutant strains exhibiting reduced PGE2 levels compared with parent strains. This work demonstrates that the synthesis of PGE2 in C. albicans proceeds via novel pathways. PMID:17470538

  11. Resveratrol potently reduces prostaglandin E2 production and free radical formation in lipopolysaccharide-activated primary rat microglia

    PubMed Central

    Candelario-Jalil, Eduardo; de Oliveira, Antonio C Pinheiro; Gräf, Sybille; Bhatia, Harsharan S; Hüll, Michael; Muñoz, Eduardo; Fiebich, Bernd L

    2007-01-01

    Background Neuroinflammatory responses are triggered by diverse ethiologies and can provide either beneficial or harmful results. Microglial cells are the major cell type involved in neuroinflammation, releasing several mediators, which contribute to the neuronal demise in several diseases including cerebral ischemia and neurodegenerative disorders. Attenuation of microglial activation has been shown to confer protection against different types of brain injury. Recent evidence suggests that resveratrol has anti-inflammatory and potent antioxidant properties. It has been also shown that resveratrol is a potent inhibitor of cyclooxygenase (COX)-1 activity. Previous findings have demonstrated that this compound is able to reduce neuronal injury in different models, both in vitro and in vivo. The aim of this study was to examine whether resveratrol is able to reduce prostaglandin E2 (PGE2) and 8-iso-prostaglandin F2α (8-iso-PGF2α) production by lipopolysaccharide (LPS)-activated primary rat microglia. Methods Primary microglial cell cultures were prepared from cerebral cortices of neonatal rats. Microglial cells were stimulated with 10 ng/ml of LPS in the presence or absence of different concentrations of resveratrol (1–50 μM). After 24 h incubation, culture media were collected to measure the production of PGE2 and 8-iso-PGF2α using enzyme immunoassays. Protein levels of COX-1, COX-2 and microsomal prostaglandin E synthase-1 (mPGES-1) were studied by Western blotting after 24 h of incubation with LPS. Expression of mPGES-1 at the mRNA level was investigated using reverse transcription-polymerase chain reaction (RT-PCR) analysis. Results Our results indicate that resveratrol potently reduced LPS-induced PGE2 synthesis and the formation of 8-iso-PGF2α, a measure of free radical production. Interestingly, resveratrol dose-dependently reduced the expression (mRNA and protein) of mPGES-1, which is a key enzyme responsible for the synthesis of PGE2 by activated

  12. Novel concepts on the role of prostaglandins on luteal maintenance and maternal recognition and establishment of pregnancy in ruminants.

    PubMed

    Arosh, Joe A; Banu, Sakhila K; McCracken, John A

    2016-07-01

    In ruminants, the corpus luteum (CL) of early pregnancy is resistant to luteolysis. Prostaglandin (PG)E2 is considered a luteoprotective mediator. Early studies indicate that during maternal recognition of pregnancy (MRP) in ruminants, a factor(s) from the conceptus or gravid uterus reaches the ovary locally through the utero-ovarian plexus (UOP) and protects the CL from luteolysis. The local nature of the embryonic antiluteolytic or luteoprotective effect precludes any direct effect of a protein transported or acting between the gravid uterus and CL in ruminants. During MRP, interferon tau (IFNT) secreted by the trophoblast of the conceptus inhibits endometrial pulsatile release of PGF2α and increases endometrial PGE2. Our recent studies indicate that (1) luteal PG biosynthesis is selectively directed toward PGF2α at the time of luteolysis and toward PGE2 at the time of establishment of pregnancy (ESP); (2) the ability of the CL of early pregnancy to resist luteolysis is likely due to increased intraluteal biosynthesis and signaling of PGE2; and (3) endometrial PGE2 is transported from the uterus to the CL through the UOP vascular route during ESP in sheep. Intrauterine co-administration of IFNT and prostaglandin E2 synthase 1 (PGES-1) inhibitor reestablishes endometrial PGF2α pulses and regresses the CL. In contrast, intrauterine co-administration of IFNT and PGES-1 inhibitor along with intraovarian administration of PGE2 rescues the CL. Together, the accumulating information provides compelling evidence that PGE2 produced by the CL in response to endometrial PGE2 induced by pregnancy may counteract the luteolytic effect of PGF2α as an additional luteoprotective mechanism during MRP or ESP in ruminants. Targeting PGE2 biosynthesis and signaling selectively in the endometrium or CL may provide luteoprotective therapy to improve reproductive efficiency in ruminants.

  13. Novel concepts on the role of prostaglandins on luteal maintenance and maternal recognition and establishment of pregnancy in ruminants.

    PubMed

    Arosh, Joe A; Banu, Sakhila K; McCracken, John A

    2016-07-01

    In ruminants, the corpus luteum (CL) of early pregnancy is resistant to luteolysis. Prostaglandin (PG)E2 is considered a luteoprotective mediator. Early studies indicate that during maternal recognition of pregnancy (MRP) in ruminants, a factor(s) from the conceptus or gravid uterus reaches the ovary locally through the utero-ovarian plexus (UOP) and protects the CL from luteolysis. The local nature of the embryonic antiluteolytic or luteoprotective effect precludes any direct effect of a protein transported or acting between the gravid uterus and CL in ruminants. During MRP, interferon tau (IFNT) secreted by the trophoblast of the conceptus inhibits endometrial pulsatile release of PGF2α and increases endometrial PGE2. Our recent studies indicate that (1) luteal PG biosynthesis is selectively directed toward PGF2α at the time of luteolysis and toward PGE2 at the time of establishment of pregnancy (ESP); (2) the ability of the CL of early pregnancy to resist luteolysis is likely due to increased intraluteal biosynthesis and signaling of PGE2; and (3) endometrial PGE2 is transported from the uterus to the CL through the UOP vascular route during ESP in sheep. Intrauterine co-administration of IFNT and prostaglandin E2 synthase 1 (PGES-1) inhibitor reestablishes endometrial PGF2α pulses and regresses the CL. In contrast, intrauterine co-administration of IFNT and PGES-1 inhibitor along with intraovarian administration of PGE2 rescues the CL. Together, the accumulating information provides compelling evidence that PGE2 produced by the CL in response to endometrial PGE2 induced by pregnancy may counteract the luteolytic effect of PGF2α as an additional luteoprotective mechanism during MRP or ESP in ruminants. Targeting PGE2 biosynthesis and signaling selectively in the endometrium or CL may provide luteoprotective therapy to improve reproductive efficiency in ruminants. PMID:27179861

  14. Trichodiene synthase. Substrate specificity and inhibition.

    PubMed

    Cane, D E; Yang, G; Xue, Q; Shim, J H

    1995-02-28

    The substrate specificity of the sesquiterpene synthase trichodiene synthase was examined by determining the Vmax and Km parameters for the natural substrate, trans,trans-farnesyl diphosphate (1), its stereoisomer, cis,trans-farnesyl diphosphate, and the tertiary allylic isomer, (3R)-nerolidyl diphosphate (3), using both the native fungal and recombinant enzymes. A series of farnesyl diphosphate analogs, 15, 16, 20, 7, 8, and 9, was also tested as inhibitors of trichodiene synthase. 10-Fluorofarnesyl diphosphate (15) was the most effective competitive inhibitor, with a K1 of 16 nM compared to the Km for 1 of 87 nM, while the ether analog of farnesyl diphosphate, 8, an extremely potent inhibitor of squalene synthase, showed only modest inhibition of trichodiene synthase, with a K1/Km of 70. PMID:7873526

  15. The roles of prostaglandin E2 and D2 in lipopolysaccharide-mediated changes in sleep.

    PubMed

    Oishi, Yo; Yoshida, Kyoko; Scammell, Thomas E; Urade, Yoshihiro; Lazarus, Michael; Saper, Clifford B

    2015-07-01

    When living organisms become sick as a result of a bacterial infection, a suite of brain-mediated responses occur, including fever, anorexia and sleepiness. Systemic administration of lipopolysaccharide (LPS), a common constituent of bacterial cell walls, increases body temperature and non-rapid eye movement (NREM) sleep in animals and induces the production of pro-inflammatory prostaglandins (PGs). PGE2 is the principal mediator of fever, and both PGE2 and PGD2 regulate sleep-wake behavior. The extent to which PGE2 and PGD2 are involved in the effect of LPS on NREM sleep remains to be clarified. Therefore, we examined LPS-induced changes in body temperature and NREM sleep in mice with nervous system-specific knockouts (KO) for the PGE2 receptors type EP3 or EP4, in mice with total body KO of microsomal PGE synthase-1 or the PGD2 receptor type DP, and in mice treated with the cyclooxygenase (COX) inhibitor meloxicam. We observed that LPS-induced NREM sleep was slightly attenuated in mice lacking EP4 receptors in the nervous system, but was not affected in any of the other KO mice or in mice pretreated with the COX inhibitor. These results suggest that the effect of LPS on NREM sleep is partially dependent on PGs and is likely mediated mainly by other pro-inflammatory substances. In addition, our data show that the main effect of LPS on body temperature is hypothermia in the absence of nervous system EP3 receptors or in the presence of a COX inhibitor.

  16. Regulation of prostaglandin production by nitric oxide; an in vivo analysis.

    PubMed Central

    Salvemini, D; Settle, S L; Masferrer, J L; Seibert, K; Currie, M G; Needleman, P

    1995-01-01

    1. Endotoxin E. Coli lipopolysaccharide (LPS)-treatment in conscious, restrained rats increased plasma and urinary prostaglandin (PG) and nitric oxide (NO) production. Inducible cyclo-oxygenase (COX-2) and nitric oxide synthase (iNOS) expression accounted for the LPS-induced PG and NO release since the glucocorticoid, dexamethasone inhibited both effects. Thus, LPS (4 mg kg-1) increased the plasma levels of nitrite/nitrate from 14 +/- 1 to 84 +/- 7 microM within 3 h and this rise was inhibited to 35 +/- 1 microM by dexamethasone. Levels of 6-keto PGF1 alpha in the plasma were below the detection limit of the assay (< 0.2 ng ml-1). However, 3 h after the injection of LPS these levels rose to 2.6 +/- 0.2 ng ml-1 and to 0.7 +/- 0.01 ng ml-1 after LPS in rats that received dexamethasone. 2. The induced enzymes were inhibited in vivo with selective COX and NOS inhibitors. Furthermore, NOS inhibitors, that did not affect COX activity in vitro markedly suppressed PG production in the LPS-treated animals. For instance, the LPS-induced increased in plasma nitrite/nitrate and 6-keto PGF1 alpha at 3 h was decreased to 18 +/- 2 microM and 0.5 +/- 0.02 ng ml-1, 23 +/- 1 microM and 0.7 +/- 0.01 ng ml-1, 29 +/- 2 microM and 1 +/- 0.01 ng ml-1 in rats treated with LPS in the presence of the NOS inhibitors NG-monomethyl-L-arginine, NG-nitro arginine methyl ester and aminoguanidine, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7542531

  17. alpha-Lipoic acid inhibits inflammatory bone resorption by suppressing prostaglandin E2 synthesis.

    PubMed

    Ha, Hyunil; Lee, Jong-Ho; Kim, Ha-Neui; Kim, Hyun-Man; Kwak, Han Bok; Lee, Seungbok; Kim, Hong-Hee; Lee, Zang Hee

    2006-01-01

    alpha-Lipoic acid (LA) has been intensely investigated as a therapeutic agent for several pathological conditions, including diabetic polyneuropathy. In the present study, we examined the effects of LA on osteoclastic bone loss associated with inflammation. LA significantly inhibited IL-1-induced osteoclast formation in cocultures of mouse osteoblasts and bone marrow cells, but LA had only a marginal effect on osteoclastogenesis from bone marrow macrophages induced by receptor activator of NF-kappaB ligand (RANKL). LA inhibited both the sustained up-regulation of RANKL expression and the production of PGE2 induced by IL-1 in osteoblasts. In addition, treatment with either prostaglandin E2 (PGE2) or RANKL rescued IL-1-induced osteoclast formation inhibited by LA or NS398, a specific cyclooxygenase-2 (COX-2) inhibitor, in cocultures. LA blocked IL-1-induced PGE2 production even in the presence of arachidonic acid, without affecting the expression of COX-2 and membrane-bound PGE2 synthase. Dihydrolipoic acid (the reduced form of LA), but not LA, attenuated recombinant COX-2 activity in vitro. LA also inhibited osteoclast formation and bone loss induced by IL-1 and LPS in mice. Our results suggest that the reduced form of LA inhibits COX-2 activity, PGE2 production, and sustained RANKL expression, thereby inhibiting osteoclast formation and bone loss in inflammatory conditions.

  18. Regulation of prostaglandin E{sub 2} synthesis after brain irradiation

    SciTech Connect

    Moore, Amy H.; Olschowka, John A.; Williams, Jacqueline P.; Okunieff, Paul; O'Banion, M. Kerry . E-mail: kerry_obanion@urmc.rochester.edu

    2005-05-01

    Purpose: A local tissue reaction, termed neuroinflammation, occurs after irradiation of brain tissue. Previous work suggested that cyclooxygenase (COX)-2 activity was important for changes in gene expression associated with neuroinflammation as well as increased prostaglandin E{sub 2} (PGE{sub 2}) levels seen after radiation treatment. Methods and materials: To begin to determine the contributions of other enzymes involved in PGE{sub 2} production, we examined protein levels of COX-1 and COX-2 as well as 2 PGE synthases (membrane and cytosolic PGES) 4 h after 35 Gy single dose irradiation to the brains of C3HeN mice. We also evaluated the effects of specific COX inhibitors on PGE{sub 2} production and PGES expression. Results: As expected, COX-2 expression increased after radiation exposure. Brain irradiation also increased tissue protein levels for both PGES isoforms. Specific COX-2 inhibition with NS398 lowered brain PGE{sub 2} levels by about 60%. Surprisingly, COX-1 inhibition with SC560 completely prevented the elevation of PGE{sub 2} seen after irradiation. Interestingly, NS398 reduced the membrane-associated PGES isoform, whereas SC560 treatment lowered cytosolic isoform levels below those seen in unirradiated controls. Conclusions: Taken together, these data indicate that both cyclooxygenases contribute to PGE{sub 2} production in irradiated brain and reveal dependence of PGES isoforms expression on specific cyclooxygenase activities.

  19. Prostaglandins, bioassay and inflammation

    PubMed Central

    Flower, R J

    2006-01-01

    The formation of the British Pharmacological Society coincided almost exactly with a series of ground-breaking studies that ushered in an entirely new field of research – that of lipid mediator pharmacology. For many years following their chemical characterisation, lipids were considered only to be of dietary or structural importance. From the 1930s, all this changed – slowly at first and then more dramatically in the 1970s and 1980s with the emergence of the prostaglandins (PGs), the first intercellular mediators to be clearly derived from lipids, in a dynamic on-demand system. The PGs exhibit a wide range of biological activities that are still being evaluated and their properties underlie the action of one of the world's all-time favourite medicines, aspirin, as well as its more modern congeners. This paper traces the development of the PG field, with particular emphasis on the skilful utilisation of the twin techniques of bioassay and analytical chemistry by U.K. and Swedish scientists, and the intellectual interplay between them that led to the award of a joint Nobel Prize to the principal researchers in the PG field, half a century after the first discovery of these astonishingly versatile mediators. PMID:16402103

  20. Multiple roles of the prostaglandin D2 signaling pathway in reproduction.

    PubMed

    Rossitto, Moïra; Ujjan, Safdar; Poulat, Francis; Boizet-Bonhoure, Brigitte

    2015-01-01

    Prostaglandins signaling molecules are involved in numerous physiological processes. They are produced by several enzyme-limited reactions upon fatty acids, which are catalyzed by two cyclooxygenases and prostaglandin synthases. In particular, the prostaglandins E2 (PGE2), D2 (PGD2), and F2 (PGF2 α) have been shown to be involved in female reproductive mechanisms. Furthermore, widespread expression of lipocalin- and hematopoietic-PGD2 synthases in the male reproductive tract supports the purported roles of PGD2 in the development of both embryonic and adult testes, sperm maturation, and spermatogenesis. In this review, we summarize the putative roles of PGD2 signaling and the roles of both PGD2 synthases in testicular formation and function. We review the data reporting the involvement of PGD2 signaling in the differentiation of Sertoli and germ cells of the embryonic testis. Furthermore, we discuss the roles of lipocalin-PGD2 synthase in steroidogenesis and spermatogenesis, in terms of lipid molecule transport and PGD2 production. Finally, we discuss the hypothesis that PGD2 signaling may be affected in certain reproductive diseases, such as infertility, cryptorchidism, and testicular cancer.

  1. Attenuation of endotoxin-induced multiple organ dysfunction by 1-amino-2-hydroxy-guanidine, a potent inhibitor of inducible nitric oxide synthase.

    PubMed Central

    Ruetten, H.; Southan, G. J.; Abate, A.; Thiemermann, C.

    1996-01-01

    1. We have investigated the effects of (i) several guanidines on the activity of the inducible isoform of nitric oxide (NO) synthase (iNOS) in murine cultured macrophages and rat aortic vascular smooth muscle cells (RASM); and (ii) 1-amino-2-hydroxy-guanidine, the most potent inhibitor of iNOS activity discovered, on haemodynamics, multiple organ (liver, renal, and pancreas) dysfunction and iNOS activity in rats with endotoxic shock. 2. The synthesized guanidine analogues caused concentration-dependent inhibitions of the increase in nitrite formation caused by lipopolysaccaride (LPS, 1 microgram ml-1) in J774.2 macrophages and RASM cells with the following rank order of potency: 1-amino-2-hydroxy-guanidine > 1-amino-2-methyl-guanidine > 1-amino-1-methyl-guanidine > 1-amino-1,2-dimethyl-guanidine. Interestingly, 1-amino-2-hydroxy-guanidine (IC50: J774.2, 68 microM; RASM, 114 microM) was more potent in inhibiting nitrite formation caused by LPS than NG-methyl-L-arginine, but less potent than aminoethyl-isothiourea. 3. In the anaesthetized rat, LPS caused a fall in mean arterial blood pressure (MAP) from 115 +/- 4 mmHg (time 0) to 98 +/- 5 mmHg at 2 h (P < 0.05, n = 10) and 69 +/- 5 mmHg at 6 h (P < 0.05, n = 10). The pressor effect of noradrenaline (NA, 1 mg kg-1, i.v.) was also significantly reduced at 1 to 6 h after LPS (vascular hyporeactivity). Treatment of LPS-rats with 1-amino-2-hydroxy-guanidine (10 mg kg-1, i.v. plus 10 mg kg-1 h-1 starting at 2 h after LPS) prevented the delayed hypotension and vascular hyporeactivity seen in LPS-rats. However, 1-amino-2-hydroxy-guanidine had no effect on either MAP or the pressor effect elicited by NA in rats infused with saline rather than LPS. 4. Endotoxaemia for 6 h caused a significant rise in the serum levels of aspartate or alanine aminotransferase (i.e. GOT or GPT) and bilirubin, and hence, liver dysfunction. Treatment of LPS-rats with 1-amino-2-hydroxy-guanidine significantly attenuated the liver dysfunction caused

  2. Prostaglandin E and F2 alpha receptors in human myometrium during the menstrual cycle and in pregnancy and labor

    SciTech Connect

    Giannopoulos, G.; Jackson, K.; Kredentser, J.; Tulchinsky, D.

    1985-12-15

    The binding of prostaglandins E1 and F2 alpha has been studied in the human myometrium and cervix during the menstrual cycle and in the myometrium of pregnant patients at term before and during labor. Tritium-labeled prostaglandin E1 and F2 alpha binding was saturable and reversible. Scatchard analysis of tritium-labeled prostaglandin E1 binding was linear, which suggests a single class of high-affinity binding sites with an estimated apparent equilibrium dissociation constant of 2.5 to 5.4 nmol/L and inhibitor affinities of 0.9, 273, 273, and 217 nmol/L for prostaglandins E2, A1, B1, and F2 alpha, respectively. Scatchard analysis of tritium-labeled prostaglandin F2 alpha, binding was also linear, but the affinity of these binding sites was much lower, with an average dissociation constant of 50 nmol/L and inhibitor affinities of 1.6, 2.2, and 11.2 nmol/L for prostaglandins E1, E2, and A1, respectively. In nonpregnant patients, the concentrations and affinities of tritium-labeled prostaglandin E1 binding sites were similar in the myometrium during the proliferative and secretory phases of the menstrual cycle, but the concentration of these sites was much lower in the cervix. The concentration of the tritium-labeled prostaglandin E1 binding sites was significantly lower in the myometrium of pregnant patients at term than in the myometrium of nonpregnant patients. The concentrations and affinities of tritium-labeled prostaglandin E1 binding sites were not significantly different in the upper and lower myometrium of pregnant patients at term or in the myometrium of such patients before and during labor. The concentrations of the tritium-labeled prostaglandin F2 alpha binding sites during the menstrual cycle and in pregnancy at term were similar to those of tritium-labeled prostaglandin E1 binding sites.

  3. Stage-dependent reduction in T colony formation in Hodgkin's disease. Coincidence with monocyte synthesis of prostaglandins.

    PubMed Central

    Bockman, R S

    1980-01-01

    Prostaglandin synthesis and T lymphocyte colony formation have been examined in previously untreated patients with Hodgkin's disease. Mononuclear cells have been isolated from peripheral blood and spleens of these patients. Significant augmentation in prostaglandin E levels were noted in the mononuclear cell cutures from Hodgkin's disease patients compared with controls (1.64 +/- 0.29 vs. 0.39 +/- 0.09 ng/10(6) cells, P < 0.005). Measured prostaglandin E levels increased with advancing stage of disease. Virtually all of the prostaglandins were synthesized by the adherent monocyte cell population. Prostaglandin E was the major product. Clonal expansion of a T lymphocyte precursor cell, which gives rise to colonies > 50 cells, was determined by a layered soft agar method. T colony formation was significantly reduced in patients with stage II, III, and IV disease. There were progressively reduced colony numbers seen with advancing stage of disease (609 +/- 209, 416 +/- 158, 207 +/- 58 compared with normals 2,274 +/- 360 colonies/10(6) cells plated; P < 0.005). The addition of inhibitors of endogenous prostaglandin synthesis resulted in significant augmentation of T colony number. However, a consistent relative decrease in T colony number was seen even when endogenous prostaglandin E synthesis was blocked. It would appear that both the prostaglandin-dependent and independent T colony precursor cells are lost with progressive stage of disease. A causative role of augmented prostaglandin synthesis in this stage-dependent reduction of T colony formation could not be established. PMID:6967491

  4. Inhibiting the neuronal isoform of nitric oxide synthase has similar effects on the compensatory choroidal and axial responses to myopic defocus in chicks as does the non-specific inhibitor L-NAME.

    PubMed

    Nickla, Debora L; Damyanova, Petya; Lytle, Grace

    2009-06-01

    In birds, the choroid plays a role in the visual regulation of eye growth, thickening in response to myopic defocus, and thinning in response to hyperopic defocus, in both cases moving the retina towards the image plane. This response is rapid, occurring within hours of the defocus stimulus. These changes are consistently associated with slower changes in the sclera, that result in the appropriate changes in axial elongation, decreasing growth in response to myopic defocus and increasing it in response to hyperopic defocus. The molecular mechanisms underlying the scleral response involve changes in the synthesis of extracellular matrix molecules, however, those underlying the changes in choroidal thickness are not known. However, evidence suggests that it may involve the gaseous signal molecule nitric oxide, as nitric oxide is a potent smooth muscle relaxant, and injections of the non-specific nitric oxide synthase inhibitor L-NAME transiently inhibits the thickening response. Interestingly, it also dis-inhibits ocular growth, in accordance with a mechanistic link between the two responses. If nitric oxide is part of the signal cascade underlying the visual regulation of eye growth, it would be important to ascertain the source of the molecule. As a first step towards doing so, we used various more specific NOS inhibitors and studied their effects on the choroidal and growth responses. Birds (7-12 days old) were fitted with +10 D lenses on one eye. On that day, single intravitreal injections (30 microl) of the following inhibitors were used: nNOS inhibitor N(omega)-propyl-L-arginine (n=12), iNOS inhibitor L-NIL (n=16), eNOS/iNOS inhibitor L-NIO (n=15), non-specific inhibitor L-NMMA (n=30) or physiological saline (n=18). Ocular dimensions were measured using high-frequency A-scan ultrasonography at the start of the experiment, and at 7, 24 and 48 h after. We found that the nNOS inhibitor N(omega)-propyl-L-arginine had the same inhibitory effects on the choroidal

  5. LPS-Challenged TNFα Production, Prostaglandin Secretion, and TNFα/TNFRs Expression in the Endometrium of Domestic Cats in Estrus or Diestrus, and in Cats with Pyometra or Receiving Medroxyprogesterone Acetate

    PubMed Central

    Jursza, Ewelina; Szóstek, Anna Z.; Kowalewski, Mariusz P.; Boos, Alois; Okuda, Kiyoshi; Siemieniuch, Marta J.

    2014-01-01

    Progesterone (P4) derivatives which are commonly used to block the cyclicity of domestic cats disturb the endocrine balance in the endometrium. The aims of this study were (i) to examine whether lipopolysaccharide (LPS) is responsible for enhancement of tumor necrosis factor-α (TNFα) secretion by the feline endometrial epithelial and stromal cells in vitro, (ii) to know whether immunolocalization of TNFα/TNFR1 and TNFR2 differs in cats at estrus or diestrus, receiving medroxyprogesterone acetate and suffering from pyometra, and (iii) to determine if TNFα-challenged prostaglandin secretion is stopped by prostaglandin synthases inhibitors. A total of 37 domestic adult cats in estrus or diestrus, receiving octane medroxyprogesterone or having clinical symptoms of pyometra, were enrolled in this study. The results obtained showed a distinct increase in LPS-challenged TNFα secretion in endometrial epithelial, but not stromal cells. TNFα augmented PG secretion was blocked by phospholipase A2 (PLA2) and cyclooxygeanase-2 (COX-2), but not by mitogen-activated protein kinase (MAPK) inhibitor. TNFα/TNFR1 and 2 protein expressions were limited mostly to the surface and glandular epithelium. TNFα/TNFRs protein was upregulated in the inflammatory uterus and hence may be involved in development of pathologic changes in the endometrial glands in cats receiving exogenous P4 as a hormonal contraceptive. PMID:25028529

  6. Pseudouridine synthases.

    PubMed

    Hamma, Tomoko; Ferré-D'Amaré, Adrian R

    2006-11-01

    Pseudouridine synthases are the enzymes responsible for the most abundant posttranscriptional modification of cellular RNAs. These enzymes catalyze the site-specific isomerization of uridine residues that are already part of an RNA chain, and appear to employ both sequence and structural information to achieve site specificity. Crystallographic analyses have demonstrated that all pseudouridine synthases share a common core fold and active site structure and that this core is modified by peripheral domains, accessory proteins, and guide RNAs to give rise to remarkable substrate versatility.

  7. Rat Mesenchymal Stromal Cells Inhibit T Cell Proliferation but Not Cytokine Production Through Inducible Nitric Oxide Synthase

    PubMed Central

    Vaage, John T.

    2012-01-01

    Mesenchymal stromal cells (MSC) have important immunomodulatory properties, they inhibit T lymphocyte allo-activation and have been used to treat graft-versus-host disease. How MSC exert their immunosuppressive functions is not completely understood but species specific mechanisms have been implicated. In this study we have investigated the mechanisms for rat MSC mediated inhibition of T lymphocyte proliferation and secretion of inflammatory cytokines in response to allogeneic and mitogenic stimuli in vitro. MSC inhibited the proliferation of T cells in allogeneic mixed lymphocyte reactions and in response to mitogen with similar efficacy. The anti-proliferative effect was mediated by the induced expression of nitric oxide (NO) synthase and production of NO by MSC. This pathway was required and sufficient to fully suppress lymphocyte proliferation and depended on proximity of MSC and target cells. Expression of inducible NO synthase by MSC was induced through synergistic stimulation with tumor necrosis factor α and interferon γ secreted by activated lymphocytes. Conversely, MSC had a pronounced inhibitory effect on the secretion of these cytokines by T cells which did not depend on NO synthase activity or cell contact, but was partially reversed by addition of the cyclooxygenase (COX) inhibitor indomethacin. In conclusion, rat MSC use different mechanisms to inhibit proliferative and inflammatory responses of activated T cells. While proliferation is suppressed by production of NO, cytokine secretion appears to be impaired at least in part by COX-dependent production of prostaglandin E2. PMID:22566943

  8. Hit Optimization of 5-Substituted-N-(piperidin-4-ylmethyl)-1H-indazole-3-carboxamides: Potent Glycogen Synthase Kinase-3 (GSK-3) Inhibitors with in Vivo Activity in Model of Mood Disorders.

    PubMed

    Furlotti, Guido; Alisi, Maria Alessandra; Cazzolla, Nicola; Dragone, Patrizia; Durando, Lucia; Magarò, Gabriele; Mancini, Francesca; Mangano, Giorgina; Ombrato, Rosella; Vitiello, Marco; Armirotti, Andrea; Capurro, Valeria; Lanfranco, Massimiliano; Ottonello, Giuliana; Summa, Maria; Reggiani, Angelo

    2015-11-25

    Novel treatments for bipolar disorder with improved efficacy and broader spectrum of activity are urgently needed. Glycogen synthase kinase 3β (GSK-3β) has been suggested to be a key player in the pathophysiology of bipolar disorder. A series of novel GSK-3β inhibitors having the common N-[(1-alkylpiperidin-4-yl)methyl]-1H-indazole-3-carboxamide scaffold were prepared taking advantage of an X-ray cocrystal structure of compound 5 with GSK-3β. We probed different substitutions at the indazole 5-position and at the piperidine-nitrogen to obtain potent ATP-competitive GSK-3β inhibitors with good cell activity. Among the compounds assessed in the in vivo PK experiments, 14i showed, after i.p. dosing, encouraging plasma PK profile and brain exposure, as well as efficacy in a mouse model of mania. Compound 14i was selected for further in vitro/in vivo pharmacological evaluation, in order to elucidate the use of ATP-competitive GSK-3β inhibitors as new tools in the development of new treatments for mood disorders.

  9. Celecoxib Improves Host Defense through Prostaglandin Inhibition during Histoplasma capsulatum Infection

    PubMed Central

    Pereira, Priscilla Aparecida Tartari; Trindade, Bruno Caetano; Secatto, Adriana; Nicolete, Roberto; Peres-Buzalaf, Camila; Ramos, Simone Gusmão; Sadikot, Ruxana; Bitencourt, Claudia da Silva

    2013-01-01

    Prostaglandins act as mediators of inflammation and, similar to cytokines, function as immune modulators during innate and adaptive immune responses. Therefore, using a pharmacological inhibitor, celecoxib, we investigated the role of prostaglandins in host defense against Histoplasma capsulatum infection in C57BL/6 mice. Our results showed that treatment with celecoxib inhibited cyclooxygenase 2, reduced the total fungal burden, and reduced the concentration of PGE2, cytokines, lymphocytes, neutrophils, and mononuclear cells in the bronchoalveolar space and lung parenchyma. In addition, celecoxib treatment increased the synthesis of nitric oxide, IFN-γ, LTB4, and the phagocytic capacity of alveolar macrophages. Moreover, celecoxib treatment increased the survival of mice after infection with a lethal inoculum of H. capsulatum. These results suggest that prostaglandins alter the host immune response and play an important role in the pathogenesis of histoplasmosis. Thus, the inhibition of prostaglandins could be a valuable immunomodulatory strategy and antifungal therapy for histoplasmosis treatment. PMID:23818746

  10. Femtomole analysis of prostaglandin pharmaceuticals.

    PubMed

    McGuffin, V L; Zare, R N

    1985-12-01

    An analytical method is described whereby the major classes of prostaglandins are fully resolved by microcolumn liquid chromatography and detected at the subfemtomole level by laser-induced fluorescence. The prostaglandins are labeled with the fluorescent reagent 4-bromo-methyl-7-methoxycoumarin and are subsequently separated on a high-efficiency fused-silica microcolumn (0.2 mm i.d., 1.06 m length, 150,000 theoretical plates). The optimal chromatographic conditions consist of a 3-micron octadecylsilica packing material and an isocratic mobile phase of 47.6% methanol, 23.8% acetonitrile, and 28.6% water. The prostaglandin derivatives are detected directly on the microcolumn by laser fluorimetry, using a helium/cadmium laser (325 nm, 15 mW) as the excitation source together with a simple filter/photo-multiplier optical detection system. In real sample matrices, the prostaglandin PGF2 alpha is readily quantifiable from the detection limit (0.3 fmol) to the formulation strength of the therapeutic agent Lutalyse (Upjohn), spanning more than six orders of magnitude in concentration. The simplicity and general applicability of the present analytical methodology and instrumentation suggest that this technique can be used to attack a wide variety of biomedically important problems with exceptional sensitivity and selectivity.

  11. Ulinastatin suppresses lipopolysaccharide-induced prostaglandin E2 synthesis and nitric oxide production through the downregulation of nuclear factor‑κB in BV2 mouse microglial cells.

    PubMed

    Sung, Yun-Hee; Shin, Mal-Soon; Ko, Il-Gyu; Kim, Sung-Eun; Kim, Chang-Ju; Ahn, Hyun-Jong; Yoon, Hye-Sun; Lee, Bong-Jae

    2013-05-01

    Ulinastatin is an intrinsic serine-protease urinary trypsin inhibitor that can be extracted and purified from human urine. Urinary trypsin inhibitors are widely used to treat patients with acute inflammatory disorders, such as shock and pancreatitis. However, although the anti-inflammatory activities of urinary trypsin inhibitors have been investigated, the mechanisms underlying their actions are not yet fully understood. In the present study, we evaluated the effect of ulinastatin on lipopolysaccharide (LPS)-induced inflammation in relation with nuclear factor-κB (NF-κB) activation using BV2 mouse microglial cells. To accomplish this, we performed a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, reverse transcription-polymerase chain reaction (RT-PCR), western blot analysis, electrophoretic mobility gel shift assay (EMSA), prostaglandin E(2) (PGE(2)) immunoassay and nitric oxide (NO) detection. The results demonstrated that ulinastatin suppressed PGE2 synthesis and NO production by inhibiting the LPS-induced mRNA and protein expression of cyclooxygenase-2 (COX-2) and inducible NO synthase (iNOS) in BV2 mouse microglial cells. Ulinastatin suppressed the activation of NF-κB in the nucleus. These findings demonstrate that ulinastatin exerts analgesic and anti-inflammatory effects that possibly occur via the suppression of COX-2 and iNOS expression through the downregulation of NF-κB activity.

  12. Prostaglandin synthesis and catabolism in the gastric mucosa: studies in normal rabbits and rabbits immunized with prostaglandin E2

    SciTech Connect

    Redfern, J.S.

    1988-09-01

    Antral and fundic mucosal homogenates obtained from prostaglandin E2-immunized rabbits converted 14C-arachidonic acid to prostaglandin E2, 6-keto prostaglandin F1 alpha, prostaglandin F2 alpha, and prostaglandin D2. Percentage conversion of 14C-arachidonic acid to these prostaglandin products was not significantly different in prostaglandin E2-immunized rabbits compared with control rabbits (thyroglobulin-immunized and unimmunized rabbits combined). Synthesis of 6-keto prostaglandin F1 alpha, prostaglandin E2 and 13,14-dihydro 15-keto prostaglandin E2 from endogenous arachidonic acid after vortex mixing fundic mucosal homogenates was similar in prostaglandin E2 immunized rabbits and control rabbits. Both in prostaglandin E2-immunized rabbits and controls, 3H-prostaglandin E2 was catabolized extensively by the fundic mucosa, whereas 3H-6-keto prostaglandin F1 alpha, 3H-prostaglandin F2 alpha, and 3H-prostaglandin D2 were not catabolized to any appreciable extent. The rate of catabolism of PGs was not significantly different in prostaglandin E2-immunized rabbits and control rabbits, with the exception of prostaglandin F2 alpha which was catabolized slightly more rapidly in prostaglandin E2-immunized rabbits. These results indicate that development of gastric ulcers in prostaglandin E2-immunized rabbits is not associated with an alteration in the capacity of the gastric mucosa to synthesize or catabolize prostaglandins.

  13. Comparison of the amnestic effects of NMDA receptor antagonist MK-801 and nitric oxide synthase inhibitors: L-NAME and L-NOARG in goldfish.

    PubMed

    Xu, X; Boshoven, W; Lombardo, B; Spranger, J

    1998-08-01

    Investigations indicate that the induction of long-term potentiation (LTP) may be mediated by postsynaptic N-methyl-D-aspartate (NMDA) receptors and that the maintenance of LTP may be initiated by nitric oxide (NO), a retrograde messenger carrying signals backward from the postsynaptic to the presynaptic neuron. The present study compared amnestic effects of dizocilpine maleate (MK-801), an NMDA receptor antagonist, and nitro-L-arginine-methyl-ester (L-NAME) and N-nitro-L-arginine (L-NOARG), nitric oxide (NO) inhibitors, in goldfish, using active-avoidance conditioning as the learning paradigm. The results showed that MK-801 and NO inhibitors produced anterograde amnesia at doses that did not impair performance processes necessary for learning to occur. Furthermore, MK-801 did not produce retrograde amnesia, whereas L-NAME did, suggesting that MK-801 impaired learning whereas NO inhibitors impaired memory consolidation and possibly also learning.

  14. Signaling of Prostaglandin E Receptors, EP3 and EP4 Facilitates Wound Healing and Lymphangiogenesis with Enhanced Recruitment of M2 Macrophages in Mice

    PubMed Central

    Hosono, Kanako; Isonaka, Risa; Kawakami, Tadashi; Narumiya, Shuh; Majima, Masataka

    2016-01-01

    Lymphangiogenesis plays an important role in homeostasis, metabolism, and immunity, and also occurs during wound-healing. Here, we examined the roles of prostaglandin E2 (PGE2) receptor (EP) signaling in enhancement of lymphangiogenesis in wound healing processes. The hole-punch was made in the ears of male C57BL/6 mice using a metal ear punch. Healing process and lymphangiogenesis together with macrophage recruitment were analyzed in EP knockout mice. Lymphangiogenesis was up-regulated in the granulation tissues at the margins of punched-hole wounds in mouse ears, and this increase was accompanied by increased expression levels of COX-2 and microsomal prostaglandin E synthase-1. Administration of celecoxib, a COX-2 inhibitor, suppressed lymphangiogenesis in the granulation tissues and reduced the induction of the pro-lymphangiogenic factors, vascular endothelial growth factor (VEGF) -C and VEGF-D. Topical applications of selective EP receptor agonists enhanced the expressions of lymphatic vessel endothelial hyaluronan receptor-1 and VEGF receptor-3. The wound-healing processes and recruitment of CD11b-positive macrophages, which produced VEGF-C and VEGF-D, were suppressed under COX-2 inhibition. Mice lacking either EP3 or EP4 exhibited reduced wound-healing, lymphangiogenesis and recruitment of M2 macrophages, compared with wild type mice. Proliferation of cultured human lymphatic endothelial cells was not detected under PGE2 stimulation. Lymphangiogenesis and recruitment of M2 macrophages that produced VEGF-C/D were suppressed in mice treated with a COX-2 inhibitor or lacking either EP3 or EP4 during wound healing. COX-2 and EP3/EP4 signaling may be novel targets to control lymphangiogenesis in vivo. PMID:27711210

  15. The endometrial expression of prostaglandin cascade components in lactating dairy cows fed different polyunsaturated fatty acids.

    PubMed

    Dirandeh, E; Towhidi, A; Pirsaraei, Z Ansari; Saberifar, T; Akhlaghi, A; Roodbari, A Rezaei

    2015-01-15

    Feeding n-6 polyunsaturated fatty acids (PUFA) increases the endometrial percentages of linoleic and arachidonic acids (AA), enhances the synthesis of prostaglandin F2α (PGF2α), and improves uterine health. In contrary, the n-3 PUFA, eicosapentaenoic acid, and docosahexaenoic acid may play pivotal roles by suppressing the synthesis of uterine PGF2α, a component being centrally involved in the control of the bovine estrous cycle and in early embryo survival. The objectives of the present study were to determine the effect of feeding a diet enriched in either α-linolenic acid (n-3) or linolenic acid (n-6) on the uterine expression of genes related to prostaglandin cascade and uterine release of PGF2α (measured as 13, 14-dihydro-15-keto PGF2α [PGFM]). From calving to 60 days in milk, cows (n = 24) were fed isonitrogenous, isocaloric, and isolipidic diets that differed in the ratio of n-3/n-6 PUFA. Treatments including palm oil ([PLM]; saturated FA, n = 8), soybean whole roast ([SOY]; n-6, n = 8), and linseed extruded ([LIN]; n-3, n = 8). At 30 days in milk, the ovulatory cycles of cows were synchronized using 2 injections of PGF2α with a 14-day interval. On day 15 postovulation, cows were injected with oxytocin and blood samples were collected to monitor the uterine release of PGF2α (measured as PGFM) and uterine endometrial biopsies were prepared to evaluate the expression of genes related to prostaglandin cascade (prostaglandin F synthase [PGFS], prostaglandin E synthase [PGES], prostaglandin endoperoxide synthase-2 [PGHS-2]), phospholipase A2 (PLA2), peroxisome proliferator-activated receptors [PPAR]). Results showed that uterine endometrial PPAR-δ genes were higher in cows fed LIN (3.17-fold) compared with cows fed PLM or SOY (P < 0.05). The messenger RNA (mRNA) level of PGES in the LIN group was threefold as high as those found in SOY and PLM diets (P < 0.05). The mean relative gene expression of PLA2 and PGFS was increased in animals fed the SOY diet (2

  16. Docking studies of flavonoid compounds as inhibitors of β-ketoacyl acyl carrier protein synthase I (Kas I) of Escherichia coli.

    PubMed

    Sabbagh, Ghalia; Berakdar, Noura

    2015-09-01

    Escherichia coli is one of the most frequent causes of many common bacterial infections, including cholecystitis, bacteremia, cholangitis, urinary tract infection (UTI), traveler's diarrhea and other clinical infections such as neonatal meningitis and pneumonia. The fatty acid biosynthesis is essential for the bacterial viability and growth. There are three types of β-ketoacyl acyl carrier protein synthase (KAS) which are important for overcoming the bacterial resistance problem. β-ketoacyl acyl carrier protein synthase I (KAS I) is member of the condensing enzyme family, which is a key catalyst in bacterial fatty acid biosynthesis, and thus an attractive target for novel antibioticsis related to the elongation of unsaturated fatty acids in bacterial fatty acid synthesis and can be a good therapeutic target of designing novel antibiotics. In this report, we performed docking study of E. coli (KAS I) and 50 flavonoids. Out of these 50 flavonoids, there are two compounds, genistein and isorhamnetin, that showed the superior binding energy while fully satisfying the conditions of drug likeliness. The predicted binding energy of genistein and isorhamnetin toward KAS I are -135.76kcal/mol and -132.42kcal/mol, respectively. These energies favorably compare to the biding energy of known drugs thiolactomicin and cerulenin that are -90.26kcal/mol and -99.64kcal/mol, respectively. The method used was docking with the selected E. coli (KAS I-PDB ID-1FJ4) using iGemdock. This was also found to obey the Lipinski's guidelines of five and to show the drug likeliness and bioavailability. PMID:26292066

  17. Acute prostaglandin reduction with indomethacin and chronic prostaglandin reduction with an essential fatty acid deficient diet both decrease plasma flow to the renal papilla in the rat.

    PubMed

    Ganguli, M; Tobian, L; Ferris, T; Johnson, M A

    1989-07-01

    Renal distribution of prostaglandin synthetase is mainly medullary, whereas the major degrading enzyme, prostaglandin dehydrogenase is primarily cortical. This suggests that prostaglandins (PG) released from the renal medulla could affect the medullary blood vessels. In two different experiments we studied the role of PG in the regulation of renal papillary plasma flow in the rat. First study: PG synthesis were stimulated in 34 adult Sprague-Dawley rats by bleeding from the femoral artery 1% of the body weight over a period of 10 minutes. Following this, indomethacin (a PG inhibitor, 10 mg/kg i.v.) was given slowly and then renal papillary plasma flow was measured 25 minutes after the end of infusion. In 17 indomethacin rats the renal papillary plasma flow averaged 18.8 ml/100 g/minute, whereas it averaged 23.0 in 17 non-indomethacin rats given diluent, an 18% reduction (p less than .025). Second study: Male Sprague-Dawley rats were made prostaglandin deficient by fasting rats for one week, followed by 10% dextrose fluid for one week and subsequent institution of an essential fatty acid (EFA) deficient diet for two weeks. With urinary PG excretion in prostaglandin deficient rats 28 ng/24 hours compared to 149 ng in control rats, they could be considered as prostaglandin deficient. When renal papillary plasma flow was measured, the 16 prostaglandin deficient rats had a 16% lower papillary plasma flow than 16 control rats, 21.6 vs 25.6 (p less than .005). These results clearly demonstrate that PG inhibition in rats decreases plasma flow to the papilla, strongly suggesting that PG are vasodilators for the vessels supplying the renal papilla.

  18. Prostaglandins inhibit lipoprotein lipase gene expression in macrophages.

    PubMed Central

    Desanctis, J B; Varesio, L; Radzioch, D

    1994-01-01

    In the present investigation of the effects of prostaglandin E2 (PGE2) on lipoprotein lipase (LPL) gene expression in macrophages, we observed that treatment of macrophages with PGE2 increased the levels of adenosine 3',5'-cyclic monophosphate (cAMP), while the addition of exogenous 5-bromo-cAMP to macrophage cultures resulted in down-regulation of LPL expression. Using indomethacin (INDO), an inhibitor of cyclo-oxygenase and prostaglandins production, we determined that PGE2 acts as a feedback inhibitor of LPL expression. We found that inhibited secretion of LPL protein in lipopolysaccharide (LPS)-treated macrophages could be restored to control levels by the addition of INDO to the medium. In contrast, INDO did not reverse the inhibition of LPL mRNA induced by LPS. Overall, our results have demonstrated that PGE2 is a potent inhibitor of LPL gene expression and indicated that its action may play an important physiological role in the regulation of LPL gene expression during bacterial infections. Images Figure 1 Figure 4 Figure 7 PMID:8039811

  19. Advances in tetrahydropyrido[1,2-a]isoindolone (valmerins) series: Potent glycogen synthase kinase 3 and cyclin dependent kinase 5 inhibitors.

    PubMed

    Boulahjar, Rajâa; Ouach, Aziz; Bourg, Stéphane; Bonnet, Pascal; Lozach, Olivier; Meijer, Laurent; Guguen-Guillouzo, Christiane; Le Guevel, Rémy; Lazar, Saïd; Akssira, Mohamed; Troin, Yves; Guillaumet, Gérald; Routier, Sylvain

    2015-08-28

    An efficient synthetic strategy was developed to modulate the structure of the tetrahydropyridine isoindolone (Valmerin) skeleton. A library of more than 30 novel final structures was generated. Biological activities on CDK5 and GSK3 as well as cellular effects on cancer cell lines were measured for each novel compound. Additionally docking studies were performed to support medicinal chemistry efforts. A strong GSK3/CDK5 dual inhibitor (38, IC50 GSK3/CDK5 32/84 nM) was obtained. A set of highly selective GSK3 inhibitors was synthesized by fine-tuning structural modifications (29 IC50 GSK3/CDK5 32/320 nM). Antiproliferative effects on cells were correlated with the in vitro kinase activities and the best effects were obtained with lung and colon cell lines. PMID:26142492

  20. Evaluation of epigallocatechin gallate and related plant polyphenols as inhibitors of the FabG and FabI reductases of bacterial type II fatty-acid synthase.

    PubMed

    Zhang, Yong-Mei; Rock, Charles O

    2004-07-23

    Epigallocatechin gallate (EGCG) is the major component of green tea extracts and possesses antibacterial, antiviral, and antitumor activity. Our study focused on validating the inhibition of the bacterial type II fatty acid synthesis system as a mechanism for the antibacterial effects of EGCG and related plant polyphenols. EGCG and the related tea catechins potently inhibited both the FabG and FabI reductase steps in the fatty acid elongation cycle with IC(50) values between 5 and 15 microm. The presence of the galloyl moiety was essential for activity, and EGCG was a competitive inhibitor of FabI and a mixed type inhibitor of FabG demonstrating that EGCG interfered with cofactor binding in both enzymes. EGCG inhibited acetate incorporation into fatty acids in vivo, although it was much less potent than thiolactomycin, a validated fatty acid synthesis inhibitor, and overexpression of FabG, FabI, or both did not confer resistance. A panel of other plant polyphenols was screened for FabG/FabI inhibition and antibacterial activity. Most of these inhibited both reductase steps, possessed antibacterial activity, and inhibited cellular fatty acid synthesis. The ability of the plant secondary metabolites to interfere with the activity of multiple NAD(P)-dependent cellular processes must be taken into account when assessing the specificity of their effects.

  1. [Role of interstitial cells in prostaglandin synthesis in the kidney medulla].

    PubMed

    Sokolova, R I; Vikhert, A M

    1976-01-01

    An electron-microscopic study of the interstitial cells of the medulla of the kidneys was carried out after the administration of the inhibitor of prostaglandine synthesis -- indometacine. Under these conditions the amount of lipid granules in the interstitial cells increased significantly. The authors consider the results of study to be an evident proof of participation of lipid granules in the synthetic function of the interstitial cells as a "depôt" of a chemical precursor of prostaglandines synthesized by the interstitial cells.

  2. Prostaglandins induce vasodilatation of the microvasculature during muscle contraction and induce vasodilatation independent of adenosine.

    PubMed

    Murrant, Coral L; Dodd, Jason D; Foster, Andrew J; Inch, Kristin A; Muckle, Fiona R; Ruiz, Della A; Simpson, Jeremy A; Scholl, Jordan H P

    2014-03-15

    Blood flow data from contracting muscle in humans indicates that adenosine (ADO) stimulates the production of nitric oxide (NO) and vasodilating prostaglandins (PG) to produce arteriolar vasodilatation in a redundant fashion such that when one is inhibited the other can compensate. We sought to determine whether these redundant mechanisms are employed at the microvascular level. First, we determined whether PGs were involved in active hyperaemia at the microvascular level. We stimulated four to five skeletal muscle fibres in the anaesthetized hamster cremaster preparation in situ and measured the change in diameter of 2A arterioles (maximum diameter 40 μm, third arteriolar level up from the capillaries) at a site of overlap with the stimulated muscle fibres before and after 2 min of contraction [stimulus frequencies: 4, 20 and 60 Hz at 15 contractions per minute (CPM) or contraction frequencies of 6, 15 or 60 CPM at 20 Hz; 250 ms train duration]. Muscle fibres were stimulated in the absence and presence of the phospholipase A2 inhibitor quinacrine. Further, we applied a range of concentrations of ADO (10(-7)-10(-5) M) extraluminally, (to mimic muscle contraction) in the absence and presence of L-NAME (NO synthase inhibitor), indomethacin (INDO, cyclooxygenase inhibitor) and L-NAME + INDO and observed the response of 2A arterioles. We repeated the latter experiment on a different level of the cremaster microvasculature (1A arterioles) and on the microvasculature of a different skeletal muscle (gluteus maximus, 2A arterioles). We observed that quinacrine inhibited vasodilatation during muscle contraction at intermediate and high contraction frequencies (15 and 60 CPM). L-NAME, INDO and L-NAME + INDO were not effective at inhibiting vasodilatation induced by any concentration of ADO tested in 2A and 1A arterioles in the cremaster muscle or 2A arterioles in the gluteus maximus muscle. Our data show that PGs are involved in the vasodilatation of the microvasculature in

  3. [Treatment of postpartal atony with prostaglandins].

    PubMed

    Heinzl, S; Hendry, M

    1986-01-01

    Uterine contraction can be brought about at any time with prostaglandins. This effect is exploited in the treatment of postpartal atonia. Prostaglandin was administered intravenously to 21 women for postpartal atonia. In 19 women the bleeding subsequently stopped. There were no side effects of the treatment. These results are presented and discussed with reference to other data in the literature.

  4. Anti-inflammatory and cytoprotective effects of a squalene synthase inhibitor, TAK-475 active metabolite-I, in immune cells simulating mevalonate kinase deficiency (MKD)-like condition.

    PubMed

    Suzuki, Nobutaka; Ito, Tatsuo; Matsui, Hisanori; Takizawa, Masayuki

    2016-01-01

    TAK-475 (lapaquistat acetate) and its active metabolite-I (TAK-475 M-I) inhibit squalene synthase, which catalyzes the conversion of farnesyl diphosphate (FPP) to squalene. FPP is a substrate for synthesis of other mevalonate-derived isoprenoids (MDIs) such as farnesol (FOH), geranlygeranyl diphosphate (GGPP), and geranylgeraniol. In patients with MKD, a rare autosomal recessive disorder, defective activity of mevalonate kinase leads to a shortage of MDIs. MDIs especially GGPP are required for prenylation of proteins, which is a posttranslation modification necessary for proper functioning of proteins like small guanosine triphosphatases. Malfunction of prenylation of proteins results in upregulation of the inflammatory cascade, leading to increased production of proinflammatory cytokines like interleukin-1β (IL-1β), eventually leading to episodic febrile attacks. In vitro, TAK-475 M-I incubation in a concentration dependent manner increased levels of FPP, GGPP, and FOH in human monocytic THP-1 cells. In subsequent experiments, THP-1 cells or human peripheral blood mononuclear cells (PBMCs) were incubated with simvastatin, which inhibits hydroxymethylglutaryl-coenzyme A reductase and thereby decreases levels of the precursors of MDIs, leading to the depletion of MDIs as expected in MKD patients. Increased levels of GGPP and FPP attenuated lipopolysaccharide (LPS)-induced IL-1β production in THP-1 cells and human PBMCs in statin-treated conditions. The MDIs also significantly reduced the damaged cell ratio in this active MKD-like condition. Moreover, TAK-475 M-I directly inhibited LPS-induced IL-1β production from statin-treated THP-1 cells. These results show anti-inflammatory and cytoprotective effects of MDIs via TAK-475 M-I treatment in statin-treated immune cells, suggesting that possible therapeutic effects of TAK-475 treatment in MKD patients. PMID:27652005

  5. Effect of the ATPase inhibitor protein IF{sub 1} on H{sup +} translocation in the mitochondrial ATP synthase complex

    SciTech Connect

    Zanotti, Franco; Gnoni, Antonio; Mangiullo, Roberto; Papa, Sergio

    2009-06-19

    The H{sup +} F{sub o}F{sub 1}-ATP synthase complex of coupling membranes converts the proton-motive force into rotatory mechanical energy to drive ATP synthesis. The F{sub 1} moiety of the complex protrudes at the inner side of the membrane, the F{sub o} sector spans the membrane reaching the outer side. The IF{sub 1} component of the mitochondrial complex is a basic 10 kDa protein, which inhibits the F{sub o}F{sub 1}-ATP hydrolase activity. The mitochondrial matrix pH is the critical factor for the inhibitory binding of the central segment of IF{sub 1} (residue 42-58) to the F{sub 1}-{alpha}/{beta} subunits. We have analyzed the effect of native purified IF{sub 1} the IF{sub 1}-(42-58) synthetic peptide and its mutants on proton conduction, driven by ATP hydrolysis or by [K{sup +}] gradients, in bovine heart inside-out submitochondrial particles and in liposome-reconstituted F{sub o}F{sub 1} complex. The results show that IF{sub 1}, and in particular its central 42-58 segment, displays different inhibitory affinity for proton conduction from the F{sub 1} to the F{sub o} side and in the opposite direction. Cross-linking of IF{sub 1} to F{sub 1}-{alpha}/{beta} subunits inhibits the ATP-driven H{sup +} translocation but enhances H{sup +} conduction in the reverse direction. These observation are discussed in terms of the rotary mechanism of the F{sub o}F{sub 1} complex.

  6. Prostaglandins, Thromboxanes and Leukotrienes in Clinical Medicine

    PubMed Central

    Zipser, Robert D.; Laffi, Giacomo

    1985-01-01

    Although prostaglandin research began about 50 years ago, many of the most important advances in understanding the biochemistry, physiology and pharmacology have taken place within the past five to ten years. There is great potential for the extension of this research to the clinical practice of medicine. At this time, the most common interaction that clinicians have with the prostaglandin field is in administering nonsteroidal anti-inflammatory drugs, which function by inhibiting prostaglandins. The uses of these drugs include treating not only inflammation, but also dysmenorrhea, some renal disease, thrombotic diseases and some metabolic disorders. Prostaglandin analogs, with their potent effects on uterine contraction, are in common use in obstetrics. Other analogs, with gastric and duodenal cytoprotective effects are useful in treating peptic ulcer disease. Future benefits from prostaglandin and leukotriene research may include new therapy for inflammatory and hypersensitivity diseases such as asthma, inflammatory bowel diseases and dermatitis. PMID:3004043

  7. Discovery of 4-Aryl-5,6,7,8-tetrahydroisoquinolines as Potent, Selective, and Orally Active Aldosterone Synthase (CYP11B2) Inhibitors: In Vivo Evaluation in Rodents and Cynomolgus Monkeys.

    PubMed

    Martin, Rainer E; Aebi, Johannes D; Hornsperger, Benoit; Krebs, Hans-Jakob; Kuhn, Bernd; Kuglstatter, Andreas; Alker, André M; Märki, Hans Peter; Müller, Stephan; Burger, Dominique; Ottaviani, Giorgio; Riboulet, William; Verry, Philippe; Tan, Xuefei; Amrein, Kurt; Mayweg, Alexander V

    2015-10-22

    Inappropriately high levels of aldosterone are associated with many serious medical conditions, including renal and cardiac failure. A focused screen hit has been optimized into a potent and selective aldosterone synthase (CYP11B2) inhibitor with in vitro activity against rat, mouse, human, and cynomolgus monkey enzymes, showing a selectivity factor of 160 against cytochrome CYP11B1 in the last species. The novel tetrahydroisoquinoline compound (+)-(R)-6 selectively reduced aldosterone plasma levels in vivo in a dose-dependent manner in db/db mice and cynomolgus monkeys. The selectivity against CYP11B1 as predicted by cellular inhibition data and free plasma fraction translated well to Synacthen challenged cynomolgus monkeys up to a dose of 0.1 mg kg(-1). This compound, displaying good in vivo potency and selectivity in mice and monkeys, is ideally suited to perform mechanistic studies in relevant rodent models and to provide the information necessary for translation to non-human primates and ultimately to man. PMID:26403853

  8. Design, synthesis, biological evaluation and X-ray crystal structure of novel classical 6,5,6-tricyclic benzo[4,5]thieno[2,3-d]pyrimidines as dual thymidylate synthase and dihydrofolate reductase inhibitors

    PubMed Central

    Zhang, Xin; Zhou, Xilin; L.Kisliuk, Roy; Piraino, Jennifer; Cody, Vivian

    2011-01-01

    Classical antifolates (4-7) with a tricyclic benzo[4,5]thieno[2,3-d]pyrimidine scaffold and a flexible and rigid benzoylglutamate were synthesized as dual thymidylate synthase (TS) and dihydrofolate reductase (DHFR) inhibitors. Oxidative aromatization of ethyl 2-amino-4-methyl-4,5,6,7-tetrahydro-1-benzothiophene-3-carboxylate (±)-9 to ethyl 2-amino-4-methyl-1-benzothiophene-3-carboxylate 10 with 10% Pd/C was a key synthetic step. Compounds with 2-CH3 substituents inhibited human (h) TS (IC50 = 0.26-0.8 μM), but not hDHFR. Substitution of the 2-CH3 with a 2-NH2 increases hTS inhibition by more than 10-fold and also affords excellent hDHFR inhibition (IC50 = 0.09-0.1 μM). This study shows that the tricyclic benzo[4,5]thieno[2,3-d]pyrimidine scaffold is highly conducive to single hTS or dual hTS-hDHFR inhibition depending on the 2-position substituents. The X-ray crystal structures of 6 and 7 with hDHFR reveal, for the first time, that tricyclics 6 and 7 bind with the benzo[4,5]thieno[2,3-d]pyrimidine ring in the folate binding mode with the thieno S mimicking the 4-amino of methotrexate. PMID:21550809

  9. Effects of the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, atorvastatin and simvastatin, on the expression of endothelin-1 and endothelial nitric oxide synthase in vascular endothelial cells.

    PubMed Central

    Hernández-Perera, O; Pérez-Sala, D; Navarro-Antolín, J; Sánchez-Pascuala, R; Hernández, G; Díaz, C; Lamas, S

    1998-01-01

    Endothelial dysfunction associated with atherosclerosis has been attributed to alterations in the L-arginine-nitric oxide (NO)-cGMP pathway or to an excess of endothelin-1 (ET-1). The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have been shown to ameliorate endothelial function. However, the physiological basis of this observation is largely unknown. We investigated the effects of Atorvastatin and Simvastatin on the pre-proET-1 mRNA expression and ET-1 synthesis and on the endothelial NO synthase (eNOS) transcript and protein levels in bovine aortic endothelial cells. These agents inhibited pre-proET-1 mRNA expression in a concentration- and time-dependent fashion (60-70% maximum inhibition) and reduced immunoreactive ET-1 levels (25-50%). This inhibitory effect was maintained in the presence of oxidized LDL (1-50 microg/ml). No significant modification of pre-proET-1 mRNA half-life was observed. In addition, mevalonate, but not cholesterol, reversed the statin-mediated decrease of pre-proET-1 mRNA levels. eNOS mRNA expression was reduced by oxidized LDL in a dose-dependent fashion (up to 57% inhibition), whereas native LDL had no effect. Statins were able to prevent the inhibitory action exerted by oxidized LDL on eNOS mRNA and protein levels. Hence, these drugs might influence vascular tone by modulating the expression of endothelial vasoactive factors. PMID:9637705

  10. Antidepressant-like effect of nitric oxide synthase inhibitors and sildenafil against lipopolysaccharide-induced depressive-like behavior in mice.

    PubMed

    Tomaz, V S; Cordeiro, R C; Costa, A M N; de Lucena, D F; Nobre Júnior, H V; de Sousa, F C F; Vasconcelos, S M M; Vale, M L; Quevedo, J; Macêdo, D

    2014-05-30

    Inflammation, oxidative and nitrosative stress underlie depression being assessed in rodents by the systemic administration of lipopolysacharide (LPS). There is an increasing body of evidence of an involvement of nitric oxide (NO) pathway in depression, but this issue was not investigated in LPS-induced model. Thus, herein we evaluated the effects of NO-pathway-modulating drugs, named aminoguanidine, l-NAME, sildenafil and l-arginine, on the behavioral (forced swimming test [FST], sucrose preference [SPT] and prepulse inhibition [PPI] of the startle) and neurochemical (glutathione [GSH], lipid peroxidation, IL-1β) alterations in the prefrontal cortex, hippocampus and striatum as well as in BDNF levels in the hippocampus 24h after LPS (0.5mg/kg, i.p.) administration, a time-point related to depressive-like behavior. Twenty-four hours post LPS there was an increase in immobility time in the FST, decrease in sucrose preference and PPI levels accompanied by a decrease in GSH levels and an increase in lipid peroxidation, IL-1β and hippocampal BDNF levels suggestive of a depressive-like state. The pretreatment with the NOS inhibitors, l-NAME and aminoguanidine as well as sildenafil prevented the behavioral and neurochemical alterations induced by LPS, although sildenafil and l-NAME were not able to prevent the increase in hippocampal BDNF levels induced by LPS. The iNOS inhibitor, aminoguanidine, and imipramine prevented all behavioral and neurochemical alterations induced by LPS. l-arginine did not prevent the alterations in immobility time, sucrose preference and GSH induced by LPS. Taken together our results show that the NO-cGMP pathway is important in the modulation of the depressive-like alterations induced by LPS. PMID:24662848

  11. Novel, potent, orally bioavailable and selective mycobacterial ATP synthase inhibitors that demonstrated activity against both replicating and non-replicating M. tuberculosis.

    PubMed

    Singh, Supriya; Roy, Kuldeep K; Khan, Shaheb R; Kashyap, Vivek Kr; Sharma, Abhisheak; Jaiswal, Swati; Sharma, Sandeep K; Krishnan, Manju Yasoda; Chaturvedi, Vineeta; Lal, Jawahar; Sinha, Sudhir; Dasgupta, Arunava; Gupta, Arnab D; Srivastava, Ranjana; Saxena, Anil K

    2015-02-15

    The mycobacterial F0F1-ATP synthase (ATPase) is a validated target for the development of tuberculosis (TB) therapeutics. Therefore, a series of eighteen novel compounds has been designed, synthesized and evaluated against Mycobacterium smegmatis ATPase. The observed ATPase inhibitory activities (IC50) of these compounds range between 0.36 and 5.45μM. The lead compound 9d [N-(7-chloro-2-methylquinolin-4-yl)-N-(3-((diethylamino)methyl)-4-hydroxyphenyl)-2,3-dichlorobenzenesulfonamide] with null cytotoxicity (CC50>300μg/mL) and excellent anti-mycobacterial activity and selectivity (mycobacterium ATPase IC50=0.51μM, mammalian ATPase IC50>100μM, and selectivity >200) exhibited a complete growth inhibition of replicating Mycobacterium tuberculosis H37Rv at 3.12μg/mL. In addition, it also exhibited bactericidal effect (approximately 2.4log10 reductions in CFU) in the hypoxic culture of non-replicating M. tuberculosis at 100μg/mL (32-fold of its MIC) as compared to positive control isoniazid [approximately 0.2log10 reduction in CFU at 5μg/mL (50-fold of its MIC)]. The pharmacokinetics of 9d after p.o. and IV administration in male Sprague-Dawley rats indicated its quick absorption, distribution and slow elimination. It exhibited a high volume of distribution (Vss, 0.41L/kg), moderate clearance (0.06L/h/kg), long half-life (4.2h) and low absolute bioavailability (1.72%). In the murine model system of chronic TB, 9d showed 2.12log10 reductions in CFU in both lung and spleen at 173μmol/kg dose as compared to the growth of untreated control group of Balb/C male mice infected with replicating M. tuberculosis H37Rv. The in vivo efficacy of 9d is at least double of the control drug ethambutol. These results suggest 9d as a promising candidate molecule for further preclinical evaluation against resistant TB strains. PMID:25614114

  12. TISSUE REGENERATION. Inhibition of the prostaglandin-degrading enzyme 15-PGDH potentiates tissue regeneration.

    PubMed

    Zhang, Yongyou; Desai, Amar; Yang, Sung Yeun; Bae, Ki Beom; Antczak, Monika I; Fink, Stephen P; Tiwari, Shruti; Willis, Joseph E; Williams, Noelle S; Dawson, Dawn M; Wald, David; Chen, Wei-Dong; Wang, Zhenghe; Kasturi, Lakshmi; Larusch, Gretchen A; He, Lucy; Cominelli, Fabio; Di Martino, Luca; Djuric, Zora; Milne, Ginger L; Chance, Mark; Sanabria, Juan; Dealwis, Chris; Mikkola, Debra; Naidoo, Jacinth; Wei, Shuguang; Tai, Hsin-Hsiung; Gerson, Stanton L; Ready, Joseph M; Posner, Bruce; Willson, James K V; Markowitz, Sanford D

    2015-06-12

    Agents that promote tissue regeneration could be beneficial in a variety of clinical settings, such as stimulating recovery of the hematopoietic system after bone marrow transplantation. Prostaglandin PGE2, a lipid signaling molecule that supports expansion of several types of tissue stem cells, is a candidate therapeutic target for promoting tissue regeneration in vivo. Here, we show that inhibition of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a prostaglandin-degrading enzyme, potentiates tissue regeneration in multiple organs in mice. In a chemical screen, we identify a small-molecule inhibitor of 15-PGDH (SW033291) that increases prostaglandin PGE2 levels in bone marrow and other tissues. SW033291 accelerates hematopoietic recovery in mice receiving a bone marrow transplant. The same compound also promotes tissue regeneration in mouse models of colon and liver injury. Tissues from 15-PGDH knockout mice demonstrate similar increased regenerative capacity. Thus, 15-PGDH inhibition may be a valuable therapeutic strategy for tissue regeneration in diverse clinical contexts.

  13. Inhibition of the Prostaglandin Degrading Enzyme 15-PGDH Potentiates Tissue Regeneration *

    PubMed Central

    Zhang, Yongyou; Desai, Amar; Yang, Sung Yeun; Bae, Ki Beom; Antczak, Monika I.; Fink, Stephen P.; Tiwari, Shruti; Willis, Joseph E.; Williams, Noelle S.; Dawson, Dawn M.; Wald, David; Chen, Wei-Dong; Wang, Zhenghe; Kasturi, Lakshmi; Larusch, Gretchen A.; He, Lucy; Cominelli, Fabio; Di Martino, Luca; Djuric, Zora; Milne, Ginger L.; Chance, Mark; Sanabria, Juan; Dealwis, Chris; Mikkola, Debra; Naidoo, Jacinth; Wei, Shuguang; Tai, Hsin-Hsiung; Gerson, Stanton L.; Ready, Joseph M.; Posner, Bruce; Willson, James K. V.; Markowitz, Sanford D.

    2015-01-01

    Tissue regeneration is a medical challenge faced in injury from disease and during medical treatments such as bone marrow transplantation. Prostaglandin PGE2, which supports expansion of several types of tissue stem cells, is a candidate therapeutic target for promoting tissue regeneration in vivo. Here we show that inhibition of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a prostaglandin-degrading enzyme, potentiates tissue regeneration in multiple organs in mice. In a chemical screen, we identify a small-molecule inhibitor of 15-PGDH (SW033291) that increases prostaglandin PGE2 levels in bone marrow and other tissues. SW033291 accelerates hematopoietic recovery in mice receiving a bone marrow transplant. SW033291 also promotes tissue regeneration in mouse models of colon and liver injury. Tissues from 15-PGDH knockout mice demonstrate similar increased regenerative capacity. These findings raise the possibility that inhibiting 15-PGDH could be a useful therapeutic strategy in several distinct clinical settings. PMID:26068857

  14. Prostaglandins in human seminal plasma. Prostaglandins and related factors 46.

    PubMed

    Hamberg, M; Samuelsson, B

    1966-01-25

    This study on human seminal plasma sought after the compounds which either possess the dienone chromophore or can be converted into it by treatment with sodium hydroxide. In addition, this investigation led to the isolation of 8 more (PGs) prostaglandins which were present in higher concentrations than the previously recognized PGs. Samples of human seminal plasma were subjected to silicic acid chromatography, reversed phase partition chromatography, thin layer chromatography, and gas liquid chromatography which isolated those 8 PGs not previously recognized. 4 of these compounds, PGE1-217, PGE2-217, PGE1-278, and PGE2-278 were known from earlier studies but had not been isolated from natural sources. The other 4 were 19 hydroxy derivatives of the 4 abovementioned compounds. The concentrations of the previously recognized PGs were recently determined and it was found that the 19 hydroxy derivatives were present in concentrations 4 times higher than the PGE compounds.

  15. Sensitivity of lymphocytes to prostaglandin E2 increases in subjects over age 70.

    PubMed Central

    Goodwin, J S; Messner, R P

    1979-01-01

    We examined the sensitivity of lymphocytes from different age groups to inhibition by prostaglandin E2. Phytohemagglutinin-stimulated cultures of peripheral blood mononuclear cells from 12 healthy subjects over the age of 70 were much more sensitive to inhibition by exogenously added prostaglandin E2 than were cells from 17 young controls (ID50 congruent to 10 nM for the subjects over 70 vs. greater than 3 micronM for the young controls). The more senstivie lymphocytes from a subject over 70 were to prostaglandin E2, the lower was his or her response to phytohemagglutinin (r = 0.75, P less than 0.01). The mean responses to phytohemagglutinin of the peripheral blood mononuclear cells from the subjects over 70 were significantly depressed compared to the young controls. Addition of indomethacin, a prostaglandin synthetase inhibitor, to the cultures resulted in an increase in [3H]thymidine incorporation of 140 +/- 16% in the cells of the subjects over 70 vs. a 36 +/- 3% increase in the young controls (mean +/- SEM, P less than 0.001). The mean phytohemagglutinin response of the subjects over 70 was 40% of the control response without indomethacin. With addition of indomethacin the response of subjects over 70 rose to 72% of control. Thus, increased sinsitivity to prostaglandin E2 appears to be responsible in part for the depressed mitogen response of peripheral blood mononuclear cells from healthy subjects over 70. PMID:457862

  16. Radiation-induced increases in sensitivity of cataleptic behavior to haloperidol: possible involvement of prostaglandins

    SciTech Connect

    Joseph, J.A.; Kandasamy, S.B.; Hunt, W.A.; Dalton, T.K.; Stevens, S.

    1988-02-01

    The effects of radiation exposure on haloperidol-induced catalepsy were examined in order to determine whether elevated prostaglandins, through an action on dopaminergic autoreceptors, could be involved in the radiation-induced increase in the potency of this neuroleptic. Cataleptic behavior was examined in animals irradiated with various doses of gamma photons (1-150 Gy) and pretreated with a subthreshold dose of haloperidol (0.1 mg/kg). This approach was chosen to maximize any synergistic effects of radiation and haloperidol. After irradiation with doses less than or equal to 30 Gy, the combined treatment of haloperidol and radiation produced catalepsy, whereas neither treatment alone had an effect. This observed catalepsy could be blocked with prior administration of indomethacin, a prostaglandin synthesis inhibitor. Animals exposed to doses of radiation less than or equal to 50 Gy and no haloperidol, however, displayed apparent catalepsy. This effect was also antagonized by indomethacin. Prostaglandins can induce catalepsy and when administered in subthreshold doses along with subthreshold doses of haloperidol, catalepsy was observed. In order to assess a possible action of prostaglandins and radiation on dopaminergic activity, the functioning of striatal dopaminergic autoreceptors was examined by determining the effects of varying concentrations of haloperidol on the K+-evoked release of dopamine from striatal slices obtained from parallel groups of animals treated as above. Results indicated that sensitivity to haloperidol increased (higher K+-evoked dopamine release) in slices from irradiated or prostaglandin-treated animals and that this increase in sensitivity was blocked by indomethacin.

  17. Prostaglandin E2 protects lower airways against bronchoconstriction.

    PubMed

    Hartney, John M; Coggins, Kenneth G; Tilley, Stephen L; Jania, Leigh A; Lovgren, Alysia Kern; Audoly, Laurent P; Koller, Beverly H

    2006-01-01

    Prostaglandin E2 (PGE2), similar to beta-adrenergic receptor agonists, can protect airways from bronchoconstriction and resulting increase in airway resistance induced by a number of agents, including cholinergic receptor agonists and antigen. We examined the impact of sustained alterations in PGE2 pathways on changes in airway resistance. Genetic methods were utilized to alter PGE2 metabolism and signal transduction in the murine lung. PGE2 levels were elevated by generating mice lacking 15-hydroxyprostaglandin (Hpgd-/-), the major catabolic enzyme of PGE2, and by generating a transgenic line in which mouse PGE2 synthase (Ptges) expression is driven by a human lung-specific promoter, hSP-C. Conversely, to determine the impact of loss of PGE2 on airway reactivity, we examined mice lacking this synthase (Ptges-/-) and receptors that mediate the actions of PGE2, particularly the PGE2 EP2 receptor (Ptger2). Diminished capacity to produce and respond to PGE2 did not alter the response of mice to cholinergic stimuli. In contrast, the responsiveness to cholinergic stimulation was dramatically altered in animals with elevated PGE2 levels. The Hpgd-/- and hSP-C-Ptges transgenic lines both showed attenuated airway responsiveness to methacholine as measured by lung resistance. Thus, whereas compromise of the Ptges/PGE2/Ptger2 pathway does not alter airway responsiveness, genetic modulation that elevates PGE2 levels in the lung attenuates airway responsiveness. PMID:16113047

  18. Role of the anterior region of the third ventricle in the cardiovascular responses produced by systemic injection of a nitric oxide synthase inhibitor

    NASA Technical Reports Server (NTRS)

    Lewis, S. J.; Whalen, E. J.; Beltz, T. G.; Johnson, A. K.

    1999-01-01

    This study examined whether a prior electrolytic lesion of the tissue surrounding the anteroventral third ventricle (AV3V) would affect the increase in mean arterial blood pressure (MAP) and the fall in heart rate (HR) produced by systemic injection of the nitric oxide synthesis (NOS) inhibitor, NG-nitro-L-arginine methyl ester (L-NAME; 25 micromol/kg, i.v.) in conscious rats. L-NAME produced a smaller increase in MAP in AV3V-lesion than in sham-lesion rats (+19+/-3 vs. +40+/-3 mmHg, respectively; P<0.05). In contrast, L-NAME produced similar falls in HR in the AV3V-lesion and sham-lesion rats (-103+/-15 vs. -97+/-8 bpm, respectively; P<0.05). These findings demonstrate that the L-NAME-induced pressor response is dependent upon the integrity of the AV3V region, whereas the L-NAME-induced bradycardia is not. Copyright 1999 Elsevier Science B. V.

  19. A nitric oxide synthase inhibitor (L-NAME) attenuates abstinence-induced withdrawal from both cocaine and a cannabinoid agonist (WIN 55212-2) in Planaria.

    PubMed

    Rawls, Scott M; Rodriguez, Tonatiu; Baron, David A; Raffa, Robert B

    2006-07-12

    We previously reported that planarians (Dugesia dorotocephala) that have been exposed to cocaine for 1 h undergo abstinence-induced withdrawal when placed into cocaine-free, but not cocaine-containing, water. We now report that planarians also display dose-related abstinence-induced withdrawal following exposure to the synthetic cannabinoid agonist WIN 55212-2, but not its inactive enantiomer (WIN 55212-3). The withdrawal from WIN 55212-2 was manifested as a significant (P < 0.05) decrease in the rate of planarian spontaneous locomotor activity over a 5-min observation period, using a recently designed metric (pLMV). We also report that withdrawal from cocaine (80 microM) or WIN 55212-2 (10 microM) was attenuated by the selective inhibitor of nitric oxide synthesis L-NAME (L-nitro-arginine methyl ester), which had no effect of its own on pLMV. These results suggest a common NO-dependent pathway of withdrawal from cocaine and WIN 55212-2 in Planaria.

  20. A Comparison of the Effects of Neuronal Nitric Oxide Synthase and Inducible Nitric Oxide Synthase Inhibition on Cartilage Damage

    PubMed Central

    Gokay, Nevzat Selim; Yilmaz, Ibrahim; Demiroz, Ahu Senem; Gokce, Alper; Dervisoglu, Sergülen; Gokay, Banu Vural

    2016-01-01

    The objective of this study was to investigate the effects of selective inducible nitric oxide synthase and neuronal nitric oxide synthase inhibitors on cartilage regeneration. The study involved 27 Wistar rats that were divided into five groups. On Day 1, both knees of 3 rats were resected and placed in a formalin solution as a control group. The remaining 24 rats were separated into 4 groups, and their right knees were surgically damaged. Depending on the groups, the rats were injected with intra-articular normal saline solution, neuronal nitric oxide synthase inhibitor 7-nitroindazole (50 mg/kg), inducible nitric oxide synthase inhibitor amino-guanidine (30 mg/kg), or nitric oxide precursor L-arginine (200 mg/kg). After 21 days, the right and left knees of the rats were resected and placed in formalin solution. The samples were histopathologically examined by a blinded evaluator and scored on 8 parameters. Although selective neuronal nitric oxide synthase inhibition exhibited significant (P = 0.044) positive effects on cartilage regeneration following cartilage damage, it was determined that inducible nitric oxide synthase inhibition had no statistically significant effect on cartilage regeneration. It was observed that the nitric oxide synthase activation triggered advanced arthrosis symptoms, such as osteophyte formation. The fact that selective neuronal nitric oxide synthase inhibitors were observed to have mitigating effects on the severity of the damage may, in the future, influence the development of new agents to be used in the treatment of cartilage disorders. PMID:27382570

  1. Enhancement of scleral macromolecular permeability with prostaglandins.

    PubMed Central

    Weinreb, R N

    2001-01-01

    PURPOSE: It is proposed that the sclera is a metabolically active and pharmacologically responsive tissue. These studies were undertaken to determine whether prostaglandin exposure can enhance scleral permeability to high-molecular-weight substances. METHODS: Topical prostaglandin F2 alpha (PGF2 alpha) was administered to monkeys to determine if this altered the amount of scleral matrix metalloproteinases (MMPs). Experiments also were performed to determine whether the prostaglandin F (FP) receptor and gene transcripts are expressed in normal human sclera. Permeability of organ-cultured human sclera following prostaglandin exposure then was studied and the amount of MMP released into the medium measured. Finally, the permeability of human sclera to basic fibroblast growth factor (FGF-2) was determined following prostaglandin exposure. RESULTS: Topical prostaglandin administration that reduced scleral collagen also increased scleral MMP-1, MMP-2, and MMP-3 by 63 +/- 35%, 267 +/- 210%, and 729 +/- 500%, respectively. FP receptor protein was localized in scleral fibroblasts, and FP receptor gene transcript was identified in sclera. Exposure to prostaglandin F2 alpha, 17-phenyltrinor, PGF2 alpha, or latanoprost acid increased scleral permeability by up to 124%, 183%, or 213%, respectively. In these cultures, MMP-1, MMP-2, and MMP-3 were increased by up to 37%, 267%, and 96%, respectively. Finally, transscleral absorption of FGF-2 was increased by up to 126% with scleral exposure to latanoprost. CONCLUSIONS: These studies demonstrate that the sclera is metabolically active and pharmacologically responsive to prostaglandins. Further, they demonstrate the feasibility of cotreatment with prostaglandin to enhance transscleral delivery of peptides, such as growth factors and high-molecular-weight substances, to the posterior segment of the eye. PMID:11797317

  2. Does Prostaglandin D2 hold the cure to male pattern baldness?

    PubMed Central

    Nieves, Ashley; Garza, Luis A.

    2014-01-01

    Lipids in the skin are the most diverse in the entire human body. Their bioactivity in health and disease is underexplored. Prostaglandin D2 has recently been identified as a factor which is elevated in the bald scalp of men with androgenetic alopecia and has the capacity to decrease hair lengthening. An enzyme which synthesizes it, prostaglandin D2 synthase (PTGDS or lipocalin-PGDS) is hormone responsive in multiple other organs. PGD2 has two known receptors, GPR44 and PTGDR. GPR44 was found to be necessary for the decrease in hair growth by PGD2. This creates an exciting opportunity to perhaps create novel treatments for androgenetic alopecia which inhibit the activity of PTGDS, PGD2 or GPR44. This review discusses the current knowledge surrounding PGD2 and future steps needed to translate these findings into novel therapies for patients with androgenetic alopecia. PMID:24521203

  3. Paracrine systems in the cardioprotective effect of angiotensin-converting enzyme inhibitors on myocardial ischemia/reperfusion injury in rats.

    PubMed

    Liu, Y H; Yang, X P; Sharov, V G; Sigmon, D H; Sabbath, H N; Carretero, O A

    1996-01-01

    After transient episodes of ischemia, benefits of thrombolytic or angioplastic therapy may be limited by reperfusion injury. Angiotensin-converting enzyme inhibitors protect the heart against ischemia/reperfusion injury, an effect mediated by kinins. We examined whether the protective effect of the angiotensin-converting enzyme inhibitor ramiprilat on myocardial ischemia/reperfusion is due to kinin stimulation of prostaglandin and/or nitric oxide release. The left anterior descending coronary artery of Lewis inbred rats was occluded for 30 minutes, followed by 120 minutes of reperfusion. Immediately before reperfusion rats were treated with vehicle, ramiprilat, or the angiotensin II type 1 receptor antagonist losartan. We tested whether pretreatment with the kinin receptor antagonist Hoe 140, the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester, or the cyclooxygenase inhibitor indomethacin blocked the effect of ramiprilat on infarct size and reperfusion arrhythmias. In controls, infarct size as a percentage of the area at risk was 79 +/- 3%; ramiprilat reduced this to 49 +/- 4% (P < .001), but losartan had little effect (74 +/- 6%, P = NS). Pretreatment with Hoe 140, NG-nitro-L-arginine methyl ester, or indomethacin abolished the beneficial effect of ramiprilat. Compared with the 30-minute ischemia/120-minute reperfusion group, nonreperfused hearts with 30 minutes of ischemia had significantly smaller infarct size as a percentage of the area at risk, whereas in the 150-minute ischemia group it was significantly larger. This suggests that reperfusion caused a significant part of the myocardial injury, but it also suggests that compared with prolonged ischemia, reperfusion salvaged some of the myocardium. Ventricular arrhythmias mirrored the changes in infarct size. Thus, angiotensin-converting enzyme inhibitors protect the myocardium against ischemia/reperfusion injury and arrhythmias; these beneficial effects are mediated primarily by a kinin-prostaglandin

  4. Synthesis and evaluation of 2,5-dihydrochorismate analogues as inhibitors of the chorismate-utilising enzymes.

    PubMed

    Payne, Richard J; Bulloch, Esther M M; Toscano, Miguel M; Jones, Michelle A; Kerbarh, Olivier; Abell, Chris

    2009-06-01

    A library of 2,5-dihydrochorismate analogues were designed as inhibitors of the chorismate-utilising enzymes including anthranilate synthase, isochorismate synthase, salicylate synthase and 4-amino-4-deoxychorismate synthase. The inhibitors were synthesised in seven or eight steps from shikimic acid, sourced from star anise. The compounds exhibited moderate but differential inhibition against the four chorismate-utilising enzymes.

  5. Treatment of vasospastic disease with prostaglandin E1.

    PubMed Central

    Clifford, P C; Martin, M F; Sheddon, E J; Kirby, J D; Baird, R N; Dieppe, P A

    1980-01-01

    Prostaglandin E1, a vasodilator and potent inhibitor of platelet aggregation, was administered to 26 patients with severe vasospastic disease of the hands. Patients tolerated infusions well and reported appreciable symptomatic improvement. Five of eight ischaemic ulcers healed in six weeks. Non-invasive studies of blood flow were used to observe haemodynamic changes during and after infusions. The Doppler-derived radial artery pulsatility index fell from 8.8 +/- 0.6 to 4.6 +/0 0.5 (mean +/- SEM), indicating hand vasodilatation. This fall was maintained 24 hours after infusion (5.9 +/- 0.9), but the index had returned to normal values at two weeks. The amplitude of finger pulse volume recordings increased (5.6 +/- 0.7 mm to 23.8 +/- 3.4 mm) and was raised two and six weeks after infusion (13.5 +/- 2.1 mm). Hand temperature measured by infrared radiometry also increased (27.4 +/- 0.7 degrees C to 31.2 +/- 1.2 degrees C). Intensity of digital vasospasm induced by cold water challenge was not objectively affected by prostaglandin E1 despite an increased finger temperature after infusion. Nevertheless, patients reported less frequent and severe attacks. Prostaglandin E1 given by central venous infusion is a safe new vasoactive agent that can produce appreciable symptomatic improvement by increasing digital perfusion, which may last for several weeks after treatment. Further study will define its mode of action and its place in the management of peripheral vascular disease. PMID:7427564

  6. Colonic Saturated Fatty Acid Concentrations and Expression of COX-1, but not Diet, Predict Prostaglandin E2 in Normal Human Colon Tissue.

    PubMed

    Sidahmed, ElKhansa; Sen, Ananda; Ren, Jianwei; Patel, Arsh; Turgeon, D Kim; Ruffin, Mack T; Brenner, Dean E; Djuric, Zora

    2016-10-01

    Prostaglandin E2 (PGE2) in the colon is a pro-inflammatory mediator that is associated with increased risk of colon cancer. In this study, expression of genes in the PGE2 pathway were quantified in colon biopsies from a trial of a Mediterranean versus a Healthy Eating diet in 113 individuals at high risk for colon cancer. Colon biopsies were obtained before and after 6 months of intervention. Quantitative, real-time PCR was used to measure mRNA expression of prostaglandin H synthases (PTGS1 and 2), prostaglandin E synthases (PTGES1 and 3), prostaglandin dehydrogenase (HPGD), and PGE2 receptors (PTGER2, PTGER4). The most highly expressed genes were HPGD and PTGS1. In multivariate linear regression models of baseline data, both colon saturated fatty acid concentrations and PTGS1 expression were significant, positive predictors of colon PGE2 concentrations after controlling for nonsteroidal anti-inflammatory drug use, gender, age, and smoking status. The effects of dietary intervention on gene expression were minimal with small increases in expression noted for PTGES3 in both arms and in PTGER4 in the Mediterranean arm. These results indicate that short-term dietary change had little effect on enzymes in the prostaglandin pathway in the colon and other factors, such as differences in fatty acid metabolism, might be more influential. PMID:27548026

  7. Expression of inducible nitric oxide synthase and cyclooxygenase-2 in pancreatic adenocarcinoma: Correlation with microvessel density

    PubMed Central

    Kasper, Hans U.; Wolf, Hella; Drebber, Uta; Wolf, Helmut K.; Kern, Michael A.

    2004-01-01

    AIM: Cyclooxygenases (COX) are key enzymes for conversion of arachidonic acid to prostaglandins. Nitric oxide synthase (NOS) is the enzyme responsible for formation of nitric oxide. Both have constitutive and inducible isoforms. The inducible isoforms (iNOS and COX-2) are of great interest as regulators of tumor angiogenesis, tumorigenesis and inflammatory processes. This study was to clarify their role in pancreatic adenocarcinomas. METHODS: We investigated the immunohistochemical iNOS and COX-2 expression in 40 pancreatic ductal adenocarcinomas of different grade and stage. The results were compared with microvessel density and clinicopathological data. RESULTS: Twenty-one (52.5%) of the cases showed iNOS expression, 15 (37.5%) of the cases were positive for COX-2. The immunoreaction was heterogeneously distributed within the tumors. Staining intensity was different between the tumors. No correlation between iNOS and COX-2 expression was seen. There was no relationship with microvessel density. However, iNOS positive tumors developed more often distant metastases and the more malignant tumors showed a higher COX-2 expression. There was no correlation with other clinicopathological data. CONCLUSION: Approximately half of the cases expressed iNOS and COX-2. These two enzymes do not seem to be the key step in angiogenesis or carcinogenesis of pancreatic adenocarcinomas. Due to a low prevalence of COX-2 expression, chemoprevention of pancreatic carcinomas by COX-2 inhibitors can only achieve a limited success. PMID:15222037

  8. Formation of the early canine CL and the role of prostaglandin E2 (PGE2) in regulation of its function: an in vivo approach.

    PubMed

    Kowalewski, M P; Ihle, S; Siemieniuch, M J; Gram, A; Boos, A; Zduńczyk, S; Fingerhut, J; Hoffmann, B; Schuler, G; Jurczak, A; Domosławska, A; Janowski, T

    2015-04-01

    The mechanisms governing corpus luteum (CL) function in domestic dogs remain not fully elucidated. The upregulated expression of cyclooxygenase 2 and prostaglandin (PG) E2 synthase (PGES) at the beginning of the canine luteal phase indicated their luteotrophic roles, and the steroidogenic activity of PGE2 in the early canine CL has been confirmed in vitro. Recently, by applying a cyclooxygenase 2 (COX2)-specific inhibitor (firocoxib [Previcox]; Merial) from the day of ovulation until the midluteal phase, the luteotrophic effects of PGs have been shown in vivo. This is a follow-up study investigating the underlying endocrine mechanisms associated with the firocoxib-mediated effects on the canine CL. Experimental groups were formed with ovariohysterectomies performed on Days 5, 10, 20, or 30 of firocoxib treatments (10 mg/kg bw/24h; TGs = treated groups). Untreated dogs served as controls. A decrease of steroidogenic acute regulatory (STAR) protein expression was observed in TGs. The expression of PGE2 synthase was significantly suppressed in TGs 5 and 10, and both PGE2 and PGF2α levels were decreased in luteal homogenates, particularly from CL in TG 5. Similarly, expression of the prolactin receptor (PRLR) was diminished in TGs 5 and 20. The expression of PGE2 receptors PTGER2 (EP2) and PTGER4 (EP4), the PG- transporter (PGT), and 15-hydroxy PG dehydrogenase (HPGD) was not affected in TGs. Our results substantiate a direct luteotrophic role of PGs in the early canine CL, i.e., by upregulating the steroidogenic machinery. Additionally, the possibility of an indirect effect on PRL function arises from the increased prolactin receptor expression in response to PGE2 treatment in canine lutein cells observed in vitro.

  9. Formation of the early canine CL and the role of prostaglandin E2 (PGE2) in regulation of its function: an in vivo approach.

    PubMed

    Kowalewski, M P; Ihle, S; Siemieniuch, M J; Gram, A; Boos, A; Zduńczyk, S; Fingerhut, J; Hoffmann, B; Schuler, G; Jurczak, A; Domosławska, A; Janowski, T

    2015-04-01

    The mechanisms governing corpus luteum (CL) function in domestic dogs remain not fully elucidated. The upregulated expression of cyclooxygenase 2 and prostaglandin (PG) E2 synthase (PGES) at the beginning of the canine luteal phase indicated their luteotrophic roles, and the steroidogenic activity of PGE2 in the early canine CL has been confirmed in vitro. Recently, by applying a cyclooxygenase 2 (COX2)-specific inhibitor (firocoxib [Previcox]; Merial) from the day of ovulation until the midluteal phase, the luteotrophic effects of PGs have been shown in vivo. This is a follow-up study investigating the underlying endocrine mechanisms associated with the firocoxib-mediated effects on the canine CL. Experimental groups were formed with ovariohysterectomies performed on Days 5, 10, 20, or 30 of firocoxib treatments (10 mg/kg bw/24h; TGs = treated groups). Untreated dogs served as controls. A decrease of steroidogenic acute regulatory (STAR) protein expression was observed in TGs. The expression of PGE2 synthase was significantly suppressed in TGs 5 and 10, and both PGE2 and PGF2α levels were decreased in luteal homogenates, particularly from CL in TG 5. Similarly, expression of the prolactin receptor (PRLR) was diminished in TGs 5 and 20. The expression of PGE2 receptors PTGER2 (EP2) and PTGER4 (EP4), the PG- transporter (PGT), and 15-hydroxy PG dehydrogenase (HPGD) was not affected in TGs. Our results substantiate a direct luteotrophic role of PGs in the early canine CL, i.e., by upregulating the steroidogenic machinery. Additionally, the possibility of an indirect effect on PRL function arises from the increased prolactin receptor expression in response to PGE2 treatment in canine lutein cells observed in vitro. PMID:25595355

  10. Stimulation of Oxytocin Receptor during Early Reperfusion Period Protects the Heart against Ischemia/Reperfusion Injury: the Role of Mitochondrial ATP-Sensitive Potassium Channel, Nitric Oxide, and Prostaglandins.

    PubMed

    Imani, Alireza; Khansari, Maryam; Azizi, Yaser; Rakhshan, Kamran; Faghihi, Mahdieh

    2015-08-01

    Postconditioning is a simple and safe strategy for cardioprotection and infarct size limitation. Our previous study showed that oxytocin (OT) exerts postconditioning effect on ischemic/reperfused isolated rat heart. The aim of this study was to investigate the involvement of OT receptor, mitochondrial ATP-sensitive potassium channel (mKATP), nitric oxide (NO) and cyclooxygenase (COX) pathways in OT postconditioning. Isolated rat hearts were divided into10 groups and underwent 30 min of regional ischemia followed by 120 min of reperfusion (n =6). In I/R (ischemia/reperfusion) group, ischemia and reperfusion were induced without any treatment. In OT group, oxytocin was perfused 5 min prior to beginning of reperfusion for 25 min. In groups 3-6, atosiban (oxytocin receptor blocker), L-NAME (N-Nitro-L-Arginine Methyl Ester, non-specific nitric oxide synthase inhibitor), 5-HD (5-hydroxydecanoate, mKATP inhibitor) and indomethacin (cyclooxygenase inhibitor) were infused prior to oxytocin administration. In others, the mentioned inhibitors were perfused prior to ischemia without oxytocin infusion. Infarct size, ventricular hemodynamic, coronary effluent, malondialdehyde (MDA) and lactate dehydrogenase (LDH) were measured at the end of reperfusion. OT perfusion significantly reduced infarct size, MDA and LDH in comparison with IR group. Atosiban, 5HD, L-NAME and indomethacin abolished the postconditioning effect of OT. Perfusion of the inhibitors alone prior to ischemia had no effect on infarct size, hemodynamic parameters, coronary effluent and biochemical markers as compared with I/R group. In conclusion, this study indicates that postconditioning effects of OT are mediated by activation of mKATP and production of NO and Prostaglandins (PGs).

  11. Aldose reductases influence prostaglandin F2α levels and adipocyte differentiation in male mouse and human species.

    PubMed

    Pastel, Emilie; Pointud, Jean-Christophe; Loubeau, Gaëlle; Dani, Christian; Slim, Karem; Martin, Gwenaëlle; Volat, Fanny; Sahut-Barnola, Isabelle; Val, Pierre; Martinez, Antoine; Lefrançois-Martinez, Anne-Marie

    2015-05-01

    Aldose reductases (AKR1B) are widely expressed oxidoreductases whose physiological function remains elusive. Some isoforms are genuine prostaglandin F2α (PGF2α) synthases, suggesting they might influence adipose homeostasis because PGF2α inhibits adipogenesis. This was shown by Akr1b7 gene ablation in the mouse, which resulted in increased adiposity related to a lower PGF2α content in fat. Yet humans have no ortholog gene for Akr1b7, so the role of aldose reductases in human adipose homeostasis remains to be explored. We analyzed expression of genes encoding human and mouse aldose reductase isoforms in adipose tissues and differentiating adipocytes to assess conserved mechanisms regulating PGF2α synthesis and adipogenesis. The Akr1b3 gene encoded the most abundant isoform in mouse adipose tissue, whereas Akr1b7 encoded the only isoform enriched in the stromal vascular fraction. Most mouse aldose reductase gene expression peaked in early adipogenesis of 3T3-L1 cells and diminished with differentiation. In contrast with its mouse ortholog Akr1b3, AKR1B1 expression increased throughout differentiation of human multipotent adipose-derived stem cells, paralleling PGF2α release, whereas PGF2α receptor (FP) levels collapsed in early differentiation. Pharmacological inhibition of aldose reductase using Statil altered PGF2α production and enhanced human multipotent adipose-derived stem adipocyte differentiation. As expected, the adipogenic effects of Statil were counteracted by an FP agonist (cloprostenol). Thus, in both species aldose reductase-dependent PGF2α production could be important in early differentiation to restrict adipogenesis. PGF2α antiadipogenic signaling could then be toned down through the FP receptor or aldose reductases down-regulation in human and mouse cells, respectively. Our data suggest that aldose reductase inhibitors could have obesogenic potential.

  12. Prostaglandin transporter mutations cause pachydermoperiostosis with myelofibrosis.

    PubMed

    Diggle, Christine P; Parry, David A; Logan, Clare V; Laissue, Paul; Rivera, Carolina; Restrepo, Carlos Martín; Fonseca, Dora J; Morgan, Joanne E; Allanore, Yannick; Fontenay, Michaela; Wipff, Julien; Varret, Mathilde; Gibault, Laure; Dalantaeva, Nadezhda; Korbonits, Márta; Zhou, Bowen; Yuan, Gang; Harifi, Ghita; Cefle, Kivanc; Palanduz, Sukru; Akoglu, Hadim; Zwijnenburg, Petra J; Lichtenbelt, Klaske D; Aubry-Rozier, Bérengère; Superti-Furga, Andrea; Dallapiccola, Bruno; Accadia, Maria; Brancati, Francesco; Sheridan, Eamonn G; Taylor, Graham R; Carr, Ian M; Johnson, Colin A; Markham, Alexander F; Bonthron, David T

    2012-08-01

    Pachydermoperiostosis, or primary hypertrophic osteoarthropathy (PHO), is an inherited multisystem disorder, whose features closely mimic the reactive osteoarthropathy that commonly accompanies neoplastic and inflammatory pathologies. We previously described deficiency of the prostaglandin-degrading enzyme 15-hydroxyprostaglandin dehydrogenase (HPGD) as a cause of this condition, implicating elevated circulating prostaglandin E(2) (PGE(2)) as causative of PHO, and perhaps also as the principal mediator of secondary HO. However, PHO is genetically heterogeneous. Here, we use whole-exome sequencing to identify recessive mutations of the prostaglandin transporter SLCO2A1, in individuals lacking HPGD mutations. We performed exome sequencing of four probands with severe PHO, followed by conventional mutation analysis of SLCO2A1 in nine others. Biallelic SLCO2A1 mutations were identified in 12 of the 13 families. Affected individuals had elevated urinary PGE(2), but unlike HPGD-deficient patients, also excreted considerable quantities of the PGE(2) metabolite, PGE-M. Clinical differences between the two groups were also identified, notably that SLCO2A1-deficient individuals have a high frequency of severe anemia due to myelofibrosis. These findings reinforce the key role of systemic or local prostaglandin excess as the stimulus to HO. They also suggest that the induction or maintenance of hematopoietic stem cells by prostaglandin may depend upon transporter activity. PMID:22553128

  13. Stilbene Synthase and Chalcone Synthase 1

    PubMed Central

    Rolfs, Claus-Henning; Kindl, Helmut

    1984-01-01

    Cultured cells of Picea excelsa capable of forming stilbenes and flavanoids have been established. Unlike needles of intact plants containing piceatannol (3,3′,4′,5-tetrahydroxystilbene) and stilbene glycosides the cultured cells converted phenylalanine and p-coumaric acid primarily into resveratrol monomethyl ether (3,4′-dihydroxy-5-methoxystilbene) and naringenin. Partially purified enzyme preparations were assayed for chalcone synthase as well as for stilbene synthase activity converting malonyl-CoA plus p-coumaroyl-CoA into 3,4′,5-trihydroxystilbene (resveratrol). Although stilbene synthase and chalcone synthase use the same substrates and exhibit similar molecular properties, i.e. molecular weight and subunit molecular weight, they are two different proteins. This difference was demonstrated by gel electrophoresis and by means of monospecific antibodies. PMID:16663649

  14. Prostaglandin endoperoxide synthetase and the activation of benzo(a)pyrene to reactive metabolites in vivo in guinea pigs

    SciTech Connect

    Garattini, E.; Coccia, P.; Romano, M.; Jiritano, L.; Noseda, A.; Salmona, M.

    1984-11-01

    The role of prostaglandin endoperoxide synthetase in the in vivo activation of benzo(a)pyrene to reactive metabolites capable of interacting irreversibly with cellular macromolecules was studied in guinea pig liver, lung, kidney, spleen, small intestine, colon, and brain. DNA and protein covalent binding experiments were made after systemic administration of acetylsalicylic acid (200 mg/kg) followed by radiolabeled benzo(a)pyrene (4 microgram/kg). Results are compared with a control situation in which the prostaglandin endoperoxide synthetase inhibitor (acetylsalicylic acid) was not administered. No decrease in the level of DNA or protein benzo(a)pyrene-derived covalent binding was observed in any of the tissues studied.

  15. CRTH2, a prostaglandin D2 receptor, mediates depression-related behavior in mice.

    PubMed

    Onaka, Yusuke; Shintani, Norihito; Nakazawa, Takanobu; Haba, Ryota; Ago, Yukio; Wang, Hyper; Kanoh, Takuya; Hayata-Takano, Atsuko; Hirai, Hiroyuki; Nagata, Kin-Ya; Nakamura, Masataka; Hashimoto, Ryota; Matsuda, Toshio; Waschek, James A; Kasai, Atsushi; Nagayasu, Kazuki; Baba, Akemichi; Hashimoto, Hitoshi

    2015-05-01

    Depression is a complex neuropsychiatric disorder with an unclear molecular etiology. Inflammatory cytokines and molecular intermediates (including prostaglandins) are suggested to be involved in depression; however, the roles of prostaglandins and their respective receptors are largely unknown in depression. Using genetic and pharmacological approaches, we show here that chemoattractant receptor-homologous molecule expressed on T helper type 2 cells (CRTH2), a second receptor for prostaglandin D2 (PGD2), mediates depression-related behavior in mice. CRTH2-deficient (CRTH2(-/-)) mice showed antidepressant-like activity in a chronic corticosterone treatment-induced depression. Consistent with this observation, the pharmacological inhibition of CRTH2 via the clinically available drug ramatroban also rescued abnormal social interaction and depression-related behavior in well-established models, including chronic corticosterone-, lipopolysaccharide-, and tumor-induced pathologically relevant depression models. Importantly, chronic stress via corticosterone treatment increased mRNA levels in PGD2-producing enzymes, such as cyclooxygenase-2 and lipocalin-type PGD2 synthase, in the brain. Furthermore, the activity of the hippocampal noradrenergic system but not the dopaminergic or serotonergic systems was increased in CRTH2(-/-) mice. Together with the observation that untreated CRTH2(-/-) mice showed antidepressant-like activity in the forced swim test, these results provide evidence that central CRTH2-mediated signaling is critically involved in depression-related behavior. PMID:25698598

  16. Production of prostaglandins in placentae and corpus luteum in pregnant hinds of red deer (Cervus elaphus).

    PubMed

    Korzekwa, A J; Szczepańska, A; Bogdaszewski, M; Nadolski, P; Malż, P; Giżejewski, Z

    2016-03-01

    Prostaglandins (PGs) are synthesized from arachidonic acid by prostaglandin synthase 2 (PTGS2) and specific terminal PG synthases such as PGES and PGFS. The role of PGs in the reproductive processes of domestic ruminants is well recognized, whereas in cervidae, it is almost unknown, although it is noteworthy because some species of this family are valued in meat production and trophies. The aim of this study was to determine an effective marker of pregnancy and investigate the production and secretion of PGs in placenta and CL tissue in pregnancy. In the preliminary experiment, the levels of progesterone and 17-β estradiol (RIA; N = 14 divided into seven pregnant and seven nonpregnant hinds) were measured in the peripheral blood. In the main experiment, a comparison of messenger RNA (real-time polymerase chain reaction) and protein expression (Western blotting) of PTGS2, PGES, and PGFS, the level of prostaglandin E2 (PGE2) and PGF2α in the placentae and CL in pregnant hinds (aged 3-4 years, ca. 100 days of pregnancy, N = 6). In pregnant hinds, the level of progesterone in the blood was higher than that in nonpregnant hinds (P < 0.05), whereas the level of E2 was similar in all animals (P > 0.05). The highest messenger RNA expression of PTGS2, PGES, and PGFS was observed in the placentae than in the CL (P < 0.05). The protein expression of PTGS2 and PGES was elevated in the placentae compared with the CL (P < 0.05). The PGE2 output was the highest in cotyledonary tissue (P < 0.05). Pregnancy development in hinds around 100 days is regulated by arachidonic acid metabolites, especially PGE2 produced by the placentae, which production increases in pregnancy. Further studies are required to unravel the mechanisms involved in the regulation of PG and biosynthetic enzymes in uteroplacental and ovarian tissues during pregnancy in red deer females. PMID:26553568

  17. Prostaglandin E2 signals white-to-brown adipogenic differentiation.

    PubMed

    García-Alonso, Verónica; Clària, Joan

    2014-01-01

    The formation of new adipocytes from precursor cells is a crucial aspect of normal adipose tissue function. During the adipogenic process, adipocytes differentiated from mesenchymal stem cells give rise to two main types of fat: white adipose tissue (WAT) characterized by the presence of adipocytes containing large unilocular lipid droplets, and brown adipose tissue (BAT) composed by multilocular brown adipocytes packed with mitochondria. WAT is not only important for energy storage but also as an endocrine organ regulating whole body homeostasis by secreting adipokines and other mediators, which directly impact metabolic functions in obesity. By contrast, BAT is specialized in dissipating energy in form of heat and has salutary effects in combating obesity and associated disorders. Unfortunately, WAT is the predominant fat type, whereas BAT is scarce and located in discrete pockets in adult humans. Luckily, another type of brown adipocytes, called beige or brite (brown-in-white) adipocytes, with similar functions to those of "classical" brown adipocytes has recently been identified in WAT. In this review, a close look is given into the role of bioactive lipid mediators in the regulation of adipogenesis, with a special emphasis on the role of the microsomal prostaglandin E (PGE) synthase-1, a terminal enzyme in PGE2 biosynthesis, as a key regulator of white-to-brown adipogenesis in WAT. PMID:26317053

  18. High dietary niacin may increase prostaglandin formation but does not increase tumor formation in ApcMin/+ mice.

    PubMed

    Kwong, Alan M; Tippin, Brigette L; Materi, Alicia M; Buslon, Virgilio S; French, Samuel W; Lin, Henry J

    2011-01-01

    High doses of niacin (nicotinic acid) used to treat dyslipidemias cause flushing, due to high levels of prostaglandin D(2) (PGD(2)). GPR109A, a G-protein coupled receptor, triggers the flushing in the skin. In addition to boosting PGD(2), niacin binding to GPR109A activates the entire prostanoid cascade. We found that GPR109A occurs throughout the gastrointestinal tract. Mice that alternated between a 1% niacin diet and a control diet had higher urinary prostaglandin E(2) (PGE(2)) metabolite levels when on niacin (2.8-fold increase; 95% confidence interval, 1.8-3.9). PGE(2) promotes tumors in the intestines, whereas PGD(2) may have an opposite effect, on the basis of our report showing that transgenic hematopoietic prostaglandin D synthase suppresses intestinal adenomas in Apc(Min/+) mice. To determine if either tumor growth or tumor suppression prevails, we fed Apc(Min/+) mice a 1% niacin diet and assessed tumor development. A 1% niacin diet did not affect the number of tumors scored histologically in Apc(Min/+) mice at 14 wk (33 mice on niacin, 33 controls). Although niacin stimulates production of various prostaglandins, our results support an interpretation that very high intakes of niacin are safe in relation to intestinal tumors in this model.

  19. Prostaglandins and their receptors in insect biology

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We treat the biological significance of prostaglandins (PGs) and their known receptors in insect biology. PGs and related eicosanoids are oxygenated derivatives of arachidonic acid (AA) and two other C20 polyunsaturated fatty acids. PGs are mostly appreciated in the context of biomedicine, but a gr...

  20. Progesterone receptor expression declines in the guinea pig uterus during functional progesterone withdrawal and in response to prostaglandins.

    PubMed

    Welsh, Toni N; Hirst, Jonathan J; Palliser, Hannah; Zakar, Tamas

    2014-01-01

    Progesterone withdrawal is essential for parturition, but the mechanism of this pivotal hormonal change is unclear in women and other mammals that give birth without a pre-labor drop in maternal progesterone levels. One possibility suggested by uterine tissue analyses and cell culture models is that progesterone receptor levels change at term decreasing the progesterone responsiveness of the myometrium, which causes progesterone withdrawal at the functional level and results in estrogen dominance enhancing uterine contractility. In this investigation we have explored whether receptor mediated functional progesterone withdrawal occurs during late pregnancy and labor in vivo. We have also determined whether prostaglandins that induce labor cause functional progesterone withdrawal by altering myometrial progesterone receptor expression. Pregnant guinea pigs were used, since this animal loses progesterone responsiveness at term and gives birth in the presence of high maternal progesterone level similarly to primates. We found that progesterone receptor mRNA and protein A and B expression decreased in the guinea pig uterus during the last third of gestation and in labor. Prostaglandin administration reduced while prostaglandin synthesis inhibitor treatment increased progesterone receptor A protein abundance. Estrogen receptor-1 protein levels remained unchanged during late gestation, in labor and after prostaglandin or prostaglandin synthesis inhibitor administration. Steroid receptor levels were higher in the non-pregnant than in the pregnant uterine horns. We conclude that the decreasing expression of both progesterone receptors A and B is a physiological mechanism of functional progesterone withdrawal in the guinea pig during late pregnancy and in labor. Further, prostaglandins administered exogenously or produced endogenously stimulate labor in part by suppressing uterine progesterone receptor A expression, which may cause functional progesterone withdrawal, promote

  1. Development and Binding Mode Assessment of N-[4-[2-propyn-1-yl[(6S)-4,6,7,8-tetrahydro-2-(hydroxymethyl)-4-oxo-3H-cyclopenta[g]quinazolin-6-yl]amino]benzoyl]-L-γ-glutamyl-D-glutamic acid (BGC 945), a Novel Thymidylate Synthase Inhibitor that Targets Tumor Cells

    PubMed Central

    Tochowicz, Anna; Dalziel, Sean; Eidam, Oliv; O’Connell, Joseph D.; Griner, Sarah; Finer-Moore, Janet S.; Stroud, Robert M.

    2013-01-01

    N-[4-[2-propyn-1-yl[(6S)-4,6,7,8-tetrahydro-2-(hydroxymethyl)-4-oxo-3H-cyclopenta[g]quinazolin-6-yl]amino]benzoyl]-L-γ-glutamyl-D-glutamic acid 1 (BGC 945, now known as ONX 0801), is a small molecule thymidylate synthase (TS) inhibitor discovered at the Institute of Cancer Research in London. It is licensed by Onyx Pharmaceuticals and is in Phase 1 clinical studies. It is a novel antifolate drug resembling TS inhibitors plevitrexed and raltitrexed that combines enzymatic inhibition of thymidylate synthase with α-folate receptor-mediated targeting of tumor cells. Thus, it has potential for efficacy with lower toxicity due to selective intracellular accumulation through α-folate receptor (α-FR) transport. The α-FR, a cell-surface receptor glycoprotein, which is over expressed mainly in ovarian and lung cancer tumors, has an affinity for 1 similar to that for its natural ligand, folic acid. This study describes a novel synthesis of 1, an X-ray crystal structure of its complex with Escherichia coli TS and 2’-deoxyuridine-5’-monophosphate, and a model for a similar complex with human TS. PMID:23710599

  2. The Role of Prostaglandins and COX-Enzymes in Chondrogenic Differentiation of ATDC5 Progenitor Cells

    PubMed Central

    Caron, Marjolein M. J.; Emans, Pieter J.; Sanen, Kathleen; Surtel, Don A. M.; Cremers, Andy; Ophelders, Daan; van Rhijn, Lodewijk W.; Welting, Tim J. M.

    2016-01-01

    Objectives NSAIDs are used to relieve pain and decrease inflammation by inhibition of cyclooxygenase (COX)-catalyzed prostaglandin (PG) synthesis. PGs are fatty acid mediators involved in cartilage homeostasis, however the action of their synthesizing COX-enzymes in cartilage differentiation is not well understood. In this study we hypothesized that COX-1 and COX-2 have differential roles in chondrogenic differentiation. Methods ATDC5 cells were differentiated in the presence of COX-1 (SC-560, Mofezolac) or COX-2 (NS398, Celecoxib) specific inhibitors. Specificity of the NSAIDs and inhibition of specific prostaglandin levels were determined by EIA. Prostaglandins were added during the differentiation process. Chondrogenic outcome was determined by gene- and protein expression analyses. Results Inhibition of COX-1 prevented Col2a1 and Col10a1 expression. Inhibition of COX-2 resulted in decreased Col10a1 expression, while Col2a1 remained unaffected. To explain this difference expression patterns of both COX-enzymes as well as specific prostaglandin concentrations were determined. Both COX-enzymes are upregulated during late chondrogenic differentiation, whereas only COX-2 is briefly expressed also early in differentiation. PGD2 and PGE2 followed the COX-2 expression pattern, whereas PGF2α and TXA2 levels remained low. Furthermore, COX inhibition resulted in decreased levels of all tested PGs, except for PGD2 and PGF2α in the COX-1 inhibited condition. Addition of PGE2 and PGF2α resulted in increased expression of chondrogenic markers, whereas TXA2 increased expression of hypertrophic markers. Conclusions Our findings point towards a differential role for COX-enzymes and PG-production in chondrogenic differentiation of ATDC5 cells. Ongoing research is focusing on further elucidating the functional partition of cyclooxygenases and specific prostaglandin production. PMID:27050768

  3. Inhibition of the Prostaglandin Transporter PGT Lowers Blood Pressure in Hypertensive Rats and Mice

    PubMed Central

    Chi, Yuling; Jasmin, Jean-Francois; Seki, Yoshinori; Lisanti, Michael P.; Charron, Maureen J.; Lefer, David J.; Schuster, Victor L.

    2015-01-01

    Inhibiting the synthesis of endogenous prostaglandins with nonsteroidal anti-inflammatory drugs exacerbates arterial hypertension. We hypothesized that the converse, i.e., raising the level of endogenous prostaglandins, might have anti-hypertensive effects. To accomplish this, we focused on inhibiting the prostaglandin transporter PGT (SLCO2A1), which is the obligatory first step in the inactivation of several common PGs. We first examined the role of PGT in controlling arterial blood pressure blood pressure using anesthetized rats. The high-affinity PGT inhibitor T26A sensitized the ability of exogenous PGE2 to lower blood pressure, confirming both inhibition of PGT by T26A and the vasodepressor action of PGE2 T26A administered alone to anesthetized rats dose-dependently lowered blood pressure, and did so to a greater degree in spontaneously hypertensive rats than in Wistar-Kyoto control rats. In mice, T26A added chronically to the drinking water increased the urinary excretion and plasma concentration of PGE2 over several days, confirming that T26A is orally active in antagonizing PGT. T26A given orally to hypertensive mice normalized blood pressure. T26A increased urinary sodium excretion in mice and, when added to the medium bathing isolated mouse aortas, T26A increased the net release of PGE2 induced by arachidonic acid, inhibited serotonin-induced vasoconstriction, and potentiated vasodilation induced by exogenous PGE2. We conclude that pharmacologically inhibiting PGT-mediated prostaglandin metabolism lowers blood pressure, probably by prostaglandin-induced natriuresis and vasodilation. PGT is a novel therapeutic target for treating hypertension. PMID:26121580

  4. Prostaglandin potentiates 5-HT responses in stomach and ileum innervating visceral afferent sensory neurons

    SciTech Connect

    Kim, Sojin; Jin, Zhenhua; Lee, Goeun; Park, Yong Seek; Park, Cheung-Seog; Jin, Young-Ho

    2015-01-02

    Highlights: • Prostaglandin E2 (PGE{sub 2}) effect was tested on visceral afferent neurons. • PGE{sub 2} did not evoke response but potentiated serotonin (5-HT) currents up to 167%. • PGE{sub 2}-induced potentiation was blocked by E-prostanoid type 4 receptors antagonist. • PGE{sub 2} effect on 5-HT response was also blocked by protein kinase A inhibitor KT5720. • Thus, PGE{sub 2} modulate visceral afferent neurons via synergistic signaling with 5-HT. - Abstract: Gastrointestinal disorder is a common symptom induced by diverse pathophysiological conditions that include food tolerance, chemotherapy, and irradiation for therapy. Prostaglandin E{sub 2} (PGE{sub 2}) level increase was often reported during gastrointestinal disorder and prostaglandin synthetase inhibitors has been used for ameliorate the symptoms. Exogenous administration of PGE{sub 2} induces gastrointestinal disorder, however, the mechanism of action is not known. Therefore, we tested PGE{sub 2} effect on visceral afferent sensory neurons of the rat. Interestingly, PGE{sub 2} itself did not evoked any response but enhanced serotonin (5-HT)-evoked currents up to 167% of the control level. The augmented 5-HT responses were completely inhibited by a 5-HT type 3 receptor antagonist, ondansetron. The PGE{sub 2}-induced potentiation were blocked by a selective E-prostanoid type4 (EP{sub 4}) receptors antagonist, L-161,982, but type1 and 2 receptor antagonist AH6809 has no effect. A membrane permeable protein kinase A (PKA) inhibitor, KT5720 also inhibited PGE{sub 2} effects. PGE{sub 2} induced 5-HT current augmentation was observed on 15% and 21% of the stomach and ileum projecting neurons, respectively. Current results suggest a synergistic signaling in visceral afferent neurons underlying gastrointestinal disorder involving PGE{sub 2} potentiation of 5-HT currents. Our findings may open a possibility for screen a new type drugs with lower side effects than currently using steroidal prostaglandin

  5. Goiter formation following prostaglandin administration in rats.

    PubMed Central

    Lupulescu, A.

    1976-01-01

    Prostaglandins (PGE1 and PGE2) induced a hyperplastic microfollicular goiter with a high radioiodine (131I) thyroid uptake, increased endocytosis, a heavy autoradiographic (125I) reaction, and a moderate increase of thyroid hormones (T4, T3), thyroxine-binding globulin (TGB), and thyrotropin (TSH) concentrations in adult rats. Ultrastructurally, both prostaglandins (E1 and E2) markedly stimulated the thyroid cell activity and increased the number of pseudopodia, the size of colloid and dense granule populations, and the number of polysomes. Conversely, a hypofunction of thyroid glands with low radioiodine (131I) thyroid uptake, a decreased autoradiographic (125I) reaction, and a moderate decrease in T4, T3, TGB, and TSH concentrations were observed following prostaglandin F 2alpha. Ultrastructurally, a decrease in size of the colloid and dense granule population and the number of degenerative mitochondria occurred infollicular cells. An intense hyperplasia of parafollicular (C) cells, with abundant population of characteristic dense granules, could be seen in PGF 2alpha-treated rats. A marked decrease of radioiodine (131I) uptake, endocytosis, and autoradiographic (125I) reaction and a sharp decline in T4, T3, and TBG were observed in hypophysectomized and chronically prostaglandin-treated rats. Light and electron microscopy revealed signs of an advanced thyroid hypofunction with flat cuboidal cells, reduced microvilli, scarce endoplasmic reticulum, and few dense droplets. The present findings demonstrate that the chronic administration of prostaglandins exerts significant effects of thyroid gland and goiter formation (goitrogenesis), radioiodine metabolism, and hormone synthesis, and that these effects are mediated by TSH secretion. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 PMID:970439

  6. Inhibiting the transient choroidal thickening response using the nitric oxide synthase inhibitor l-NAME prevents the ameliorative effects of visual experience on ocular growth in two different visual paradigms.

    PubMed

    Nickla, Debora L; Wilken, Erika; Lytle, Grace; Yom, Sung; Mertz, James

    2006-08-01

    It is generally accepted that the increase in choroidal thickness in response to myopic defocus in chicks acts to move the retina towards the image plane. It may also constitute part of the signal cascade in the visual regulation of eye growth. To test this, we used the nitric oxide synthase inhibitor l-NAME to inhibit the defocus induced choroidal thickening under two different visual conditions, and looked at the effects on ocular growth rate. Exp. 1: Deprivation/Vision: chicks were monocularly deprived of form vision with translucent diffusers from day 6 to day 9. In the middle of each day the diffusers were removed for 2 h. One group received an intravitreal injection of 30 microl l-NAME (16 micromole; n=12) prior to the vision, a second group received injections of physiological saline (n=11). Exp. 2: Recovery/Vision: chicks were made myopic by form deprivation from day 6 to day 10. On days 11 to 14 the diffusers were removed for 2 h per day for 4 days to allow eyes to "recover" from the myopia. One group received an injection of l-NAME prior to vision (n=8), the other saline (n=6). Refractive errors were measured with a refractometer at the start (days 6 and 11) and end (days 10 and 15, respectively) of both experiments. Ocular dimensions were measured with high frequency A-scan ultrasonography at the start and end, and on the third experimental day immediately before and after the period of vision. Choroidal retinoic acid synthesis was measured by HPLC. Finally, NO production and scleral proteoglycan synthesis were measured in eyes wearing positive lenses 6 and 24h after an injection of l-NAME. l-NAME prevented the transient vision-induced choroidal thickening in both experiments. Furthermore, l-NAME inhibited the protective effect of brief daily vision: eyes became significantly more myopic than saline controls (exp. 1: -9 D vs -2.7D; exp. 2: -0.9 D vs +4.3 D; p<0.005 for both) and grew faster (change in lens-sclera: exp. 1: 295 vs 158 microm; exp. 2: 147

  7. The prostaglandin transporter (PGT) as a potential mediator of ovulation.

    PubMed

    Yerushalmi, Gil M; Markman, Svetlana; Yung, Yuval; Maman, Ettie; Aviel-Ronen, Sarit; Orvieto, Raoul; Adashi, Eli Y; Hourvitz, Ariel

    2016-05-11

    Prostaglandins (PGs) play an important role in the ovulatory process. However, the role of the PG transporter (PGT) in this context remains unknown. We report that the expression of PGT, a transmembrane PG carrier protein, is markedly up-regulated in preovulatory human granulosa cells (GCs). Treatment with human chorionic gonadotropin (hCG), an ovulatory trigger, significantly increases the expression of PGT mRNA and protein in human GCs both in vivo and in vitro. The hCG-induced increase in the expression of PGT in cultured human GCs is mediated via protein kinase A and protein kinase C by way of the extracellular signal-regulated kinase pathway. PGT in cultured human GCs mediates the uptake of PGE2, thereby regulating its extracellular concentration. In vivo treatment of mice with PGT inhibitors effectively blocks ovulation and markedly attenuates the expression of key ovulatory genes. We hypothesize that the inhibition of PGT activity in GCs increases the extracellular concentration of PGE2, the ability of which to exert its ovulatory effect is compromised by desensitization of its cognate receptors. Together, these findings support the idea that PGT is an important mediator of ovulation and that its inhibitors may be viewed as potential candidates for nonhormonal contraception. These findings may also fill the gap in the understanding of PGT signaling, enhance the understanding of ovulatory disorders, and facilitate the treatment of infertility or subfertility in women by using nonsteroidal PG-based therapeutic approaches. PMID:27169804

  8. The prostaglandin E2 receptor EP4 is integral to a positive feedback loop for prostaglandin E2 production in human macrophages infected with Mycobacterium tuberculosis.

    PubMed

    Nishimura, Tomoyasu; Zhao, Xiaomin; Gan, Huixian; Koyasu, Shigeo; Remold, Heinz G

    2013-09-01

    Prostaglandin E2 (PGE2) is an important biological mediator involved in the defense against Mycobacterium tuberculosis (Mtb) infection. Previously, we reported that in macrophages (Mϕs), infection with avirulent Mtb H37Ra resulted in inhibition of necrosis by an inhibitory effect on mitochondrial permeability transition via the PGE2 receptor EP2. However, human Mϕs also express EP4, a PGE2 receptor functionally closely related to EP2 that also couples to stimulatory guanine nucleotide binding protein, but the functional differences between EP2 and EP4 in Mtb-infected Mϕs have been unclear. EP4 antagonist addition to H37Ra-infected Mϕs inhibited the expression of cyclooxygenase 2 (COX2) and microsomal prostaglandin E synthase-1 (mPGES-1), which are involved in PGE2 production. Moreover, H37Ra infection induced PGE2 production through the Toll-like receptor (TLR) 2/p38 mitogen-activated protein kinase (MAPK) signaling pathway. Induction of COX2 and mPGES-1 expression by TLR2 stimulation or Mtb infection was increased after additional stimulation with EP4 agonist. Hence, in Mtb-infected Mϕs, PGE2 production induced by pathogen recognition receptors/p38 MAPK signaling is up-regulated by EP4-triggered signaling to maintain an effective PGE2 concentration.

  9. Prostaglandins and the regulation of parturition in mares.

    PubMed

    Ousey, J C; Fowden, A L

    2012-02-01

    Prostaglandins play an essential role during the perinatal period in the mare. Prostaglandin concentrations are low for the majority of pregnancy due to the regulatory action of progestagens on those enzymes responsible for metabolism of prostaglandins. Towards term, prostaglandin concentrations gradually increase, closely associated with upregulation of the fetal hypothalamo-pituitary-adrenal axis, stimulation of the prostaglandin synthesising enzyme PGHS-2 and changes in the ratio of progestagens and oestrogens. Recent evidence in the mare indicates that proinflammatory cytokines are key mediators of prostaglandin synthesis both at term parturition in healthy mares and at preterm parturition associated with placental infection. Prostaglandin concentrations rise substantially during active labour and decline after birth, associated with delivery of the placenta. During induced labour, prostaglandin concentrations are variable depending on the proximity to spontaneous parturition at term. Once the proinflammatory endocrine cascade is initiated, it is difficult to prevent active labour by administration of drugs that reduce prostaglandin concentrations in peripheral plasma. Further work is needed to establish the inter-relationships between prostaglandin production and other endocrine changes associated with labour at term and preterm in the mare.

  10. Inhibition of glycogen synthase kinase-3β by lithium chloride suppresses 6-hydroxydopamine-induced inflammatory response in primary cultured astrocytes.

    PubMed

    Wang, Hong-Mei; Zhang, Ting; Li, Qiang; Huang, Jian-Kang; Chen, Rong-Fu; Sun, Xiao-Jiang

    2013-11-01

    An increasing amount of evidence has emerged to suggest that neuroinflammatory process is involved in the pathogenesis of Parkinson's disease (PD). Activated microglia and astrocytes are found in the substantia nigra (SN) of Parkinson's disease brains as well as in animal models of Parkinson's disease. Although reactive astrocytes are involved in the progression of PD, the role of reactive astrocytes in neuroinflammation of PD has received limited attention to date. Recently, Glycogen synthase kinase-3β (GSK-3β) was identified as a crucial regulator of the inflammatory response. The purpose of this study was to explore the mechanism by which 6-hydroxydopamine (6-OHDA) induces inflammatory response in astrocytes and observe the anti-inflammatory effect of lithium chloride (LiCl) on 6-OHDA-treated astrocytes. In the present study, we found that glial fibrillary acidic protein (GFAP) was markedly upregulated in the presence of 6-OHDA. Moreover, our results revealed that proinflammatory molecules including inducible nitric oxide synthase (iNOS), nitric oxide (NO), cyclooxygenase-2(COX-2), prostaglandins E2 (PGE2), and tumor necrosis factor-α (TNF-α) were obviously increased in astrocytes exposed to 6-OHDA. Western blot analysis revealed that 6-OHDA significantly increased dephosphorylation/activation of GSK-3β as well as the nuclear translocation of nuclear factor-κB (NF-κB) p65. Besides, GSK-3β inhibitor LiCl and SB415286 inhibited the GSK-3β/NF-κB signaling pathway, leading to the reduction of proinflammatory molecules in 6-OHDA-activated astrocytes. These results confirmed that GSK-3β inhibitor LiCl and SB415286 provide protection against neuroinflammation in 6-OHDA-treated astrocytes. Therefore, GSK-3β may be a potential therapeutic target for the treatment of PD.

  11. Prostaglandin Endoperoxides. A New Concept Concerning the Mode of Action and Release of Prostaglandins*

    PubMed Central

    Hamberg, Mats; Svensson, Jan; Samuelsson, Bengt

    1974-01-01

    Methods were developed for quantitative determination of the three major metabolites of arachidonic acid in human platelets, i.e., 12L-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE), 12L-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 8-(1-hydroxy-3-oxopropyl)-9,12L-dihydroxy-5,10-heptadecadienoic acid (PHD). Aggregation of washed platelets by thrombin was accompanied by release of 1163-2175 ng/ml of HETE, 1129-2430 ng/ml of HHT, and 998-2299 ng/ml of PHD. The amount of PGG2 (prostaglandin G2) produced as calculated from the sum of the amounts of its metabolites (HHT and PHD) was 2477-5480 ng/ml. In contrast, the amounts of PGE2 (prostaglandin E2) and PGF2α (prostaglandin F2α) released were approximately two orders of magnitude lower. In this system, the prostaglandins thus exert their biological action through the endoperoxides, which are almost exclusively metabolized to nonprostanoate structures and only to a small extent to the classical prostaglandins. Platelets from subjects given aspirin produced less than 5% of the above mentioned amounts of HHT and PHD, whereas the production of HETE was stimulated about 3-fold. This provides additional evidence for our earlier proposal [Hamberg, M., Svensson, J., Wakabayashi, T. & Samuelsson, B. (1974) Proc. Nat. Acad. Sci. USA 71, 345-349] that the anti-aggregating effect of aspirin is through inhibition of PGG2 formation. PMID:4530264

  12. Prostaglandin-E2 Mediated Increase in Calcium and Phosphate Excretion in a Mouse Model of Distal Nephron Salt Wasting

    PubMed Central

    Soleimani, Manoocher; Barone, Sharon; Xu, Jie; Alshahrani, Saeed; Brooks, Marybeth; McCormack, Francis X.; Smith, Roger D.; Zahedi, Kamyar

    2016-01-01

    Contribution of salt wasting and volume depletion to the pathogenesis of hypercalciuria and hyperphosphaturia is poorly understood. Pendrin/NCC double KO (pendrin/NCC-dKO) mice display severe salt wasting under basal conditions and develop profound volume depletion, prerenal renal failure, and metabolic alkalosis and are growth retarded. Microscopic examination of the kidneys of pendrin/NCC-dKO mice revealed the presence of calcium phosphate deposits in the medullary collecting ducts, along with increased urinary calcium and phosphate excretion. Confirmatory studies revealed decreases in the expression levels of sodium phosphate transporter-2 isoforms a and c, increases in the expression of cytochrome p450 family 4a isotypes 12 a and b, as well as prostaglandin E synthase 1, and cyclooxygenases 1 and 2. Pendrin/NCC-dKO animals also had a significant increase in urinary prostaglandin E2 (PGE-2) and renal content of 20-hydroxyeicosatetraenoic acid (20-HETE) levels. Pendrin/NCC-dKO animals exhibit reduced expression levels of the sodium/potassium/2chloride co-transporter 2 (NKCC2) in their medullary thick ascending limb. Further assessment of the renal expression of NKCC2 isoforms by quantitative real time PCR (qRT-PCR) reveled that compared to WT mice, the expression of NKCC2 isotype F was significantly reduced in pendrin/NCC-dKO mice. Provision of a high salt diet to rectify volume depletion or inhibition of PGE-2 synthesis by indomethacin, but not inhibition of 20-HETE generation by HET0016, significantly improved hypercalciuria and salt wasting in pendrin/NCC dKO mice. Both high salt diet and indomethacin treatment also corrected the alterations in NKCC2 isotype expression in pendrin/NCC-dKO mice. We propose that severe salt wasting and volume depletion, irrespective of the primary originating nephron segment, can secondarily impair the reabsorption of salt and calcium in the thick ascending limb of Henle and/or proximal tubule, and reabsorption of sodium and

  13. Evaluation of genes involved in prostaglandin action in equine endometrium during estrous cycle and early pregnancy.

    PubMed

    Atli, Mehmet O; Kurar, Ercan; Kayis, Seyit A; Aslan, Selim; Semacan, Ahmet; Celik, Sefa; Guzeloglu, Aydin

    2010-10-01

    The aim was to evaluate expression of genes involved in the biosynthesis of prostaglandins (PTG), Prostaglandin H Synthase-1 (PTGS1) and PTGS2, PGF synthase (PTGFS), and PGE synthase (PTGES), PGF receptor (PTGFR), PGE receptors (PTGER2 and PTGER4), prostaglandin transporter (SLCO2A1) and hydroxyprostaglandin dehydrogenase-15 (HPGD). Endometrial biopsies were obtained from mares on day of ovulation (d0, n=4), late diestrus (LD, n=4), early luteolysis (EL, n=4) and after luteolysis (AL, n=4) during the cycle. Stages of the cycle were confirmed by plasma progesterone concentrations measured daily and ultrasound examinations. Biopsies were also taken on days 14 (P14; n=4), 15 (P15, n=4), 18 (P18, n=4) and 22 (P22; n=4) of pregnancy. Relative mRNA expressions were quantified using real-time RT-PCR. A mixed model was fitted on the normalized data and least significant difference test (α=0.05) was employed. Expression of PTGS1 mRNA was low throughout the estrous cycle and early days of pregnancy, but upregulated on P18 and P22. PTGS2 expression was increased on EL, but it was suppressed by pregnancy on P15, P18, and P22. PTGFS expression was upregulated in both cyclic and pregnant mares compared to d0 and its level was the highest on LD. PTGFR expression was transiently increased on LD and EL and was suppressed during early pregnancy. Both PTGES and PTGER2 expressions were increased on LD, EL, and early pregnancy, but were decreased after the luteolysis in cyclic mares as they remained high on P18 and P22. PTGER4 expression did not change throughout the cycle and early pregnancy. Levels of HPGD and SLCO2A1 were significantly increased only on P22. In conclusion, PTGS2 expression increases around the time of luteolysis and concurrent upregulation of PTGFS and PTGES indicates that equine endometrium has increased capability of PTG production around the time of luteolysis. However, pregnancy reduces PTGS2 expression, but maintains the high levels of PTGES during early

  14. Inhibitory effects of β-chamigrenal, isolated from the fruits of Schisandra chinensis, on lipopolysaccharide-induced nitric oxide and prostaglandin E2 production in RAW 264.7 macrophages [corrected].

    PubMed

    Shin, Ji-Sun; Ryu, Suran; Cho, Young-Wuk; Kim, Hyun Ji; Jang, Dae Sik; Lee, Kyung-Tae

    2014-06-01

    Much is known about the bioactive properties of lignans from the fruits of Schisandra chinensis. However, very little work has been done to determine the properties of sesquiterpenes in the fruits of S. chinensis. The aim of the present study was to investigate the anti-inflammatory potential of new sesquiterpenes (β-chamigrenal, β-chamigrenic acid, α-ylangenol, and α-ylangenyl acetate) isolated from the fruits of S. chinensis and to explore their effect on macrophages stimulated with lipopolysaccharide. Of these four sesquiterpenes, β-chamigrenal most significantly suppressed lipopolysaccharide-induced nitric oxide and prostaglandin E2 production in RAW 264.7 macrophages (47.21 ± 4.54 % and 51.61 ± 3.95 % at 50 µM, respectively). Molecularly, the inhibitory activity of β-chamigrenal on nitric oxide production was mediated by suppressing inducible nitric oxide synthase activity but not its expression. In the prostaglandin E2 synthesis pathway, β-chamigrenal prevented the upregulation of inducible microsomal prostaglandin E synthase-1 expression after stimulation with lipopolysaccharide. Conversely, β-chamigrenal had no effect on the expression and enzyme activity of cyclooxygenase-2. In addition, the expression of early growth response factor-1, a key transcription factor of microsomal prostaglandin E synthase-1 expression, was inhibited by β-chamigrenal. These results may suggest a possible anti-inflammatory activity of β-chamigrenal which has to be proven in in vivo experiments.

  15. Cyclooxygenase-1-Selective Inhibitors Based on the (E)-2′-Des-methyl-sulindac Sulfide Scaffold

    PubMed Central

    2012-01-01

    Prostaglandins (PGs) are powerful lipid mediators in many physiological and pathophysiological responses. They are produced by oxidation of arachidonic acid (AA) by cyclooxygenases (COX-1 and COX-2) followed by metabolism of endoperoxide intermediates by terminal PG synthases. PG biosynthesis is inhibited by nonsteroidal anti-inflammatory drugs (NSAIDs). Specific inhibition of COX-2 has been extensively investigated, but relatively few COX-1-selective inhibitors have been described. Recent reports of a possible contribution of COX-1 in analgesia, neuroinflammation, or carcinogenesis suggest that COX-1 is a potential therapeutic target. We designed, synthesized, and evaluated a series of (E)-2′-des-methyl-sulindac sulfide (E-DMSS) analogues for inhibition of COX-1. Several potent and selective inhibitors were discovered, and the most promising compounds were active against COX-1 in intact ovarian carcinoma cells (OVCAR-3). The compounds inhibited tumor cell proliferation but only at concentrations >100-fold higher than the concentrations that inhibit COX-1 activity. E-DMSS analogues may be useful probes of COX-1 biology in vivo and promising leads for COX-1-targeted therapeutic agents. PMID:22263894

  16. Prostaglandin I2 and prostaglandin E2 modulate human intrarenal artery contractility through prostaglandin E2-EP4, prostacyclin-IP, and thromboxane A2-TP receptors.

    PubMed

    Eskildsen, Morten P; Hansen, Pernille B L; Stubbe, Jane; Toft, Anja; Walter, Steen; Marcussen, Niels; Rasmussen, Lars M; Vanhoutte, Paul M; Jensen, Boye L

    2014-09-01

    Cyclooxygenase inhibitors decrease renal blood flow in settings with decreased effective circulating volume. The present study examined the hypothesis that prostaglandins, prostaglandin E2 (PGE2) and prostacyclin (PGI2), induce relaxation of human intrarenal arteries through PGE2-EP and PGI2-IP receptors. Intrarenal arteries were microdissected from human nephrectomy samples (n=53, median diameter ≈362 μm, 88% viable, 76% relaxed in response to acetylcholine). Rings were suspended in myographs to record force development. In vessels with K(+)-induced tension (EC70: -log [mol/L]=1.36±0.03), PGE2 and PGI2 induced concentration-dependent relaxation (-log EC50: PGE2=7.1±0.3 and PGI2=7.7). The response to PGE2 displayed endothelium dependence and desensitization. Relaxation by PGE2 was mimicked by an EP4 receptor agonist (CAY10598, EC50=6.7±0.2). The relaxation after PGI2 was abolished by an IP receptor antagonist (BR5064, 10(-8) mol/L). Pretreatment of quiescent arteries with PGE2 for 5 minutes (10(-6) mol/L) led to a significant right shift of the concentration-response to norepinephrine (EC50 from 6.6±0.1-5.9±0.1). In intrarenal arteries with K(+)-induced tone, PGE2 and PGI2 at 10(-5) mol/L elicited increased tension. This was abolished by thromboxane receptor (TP) antagonist (S18886, 10(-6) mol/L). A TP agonist (U46619, n=6) evoked tension (EC50=8.1±0.2) that was inhibited by S18886. Polymerase chain reaction and immunoblotting showed EP4, IP, and TP receptors in intrarenal arteries. In conclusion, PGE2 and PGI2 may protect renal perfusion by activating cognate IP and EP4 receptors associated with smooth muscle cells and endothelium in human intrarenal arteries and contribute to increased renal vascular resistance at high pathological concentrations mediated by noncognate TP receptor.

  17. Captopril-induced Changes in Prostaglandin Production

    PubMed Central

    Swartz, Stephen L.; Williams, Gordon H.; Hollenberg, Norman K.; Levine, Lawrence; Dluhy, Robert G.; Moore, Thomas J.

    1980-01-01

    Captopril is a potent hypotensive agent whose efficacy has hitherto been attributed to its ability to alter either angiotensin II formation or kinin degradation. Our purpose was to examine captopril's acute effect on prostaglandin production, because changes in neither the renin-angiotensin nor the kallikrein-kinin systems appear adequate to account for the fall in arterial pressure. The plasma levels of angiotensin II, kinins, and prostaglandins were determined in response to increasing doses (5, 12.5, and 25 mg) of captopril and these responses were compared with the change in arterial pressure observed in nine supine normal male subjects studied on both a high (200 meq) and low (10 meq) sodium intake. Captopril significantly (P < 0.01) increased the levels of the 13,14-dihydro-15-keto metabolite of prostaglandin E2 (PGE2-M), a potent vasodilator, with similar responses being observed on both a high and a low sodium intake. No significant changes in the plasma levels of 6-keto-prostaglandin F 1α, or thromboxane B2, the stable products of prostacyclin and thromboxane A2, respectively, occurred. The depressor response to captopril correlated with the change in PGE2-M (r = 0.52, t = 5.44, P < 0.0001). On the other hand, although significant (P < 0.02) decrements in angiotensin II and increments in plasma kinins accompanied the hypotensive response in sodium-restricted subjects, in sodium-loaded subjects where the renin-angiotensin system was suppressed, no change in angiotensin II, and only a modest change in kinins was noted, even though significant (P < 0.01) decrements in diastolic blood pressure occurred (−10±2 mm Hg). Thus, changes in depressor prostaglandin production can better account for the hypotensive response to captopril, thereby extending to yet another vasoactive system an influence by this class of drugs and providing a new approach to dissecting the abnormality in the control of vascular tone in patients with hypertension. PMID:6997332

  18. Cell cycle arrest by prostaglandin A1 at the G1/S phase interface with up-regulation of oncogenes in S-49 cyc- cells

    NASA Technical Reports Server (NTRS)

    Hughes-Fulford, M.

    1994-01-01

    Our previous studies have implied that prostaglandins inhibit cell growth independent of cAMP. Recent reports, however, have suggested that prostaglandin arrest of the cell cycle may be mediated through protein kinase A. In this report, in order to eliminate the role of c-AMP in prostaglandin mediated cell cycle arrest, we use the -49 lymphoma variant (cyc-) cells that lack adenylate cyclase activity. We demonstrate that dimethyl prostaglandin A1 (dmPGA1) inhibits DNA synthesis and cell growth in cyc- cells. DNA synthesis is inhibited 42% by dmPGA1 (50 microM) despite the fact that this cell line lacks cellular components needed for cAMP generation. The ability to decrease DNA synthesis depends upon the specific prostaglandin structure with the most effective form possessing the alpha, beta unsaturated ketone ring. Dimethyl PGA1 is most effective in inhibiting DNA synthesis in cyc- cells, with prostaglandins PGE1 and PGB1 being less potent inhibitors of DNA synthesis. DmPGE2 caused a significant stimulation of DNA synthesis. S-49 cyc- variant cells exposed to (30-50 microns) dmPGA1, arrested in the G1 phase of the cell cycle within 24 h. This growth arrest was reversed when the prostaglandin was removed from the cultured cells; growth resumed within hours showing that this treatment is not toxic. The S-49 cyc- cells were chosen not only for their lack of adenylate cyclase activity, but also because their cell cycle has been extensively studied and time requirements for G1, S, G2, and M phases are known. Within hours after prostaglandin removal the cells resume active DNA synthesis, and cell number doubles within 15 h suggesting rapid entry into S-phase DNA synthesis from the G1 cell cycle block.(ABSTRACT TRUNCATED AT 250 WORDS).

  19. Pro-oxidant activity of indicaxanthin from Opuntia ficus indica modulates arachidonate metabolism and prostaglandin synthesis through lipid peroxide production in LPS-stimulated RAW 264.7 macrophages.

    PubMed

    Allegra, M; D'Acquisto, F; Tesoriere, L; Attanzio, A; Livrea, M A

    2014-01-01

    Macrophages come across active prostaglandin (PG) metabolism during inflammation, shunting early production of pro-inflammatory towards anti-inflammatory mediators terminating the process. This work for the first time provides evidence that a phytochemical may modulate the arachidonate (AA) metabolism in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages, promoting the ultimate formation of anti-inflammatory cyclopentenone 15deoxy-PGJ2. Added 1 h before LPS, indicaxanthin from Opuntia Ficus Indica prevented activation of nuclear factor-κB (NF-κB) and over-expression of PGE2 synthase-1 (mPGES-1), but up-regulated cyclo-oxygenase-2 (COX-2) and PGD2 synthase (H-PGDS), with final production of the anti-inflammatory cyclopentenone. The effects were positively related with concentration between 50 and 100 µM. Indicaxanthin did not have any effect in the absence of LPS. A kinetic study investigating the redox status of LPS-stimulated macrophages between 0.5 and 12 h, either in the absence or in the presence of 50-100 µM indicaxanthin, revealed a differential control of ROS production, with early (0.5-3 h) modest inhibition, followed by a progressive (3-12 h) concentration-dependent enhancement over the level induced by LPS alone. In addition, indicaxanthin caused early (0.5-3 h) concentration-dependent elevation of conjugated diene lipid hydroperoxides, and production of hydroxynonenal-protein adducts, over the amount induced by LPS. In LPS-stimulated macrophages indicaxanthin did not affect PG metabolism when co-incubated with either an inhibitor of NADPH oxidase or vitamin E. It is concluded that LPS-induced pro-oxidant activity of indicaxanthin at the membrane level allows formation of signaling intermediates whose accumulation modulates PG biosynthetic pathway in inflamed macrophages.

  20. Role of IL-1 beta and prostaglandins in beta 2-microglobulin-induced bone mineral dissolution.

    PubMed

    Moe, S M; Hack, B K; Cummings, S A; Sprague, S M

    1995-02-01

    beta 2-microglobulin (beta 2m) induces an osteoclast-mediated net calcium efflux from neonatal mouse calvariae which occurs only after 48 hours of incubation, suggesting that beta 2m acts via other growth factors. To further test this hypothesis, calvariae were incubated with and without beta 2m in the presence of the prostaglandin inhibitor indomethacin, anti-interleukin-1 beta antibody (anti-IL-1 beta), or interleukin-1 beta receptor antagonist (IL-1 beta RA). The addition of beta 2m to the culture medium stimulated, whereas indomethacin inhibited basal calcium efflux following 48 hours. However, the difference (delta) between the calcium efflux induced in calvariae incubated with and without beta 2m in basal medium and that in calvariae incubated with and without beta 2m in indomethacin supplemented medium was similar, suggesting a prostaglandin independent mechanism. There was a time dependent increase in PGE2 in basal medium which was unaffected by beta 2m. In contrast, pre-incubating calvariae with either anti-IL-1 beta or IL-1 beta RA did not alter basal calcium efflux but completely blocked the beta 2m induced calcium efflux. Anti-IL-1 beta had no effect on the basal release of beta-glucuronidase but partially blocked the beta 2m induced release of beta-glucuronidase. Thus, the beta 2m-induced calcium efflux observed in neonatal mouse calvariae is dependent on interleukin-1 beta but not prostaglandins.

  1. Major role of adipocyte prostaglandin E2 in lipolysis-induced macrophage recruitment.

    PubMed

    Hu, Xiaoqian; Cifarelli, Vincenza; Sun, Shishuo; Kuda, Ondrej; Abumrad, Nada A; Su, Xiong

    2016-04-01

    Obesity induces accumulation of adipose tissue macrophages (ATMs), which contribute to both local and systemic inflammation and modulate insulin sensitivity. Adipocyte lipolysis during fasting and weight loss also leads to ATM accumulation, but without proinflammatory activation suggesting distinct mechanisms of ATM recruitment. We examined the possibility that specific lipid mediators with anti-inflammatory properties are released from adipocytes undergoing lipolysis to induce macrophage migration. In the present study, we showed that conditioned medium (CM) from adipocytes treated with forskolin to stimulate lipolysis can induce migration of RAW 264.7 macrophages. In addition to FFAs, lipolytic stimulation increased release of prostaglandin E2(PGE2) and prostaglandin D2(PGD2), reflecting cytosolic phospholipase A2α activation and enhanced cyclooxygenase (COX) 2 expression. Reconstituted medium with the anti-inflammatory PGE2potently induced macrophage migration while different FFAs and PGD2had modest effects. The ability of CM to induce macrophage migration was abolished by treating adipocytes with the COX2 inhibitor sc236 or by treating macrophages with the prostaglandin E receptor 4 antagonist AH23848. In fasted mice, macrophage accumulation in adipose tissue coincided with increases of PGE2levels and COX1 expression. Collectively, our data show that adipocyte-originated PGE2with inflammation suppressive properties plays a significant role in mediating ATM accumulation during lipolysis. PMID:26912395

  2. Muscle sympathetic nerve responses to physiological changes in prostaglandin production in humans

    NASA Technical Reports Server (NTRS)

    Doerzbacher, K. J.; Ray, C. A.

    2001-01-01

    Previous studies suggest that prostaglandins may contribute to exercise-induced increases in muscle sympathetic nerve activity (MSNA). To test this hypothesis, MSNA was measured at rest and during exercise before and after oral administration of ketoprofen, a cyclooxygenase inhibitor, or placebo. Twenty-one subjects completed two bouts of graded dynamic and isometric handgrip to fatigue. Each exercise bout was followed by 2 min of postexercise muscle ischemia. The second exercise bouts were performed after 60 min of rest in which 11 subjects were given ketoprofen (300 mg) and 10 subjects received a placebo. Ketoprofen significantly lowered plasma thromboxane B(2) in the drug group (from 36 +/- 6 to 22 +/- 3 pg/ml, P < 0.04), whereas thromboxane B(2) in the placebo group increased from 40 +/- 5 to 61 +/- 9 pg/ml from trial 1 to trial 2 (P < 0.008). Ketoprofen and placebo did not change sympathetic and cardiovascular responses to dynamic handgrip, isometric handgrip, and postexercise muscle ischemia. There was no relationship between thromboxane B(2) concentrations and MSNA or arterial pressure responses during both exercise modes. The data indicate that physiological increases or decreases in prostaglandins do not alter exercise-induced increases in MSNA and arterial pressure in humans. These findings suggest that contraction-induced metabolites other than prostaglandins mediate MSNA responses to exercise in humans.

  3. Release of prostaglandins from the isolated frog ventricle and associated changes in endogenous cyclic nucleotide levels.

    PubMed Central

    Flitney, F W; Singh, J

    1980-01-01

    1. A study has been made of the decline in contractility and some associated metabolic changes which occur in the isolated frog ventricle during the development of hypodynamic depression. 2. The release of two identified prostaglandins (PG), E1 and E2, together with several as yet unknown prostaglandin-related substances (PRS), accompanies the development of hypodynamic depression. There is a close correlation between the extent to which the isometric twitch is depressed and the quantity of prostaglandin released into the superfusate. 3. Fractionation of extracts of 'used' superfusates, using preparative-scale thin-layer chromatography, revealed the presence of six major components, four of which (PGE1 and PGE2 and two unidentified components) were found to be cardioactive and potentiated contraction when tested subsequently on hypodynamic preparations. 4. Two agents which influence prostaglandin biosynthesis, arachidonic acid and indomethacin, are found to affect both the rate at which the hypodynamic state develops and the extent to which the 'steady-state' twitch tension is depressed, in a dose-dependent manner. Indomethacin, a PG-synthetase inhibitor, accelerates the decay and depresses the final 'steady-state' tension attained, whereas arachidonic acid, the principal precursor for prostaglandin biosynthesis, has the converse effects. 5. Measurements of endogenous 3'5'-cyclic nucleotide levels reveal a time-dependent decrease in intracellular adenosine 3'5'-cyclic monophosphate (3'5'-cyclic AMP) and a concomitant increase in guanosine 3'5' cyclic monophosphate (3'5'-cyclic GMP). The decline in isometric twitch tension is paralleled almost exactly by an equivalent reduction in the ratio 3'5'-cyclic AMP: 3'5'-cyclic GMP. 6. Superfusion of isolated ventricles with Ringer solution containing exogenous, lipid-soluble derivatives of 3'5'-cyclic AMP and 3'5'-cyclic GMP affects both the rate of decline of the isometric twitch and the steady-state tension ultimately

  4. ATP Synthase: A Molecular Therapeutic Drug Target for Antimicrobial and Antitumor Peptides

    PubMed Central

    Ahmad, Zulfiqar; Okafor, Florence; Azim, Sofiya; Laughlin, Thomas F.

    2015-01-01

    In this review we discuss the role of ATP synthase as a molecular drug target for natural and synthetic antimi-crobial/antitumor peptides. We start with an introduction of the universal nature of the ATP synthase enzyme and its role as a biological nanomotor. Significant structural features required for catalytic activity and motor functions of ATP synthase are described. Relevant details regarding the presence of ATP synthase on the surface of several animal cell types, where it is associated with multiple cellular processes making it a potential drug target with respect to antimicrobial peptides and other inhibitors such as dietary polyphenols, is also reviewed. ATP synthase is known to have about twelve discrete inhibitor binding sites including peptides and other inhibitors located at the interface of α/β subunits on the F1 sector of the enzyme. Molecular interaction of peptides at the β DEELSEED site on ATP synthase is discussed with specific examples. An inhibitory effect of other natural/synthetic inhibitors on ATP is highlighted to explore the therapeutic roles played by peptides and other inhibitors. Lastly, the effect of peptides on the inhibition of the Escherichia coli model system through their action on ATP synthase is presented. PMID:23432591

  5. Cutaneous vascular and sweating responses to intradermal administration of prostaglandin E1 and E2 in young and older adults: a role for nitric oxide?

    PubMed

    Fujii, Naoto; Singh, Maya Sarah; Halili, Lyra; Boulay, Pierre; Sigal, Ronald J; Kenny, Glen P

    2016-06-01

    Cyclooxygenase (COX) contributes to cutaneous vasodilation and sweating responses; however, the mechanisms underpinning these responses remain unknown. We hypothesized that prostaglandin E1 (PGE1) and E2 (PGE2) (COX-derived vasodilator products) directly mediate cutaneous vasodilation and sweating through nitric oxide synthase (NOS)-dependent mechanisms in young adults. Furthermore, we hypothesized that this response is diminished in older adults, since aging attenuates COX-dependent cutaneous vasodilation and sweating. In 9 young (22 ± 5 yr) and 10 older (61 ± 6 yr) adults, cutaneous vascular conductance (CVC) and sweat rate were evaluated at four intradermal forearm skin sites receiving incremental doses (0.05, 0.5, 5, 50, 500 μM each for 25 min) of PGE1 or PGE2 with and without coadministration of 10 mM N(ω)-nitro-l-arginine, a nonspecific NOS inhibitor. N(ω)-nitro-l-arginine attenuated PGE1-mediated increases in CVC at all concentrations in young adults, whereas it reduced PGE2-mediated increases in CVC at lower concentrations (0.05-0.5 μM) in older adults (all P < 0.05). However, the magnitude of the PGE1- and PGE2-mediated increases in CVC did not differ between groups (all P > 0.05). Neither PGE1 nor PGE2 increased sweat rate at any of the administered concentrations for either the young or older adults (all P > 0.05). We show that although cutaneous vascular responsiveness to PGE1 and PGE2 is similar between young and older adults, the cutaneous vasodilator response is partially mediated through NOS albeit via low-to-high concentrations of PGE1 in young adults and low concentrations of PGE2 in older adults, respectively. We also show that in both young and older adults, PGE1 and PGE2 do not increase sweat rate under normothermic conditions.

  6. Prostaglandin-secreting cells: a portable first aid kit for tissue repair

    PubMed Central

    Rakoff-Nahoum, Seth; Medzhitov, Ruslan

    2007-01-01

    After intestinal injury, both the number and type of intestinal epithelial cells must be restored. Intestinal stem cells, located at the base of the intestinal crypt, repopulate the depleted crypt in a process known as compensatory proliferation. In this issue of the JCI, Brown et al. describe a new mechanism by which this process is regulated (see the related article beginning on page 258). Surprisingly, they find that a subset of stromal cells present within the intestinal tissue and expressing the proliferative factor prostaglandin-endoperoxidase synthase 2 (Ptgs2) is repositioned next to the intestinal stem cell compartment where local production of PGE2 controls injury-induced epithelial cell proliferation. PMID:17200710

  7. Interaction between inducible nitric oxide synthase and cyclooxygenase-2 after cerebral ischemia.

    PubMed

    Nogawa, S; Forster, C; Zhang, F; Nagayama, M; Ross, M E; Iadecola, C

    1998-09-01

    Focal cerebral ischemia is associated with expression of both inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), enzymes whose reaction products contribute to the evolution of ischemic brain injury. We tested the hypothesis that, after cerebral ischemia, nitric oxide (NO) produced by iNOS enhances COX-2 activity, thereby increasing the toxic potential of this enzyme. Cerebral ischemia was produced by middle cerebral artery occlusion in rats or mice. Twenty-four hours after ischemia in rats, iNOS-immunoreactive neutrophils were observed in close proximity (<20 micrometer) to COX-2-positive cells at the periphery of the infarct. In the olfactory bulb, only COX-2 positive cells were observed. Cerebral ischemia increased the concentration of the COX-2 reaction product prostaglandin E2 (PGE2) in the ischemic area and in the ipsilateral olfactory bulb. The iNOS inhibitor aminoguanidine reduced PGE2 concentration in the infarct, where both iNOS and COX-2 were expressed, but not in the olfactory bulb, where only COX-2 was expressed. Postischemic PGE2 accumulation was reduced significantly in iNOS null mice compared with wild-type controls (C57BL/6 or SV129). The data provide evidence that NO produced by iNOS influences COX-2 activity after focal cerebral ischemia. Pro-inflammatory prostanoids and reactive oxygen species produced by COX-2 may be a previously unrecognized factor by which NO contributes to ischemic brain injury. The pathogenic effect of the interaction between NO, or a derived specie, and COX-2 is likely to play a role also in other brain diseases associated with inflammation.

  8. Investigation of potential inhibitors of chorismate-utilizing enzymes.

    PubMed

    Švarcová, Markéta; Krátký, Martin; Vinšova, Jarmila

    2015-01-01

    Chorismate-utilizing enzymes (CUE) such as chorismate mutase, anthranilate synthase, chorismate pyruvate-lyase, 4-amino-4-deoxychorismate synthase, isochorismate synthase and salicylate synthase are responsible for converting chorismate into various products necessary for the survival of bacteria. The absence of these enzymes in humans and their importance in the virulence and survival of bacteria make them suitable targets for potential antimicrobial compounds. Furthermore, the CUE have significant structural homology and similar catalytic mechanisms, enabling the strategy of affecting multiple enzymes with one single inhibitor. This review follows up the investigation of mechanisms of CUE-catalysed reactions and the concurrent development of CUE inhibitors. Many active compounds were found amongst the structures mimicking the transition state of chorismate during the reaction. Most recently, high nanomolar and low micromolar inhibitors against isochorismate-pyruvate lyase were identified, which were also effective against chorismate mutase and salicylate synthase and belong to the most active inhibitors reported up to date.

  9. Therapeutic targets in prostaglandin E2 signaling for neurologic disease

    PubMed Central

    Cimino, P.J.; Keene, C. Dirk; Breyer, Richard M.; Montine, Kathleen S.; Montine, Thomas J.

    2009-01-01

    Prostaglandins (PGs) are potent autocrine and paracrine oxygenated lipid molecules that contribute appreciably to physiologic and pathophysiologic responses in almost all organs, including brain. Emerging data indicate that the PGs, and more specifically PGE2, play a central role in brain diseases including ischemic injury and several neurodegenerative diseases. Given concerns over the potential toxicity from protracted use of cyclooxygenase inhibitors in the elderly, attention is now focused on blocking PGE2 signaling that is mediated by interactions with four distinct G protein-coupled receptors, EP1-4, which are differentially expressed on neuronal and glial cells throughout the central nervous system. EP1 activation has been shown to mediate Ca2+-dependent neurotoxicity in ischemic injury. EP2 activation has been shown to mediate microglial-induced paracrine neurotoxicity as well as suppress microglia internalization of aggregated neurotoxic peptides. Animal models support the potential efficacy of targeting specific EP receptor subtypes in Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and ischemic stroke. However promising these preclinical studies are, they have yet to be followed by clinical trials targeting any EP receptor in neurologic diseases. PMID:18691044

  10. Hangover headache and prostaglandins: prophylactic treatment with tolfenamic acid.

    PubMed

    Kaivola, S; Parantainen, J; Osterman, T; Timonen, H

    1983-03-01

    Tolfenamic acid (TA), a potent inhibitor of prostaglandin (PG) biosynthesis and action, was tested prophylactically against hangover symptoms in 30 healthy volunteers in a double-blind cross-over study. One capsule of TA (200 mg) or placebo was taken before starting to drink alcohol and another before going to bed. The hangover symptoms were evaluated in the morning. TA was found significantly better than placebo in the subjective evaluation of drug efficacy (p less than 0.001) and in reducing the reported hangover symptoms in general (p less than 0.01). In the TA group, significantly lower symptom scores were obtained for headache (p less than 0.01), and for nausea, vomiting, irritation, tremor, thirst and dryness of mouth (all p less than 0.05). In a separate study with eight participants, plasma levels of PGs were followed during ingestion of alcohol with or without TA. The plasma concentrations of PGE2 and TXB2 (a metabolite of thromboxane A2) were lower in the TA group during alcohol ingestion, while PGF2 alpha and 6-keto-PGF1 alpha (a metabolite of prostacyclin) were unaffected. TXB2 correlated with blood alcohol levels in a U-shaped manner.

  11. Distribution of Prostaglandin E2 in Gastric and Duodenal Mucosa: Possible Role in the Pathogenesis of Peptic Ulcer

    PubMed Central

    Park, Sill Moo; Yoo, Byung Chul; Lee, Hyo Rang; Chung, Hyuk; Lee, Young Soon

    1992-01-01

    Background Prostaglandin E which is present abundantly in the gastric mucosa is a powerful inhibitor of gastric acid secretion and a stimulus to gastric mucus production. In addition, prostaglandin E2 inhibits ulcer formation in animals, and the synthetic analogues of prostaglandin E have successfully been used in the treatment of patients with gastric and duodenal ulcer disease. To evaluate the role of endogenous prostaglandin E2 in the pathogenesis of the peptic ulcer disease, we measured mucosal prostaglandin E2 levels in patients with gastric and duodenal ulcer disease and compared with that of non-ulcer control persons. Methods The study population was made up of 44 non-ulcer persons, 36 patients with a benign gastric ulcer, and 48 with a duodenal ulcer. Every mucosai specimen, taken from the antrum and from the duodenal bulb, were homogenized, mixed with 1 M HCI, and centrifuged. After removal of the supernatant, precipitate was eluted with ethyl acetate in the Amprep C18 minicolumn. Then the extracted prostaglandin E2 in the ethyl acetate fractions was converted into its methyl oximate derivatives, and the prostaglandin E2 level was measured by radioimmunoassay. During the procedure any homogenized specimen which was looking grossly bloody was removed from the assay in order to avoid any possible contamination or prostaglandin E2 in blood. Results In non-ulcer persons, the mean values was 258.17±127.03 pg/mg. tissue in antrum and 121.07±67.46 pg/mg. tissue in duodenal bulb. The corresponding values were 186.42±70.51 pg/mg. tissue, 79.44±39.04 pg/mg. tissue in gastric ulcer patients and 204. 94 92.03 pg/mg. tissue, 99.66±56.10 pg/mgl. tissue in duodenal ulcer patients respectively. Gastric ulcer patients have the significantly lower level of the antral and duodenal prostaglandin E2 (p<0.005). Those levels of duodenal ulcer patients were also significantly lower than those of non-ulcer persons (p<0.025 & 0.05). Antral prostaglandin E2 level increased to

  12. Prostaglandin E Receptor Subtype 4 Signaling in the Heart: Role in Ischemia/Reperfusion Injury and Cardiac Hypertrophy

    PubMed Central

    Cai, Yin; Tang, Eva Hoi Ching; Ma, Haichun

    2016-01-01

    Prostaglandin E2 (PGE2) is an endogenous lipid mediator, produced from the metabolism of arachidonic acids, upon the sequential actions of phospholipase A2, cyclooxygenases, and prostaglandin E synthases. The various biological functions governed by PGE2 are mediated through its four distinct prostaglandin E receptors (EPs), designated as EP1, EP2, EP3, and EP4, among which the EP4 receptor is the one most widely distributed in the heart. The availability of global or cardiac-specific EP4 knockout mice and the development of selective EP4 agonists/antagonists have provided substantial evidence to support the role of EP4 receptor in the heart. However, like any good drama, activation of PGE2-EP4 signaling exerts both protective and detrimental effects in the ischemic heart disease. Thus, the primary object of this review is to provide a comprehensive overview of the current progress of the PGE2-EP4 signaling in ischemic heart diseases, including cardiac hypertrophy and myocardial ischemia/reperfusion injury. A better understanding of PGE2-EP4 signaling should promote the development of more effective therapeutic approaches to treat the ischemic heart diseases without triggering unwanted side effects. PMID:27190998

  13. Targeted prostaglandin E2 inhibition enhances antiviral immunity through induction of type I interferon and apoptosis in macrophages.

    PubMed

    Coulombe, François; Jaworska, Joanna; Verway, Mark; Tzelepis, Fanny; Massoud, Amir; Gillard, Joshua; Wong, Gary; Kobinger, Gary; Xing, Zhou; Couture, Christian; Joubert, Philippe; Fritz, Jörg H; Powell, William S; Divangahi, Maziar

    2014-04-17

    Aspirin gained tremendous popularity during the 1918 Spanish Influenza virus pandemic, 50 years prior to the demonstration of their inhibitory action on prostaglandins. Here, we show that during influenza A virus (IAV) infection, prostaglandin E2 (PGE2) was upregulated, which led to the inhibition of type I interferon (IFN) production and apoptosis in macrophages, thereby causing an increase in virus replication. This inhibitory role of PGE2 was not limited to innate immunity, because both antigen presentation and T cell mediated immunity were also suppressed. Targeted PGE2 suppression via genetic ablation of microsomal prostaglandin E-synthase 1 (mPGES-1) or by the pharmacological inhibition of PGE2 receptors EP2 and EP4 substantially improved survival against lethal IAV infection whereas PGE2 administration reversed this phenotype. These data demonstrate that the mPGES-1-PGE2 pathway is targeted by IAV to evade host type I IFN-dependent antiviral immunity. We propose that specific inhibition of PGE2 signaling might serve as a treatment for IAV.

  14. Effect of forskolin on alterations of vascular permeability induced with bradykinin, prostaglandin E1, adenosine, histamine and carrageenin in rats.

    PubMed

    Sugio, K; Daly, J W

    1983-07-01

    The effect of the diterpene forskolin on vascular permeability alone and in combination with bradykinin, prostaglandin E1, adenosine or histamine has been investigated in rats. Vascular permeability in rat skin was measured using [125I]-labelled bovine serum albumin ([125I]BSA) as a tracer. In addition, the effect of forskolin on footpad edema induced by the injection of a mixture of 2% carrageenin was determined. Forskolin caused a marked potentiation of the increase in vascular permeability in rat skin elicited by the intradermal injection of histamine or bradykinin. However, forskolin caused a significant suppression of the prostaglandin E1-induced vascular permeability response and at a low concentration suppressed the response to adenosine. Forskolin greatly potentiated the footpad edema induced with carrageenin in rats. Intravenous administration of the enzyme bromelain, which reduces plasma kininogen levels, inhibited the footpad edema induced with carrageenin or with a mixture of carrageenin and forskolin. Parenteral administration of a prostaglandin synthetase inhibitor, indomethacin, suppressed the footpad edema induced with carrageenin, but did not inhibit the footpad edema induced with a mixture of carrageenin and forskolin. An antihistamine, cyproheptadine, had no effect on carrageenin-induced footpad edema either in the presence or absence of forskolin. These results suggest that both bradykinin and prostaglandins are essential for the development of carrageenin-induced footpad edema and that bradykinin plays an important role in the potentiative effect of forskolin on footpad edema induced with carrageenin in rats.

  15. Gastroprotective effects of thymol on acute and chronic ulcers in rats: The role of prostaglandins, ATP-sensitive K(+) channels, and gastric mucus secretion.

    PubMed

    Ribeiro, Ana Roseli S; Diniz, Polyana B F; Pinheiro, Malone S; Albuquerque-Júnior, Ricardo L C; Thomazzi, Sara M

    2016-01-25

    Thymol, a monoterpene phenol derivative of cymene, is found in abundance in the essential oils of Thymus, Origanum, and Lippia species. The present study investigated the gastroprotective actions of thymol (10, 30, and 100 mg/kg, p.o.) in the acute (ethanol- and nonsteroidal anti-inflammatory drug-induced ulcers) and chronic (acetic acid-induced ulcers) ulcer models in rats. Some of the mechanisms underlying to the gastroprotective effect of thymol were investigated in the ethanol-induced ulcer model. Gastric secretion parameters (volume, pH, and total acidity) were also evaluated by the pylorus ligature model, and the mucus in the gastric content was determined. The anti-Helicobacter pylori activity of thymol was performed using the agar-well diffusion method. Thymol (10, 30, and 100 mg/kg) produced dose dependent reduction (P < 0.01) on the total lesion area in the ethanol-induced ulcer model. The gastroprotective response caused by thymol (30 mg/kg) was significantly attenuated (P < 0.001) by intraperitoneal treatment of rats with indomethacin (a non-selective inhibitor of cyclo-oxygenase, 10 mg/kg) and glibenclamide (ATP-sensitive K(+) channel blocker, 10 mg/kg), but not by DL-Propargylglycine (PAG, a cystathionine-γ-lyase inhibitor, 25 mg/kg) and Nw-nitro-L-arginine methyl ester hydrochloride (L-NAME, a non-selective inhibitor of nitric oxide synthase, 70 mg/kg). Thymol (30 and 100 mg/kg) also reduced the ulcer index (P < 0.05) and the total lesion area (P < 0.001) in the indomethacin- and acetic-acid-induced ulcer models, respectively. In the model pylorus ligature, the treatment with thymol failed to significantly change the gastric secretion parameters. However, after treatment with thymol (30 and 100 mg/kg), there was a significant increase (P < 0.01) in mucus production. Thymol no showed anti-H. pylori activity in vitro. Collectively, the present results provide convincing evidence that thymol displays gastroprotective actions on the acute and chronic

  16. Gastroprotective effects of thymol on acute and chronic ulcers in rats: The role of prostaglandins, ATP-sensitive K(+) channels, and gastric mucus secretion.

    PubMed

    Ribeiro, Ana Roseli S; Diniz, Polyana B F; Pinheiro, Malone S; Albuquerque-Júnior, Ricardo L C; Thomazzi, Sara M

    2016-01-25

    Thymol, a monoterpene phenol derivative of cymene, is found in abundance in the essential oils of Thymus, Origanum, and Lippia species. The present study investigated the gastroprotective actions of thymol (10, 30, and 100 mg/kg, p.o.) in the acute (ethanol- and nonsteroidal anti-inflammatory drug-induced ulcers) and chronic (acetic acid-induced ulcers) ulcer models in rats. Some of the mechanisms underlying to the gastroprotective effect of thymol were investigated in the ethanol-induced ulcer model. Gastric secretion parameters (volume, pH, and total acidity) were also evaluated by the pylorus ligature model, and the mucus in the gastric content was determined. The anti-Helicobacter pylori activity of thymol was performed using the agar-well diffusion method. Thymol (10, 30, and 100 mg/kg) produced dose dependent reduction (P < 0.01) on the total lesion area in the ethanol-induced ulcer model. The gastroprotective response caused by thymol (30 mg/kg) was significantly attenuated (P < 0.001) by intraperitoneal treatment of rats with indomethacin (a non-selective inhibitor of cyclo-oxygenase, 10 mg/kg) and glibenclamide (ATP-sensitive K(+) channel blocker, 10 mg/kg), but not by DL-Propargylglycine (PAG, a cystathionine-γ-lyase inhibitor, 25 mg/kg) and Nw-nitro-L-arginine methyl ester hydrochloride (L-NAME, a non-selective inhibitor of nitric oxide synthase, 70 mg/kg). Thymol (30 and 100 mg/kg) also reduced the ulcer index (P < 0.05) and the total lesion area (P < 0.001) in the indomethacin- and acetic-acid-induced ulcer models, respectively. In the model pylorus ligature, the treatment with thymol failed to significantly change the gastric secretion parameters. However, after treatment with thymol (30 and 100 mg/kg), there was a significant increase (P < 0.01) in mucus production. Thymol no showed anti-H. pylori activity in vitro. Collectively, the present results provide convincing evidence that thymol displays gastroprotective actions on the acute and chronic

  17. Multifactorial regulation of prostaglandin synthesis in preovulatory goldfish ovarian follicles.

    PubMed

    Kellner, R G; Van der Kraak, G

    1992-04-01

    Goldfish preovulatory ovarian follicles (prior to germinal vesicle breakdown) were utilized for studies investigating the actions of activators of different signal transduction pathways on prostaglandin (PG) production. The protein kinase C (PKC) activators phorbol 12-myristate 13-acetate (PMA; 100-400 nM), 1-oleoyl-2-acetylglycerol (5 and 25 micrograms/ml), and 1,2-dioctanoylglycerol (10 and 50 micrograms/ml) stimulated PGE production; the inactive phorbol 4 alpha-phorbol didecanoate, which does not activate PKC, had no effect. Calcium ionophore A23187 (0.25-4.0 microM) stimulated PGE production and acted in a synergistic manner with activators of PKC. Although produced in lower amounts than PGE, PGF was stimulated by PMA and A23187. The direct activator of phospholipase A2, melittin (0.1-1.0 microM), stimulated a dose-related increase in PGE production, whereas chloroquine (100 microM), a putative inhibitor of phospholipase A2, blocked basal and PMA + A23187-stimulated PGE production. Several drugs known to elevate intracellular levels of cAMP including the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.1-1.0 mM), forskolin (10 microM), and dibutyryl cAMP (dbcAMP; 5 mM) attenuate PMA + A23187-stimulated PGE production. Melittin-stimulated production of PGE was inhibited by dbcAMP, suggesting that the action of cAMP was distal to the activation of phospholipase A2. In summary, these studies demonstrate that activation of PKC and elevation of intracellular calcium levels stimulate PG production, in part, through activation of phospholipase A2. The adenylate cyclase/cAMP signalling pathway is inhibitory to PG production by goldfish ovarian follicles. PMID:1315582

  18. Pulmonary biosynthesis and metabolism of prostaglandins and related substances.

    PubMed Central

    Eling, T E; Ally, A I

    1984-01-01

    On passage through the lung vascular bed, prostaglandins are removed from the circulation by a transport carrier and subsequently inactivated by intracellular enzymes. However, PGI2 is not inactivated by the lung in vivo. Although PGI2 is an excellent substrate for the intracellular enzymes in vitro, PGI2 is not a substrate for the carrier system. Thus, the transport carrier determines which circulating prostaglandin is inactivated by the pulmonary vascular bed. Also, the lung has a high capacity for forming prostaglandins from arachidonic acid. Considerable differences exist between species in relation to amount and specific prostaglandin formed as determined by incubation of 11C-PGH2 with pulmonary microsomes. The pulmonary biosynthesis and metabolism of these prostaglandins and related substances are discussed. PMID:6428876

  19. Effects of flavonoids on prostaglandin E2 production and on COX-2 and mPGES-1 expressions in activated macrophages.

    PubMed

    Hämäläinen, Mari; Nieminen, Riina; Asmawi, M Zaini; Vuorela, Pia; Vapaatalo, Heikki; Moilanen, Eeva

    2011-09-01

    Prostaglandin E2 (PGE2) has a central role in inflammation and both cyclooxygenase-2 (COX-2) and prostaglandin E synthases are critical enzymes in its synthesis. In inflammation, bacterial products and cytokines enhance the expression of COX-2 and inducible microsomal prostaglandin E synthase-1 (mPGES-1) which are functionally coupled to result in increased PGE2 formation in macrophages and tissue cells. In the present study, we systematically investigated the effects of 26 naturally occurring flavonoids on PGE2 production and on COX-2 and mPGES-1 expression in activated macrophages