Science.gov

Sample records for nad synthesis inhibitor

  1. NAD[S], an NAD analogue with reduced susceptibility to phosphodiesterase. Chemical synthesis and enzymic properties.

    PubMed

    Meyer, T; Wielckens, K; Thiem, J; Hilz, H

    1984-05-01

    The chemical synthesis of adenosine(5') [alpha-thio]diphospho(5')ribofuranosyl-nicotinamide (NAD[S]) is described. The product occurs as a pair of diastereomers with different configuration at the sulfur-bearing phosphorus atom. The diastereomers were separated by high-performance liquid chromatography and their absolute configuration was determined after chemical degradation to the ADP[alpha S] diastereomers and chromatographic comparison with enzymically synthesized ADP[alpha S] diastereomers of known absolute configuration. Additional support for this assignment is based on different rates in the phosphodiesterase-catalyzed hydrolysis. Furthermore the synthesis of [14C]NAD[S] is described. The coenzyme activity of NAD[S] in the reaction with alcohol dehydrogenase from baker's yeast and lactate dehydrogenase from pig heart is very similar to that of beta-NAD. Also, NAD and NAD[S] serve equally well as substrates for NAD glycohydrolase from calf spleen. In contrast, no reaction was detected with NAD pyrophosphorylase, and hydrolysis of the separated NAD[S] diastereomers with snake venom phosphodiesterase showed a 26-fold and a 33-fold slower reaction rate than that of NAD. Nucleotide pyrophosphatase was less sensitive to the S substitution, hydrolyzing NAD[S] 14-times slower than NAD. Poly(ADP-ribose) polymerase from Ehrlich ascites tumor cell nuclei accepted NAD[S] as a substrate but the reaction was significantly slower and approached saturation at much lower values than with NAD. Alkaline hydrolysis of the products insoluble in trichloroacetic acid yielded AMP[S] as the main derivative. It is concluded that with NAD[S] as a substrate the nuclear acceptors were nearly exclusively mono(ADP-ribosyl) ated . PMID:6144544

  2. Vitamins and aging: pathways to NAD+ synthesis.

    PubMed

    Denu, John M

    2007-05-01

    Recent genetic evidence reveals additional salvage pathways for NAD(+) synthesis. In this issue, Belenky et al. (2007) report that nicotinamide riboside, a new NAD(+) precursor, regulates Sir2 deacetylase activity and life span in yeast. The ability of nicotinamide riboside to enhance life span does not depend on calorie restriction. PMID:17482537

  3. Virtual Screening to Identify Lead Inhibitors for Bacterial NAD Synthetase (NADs)

    PubMed Central

    Moro, Whitney Beysselance; Yang, Zhengrong; Kane, Tasha; Brouillette, Christie G.; Brouillette, Wayne J.

    2009-01-01

    Virtual screening was employed to identify new drug-like inhibitors of NAD synthetase (NADs) as antibacterial agents. Four databases of commercially available compounds were docked against three subsites of the NADs active site using FlexX in conjunction with CScore. Over 200 commercial compounds were purchased and evaluated in enzyme inhibition and antibacterial assays. 18 compounds inhibited NADs at or below 100 μM (7.6% hit rate), and two were selected for future SAR studies. PMID:19249205

  4. NAD

    PubMed Central

    Pétriacq, Pierre; de Bont, Linda; Tcherkez, Guillaume; Gakière, Bertrand

    2013-01-01

    Many metabolic processes that occur in living cells involve oxido-reduction (redox) chemistry underpinned by redox compounds such as glutathione, ascorbate and/or pyridine nucleotides. Among these redox carriers, nicotinamide adenine dinucleotide (NAD) is the cornerstone of cellular oxidations along catabolism and is therefore essential for plant growth and development. In addition to its redox role, there is now compelling evidence that NAD is a signal molecule controlling crucial functions like primary and secondary carbon metabolism. Recent studies using integrative -omics approaches combined with molecular pathology have shown that manipulating NAD biosynthesis and recycling lead to an alteration of metabolites pools and developmental processes, and changes in the resistance to various pathogens. NAD levels should now be viewed as a potential target to improve tolerance to biotic stress and crop improvement. In this paper, we review the current knowledge on the key role of NAD (and its metabolism) in plant responses to pathogen infections. PMID:23104110

  5. In vitro metabolic engineering for the salvage synthesis of NAD(.).

    PubMed

    Honda, Kohsuke; Hara, Naoya; Cheng, Maria; Nakamura, Anna; Mandai, Komako; Okano, Kenji; Ohtake, Hisao

    2016-05-01

    Excellent thermal and operational stabilities of thermophilic enzymes can greatly increase the applicability of biocatalysis in various industrial fields. However, thermophilic enzymes are generally incompatible with thermo-labile substrates, products, and cofactors, since they show the maximal activities at high temperatures. Despite their pivotal roles in a wide range of enzymatic redox reactions, NAD(P)(+) and NAD(P)H exhibit relatively low stabilities at high temperatures, tending to be a major obstacle in the long-term operation of biocatalytic chemical manufacturing with thermophilic enzymes. In this study, we constructed an in vitro artificial metabolic pathway for the salvage synthesis of NAD(+) from its degradation products by the combination of eight thermophilic enzymes. The enzymes were heterologously produced in recombinant Escherichia coli and the heat-treated crude extracts of the recombinant cells were directly used as enzyme solutions. When incubated with experimentally optimized concentrations of the enzymes at 60°C, the NAD(+) concentration could be kept almost constant for 15h. PMID:26912312

  6. SAR Studies for a New Class of Antibacterial NAD Biosynthesis Inhibitors

    PubMed Central

    Moro, Whitney Beysselance; Yang, Zhengrong; Kane, Tasha A.; Zhou, Qingxian; Harville, Steve; Brouillette, Christie G.; Brouillette, Wayne J.

    2009-01-01

    A new lead class of antibacterial drug-like NAD synthetase (NADs) inhibitors was previously identified from a virtual screening study. Here a solution-phase synthetic library of 76 compounds, analogs of the urea-sulfonamide 5838, was synthesized in parallel to explore SAR on the sulfonamide aryl group. All library members were tested for enzyme inhibition against NADs and nicotinic acid mononucleotide adenylyltransferase (NaMNAT), the last two enzymes in the biosynthesis of NAD, and for growth inhibition in a B. anthracis antibacterial assay. Most compounds that inhibited bacterial growth also showed inhibition against one of the enzymes tested. While only modest enhancements in the enzyme inhibition potency against NADs were observed, of significance was the observation that the antibacterial urea-sulfonamides more consistently inhibited NaMNAT. PMID:19408950

  7. Bioluminescent Cell-Based NAD(P)/NAD(P)H Assays for Rapid Dinucleotide Measurement and Inhibitor Screening

    PubMed Central

    Leippe, Donna; Sobol, Mary; Vidugiris, Gediminas; Zhou, Wenhui; Meisenheimer, Poncho; Gautam, Prson; Wennerberg, Krister; Cali, James J.

    2014-01-01

    Abstract The central role of nicotinamide adenine dinucleotides in cellular energy metabolism and signaling makes them important nodes that link the metabolic state of cells with energy homeostasis and gene regulation. In this study, we describe the implementation of cell-based bioluminescence assays for rapid and sensitive measurement of those important redox cofactors. We show that the sensitivity of the assays (limit of detection ∼0.5 nM) enables the selective detection of total amounts of nonphosphorylated or phosphorylated dinucleotides directly in cell lysates. The total amount of NAD+NADH or NADP+NADPH levels can be detected in as low as 300 or 600 cells/well, respectively. The signal remains linear up to 5,000 cells/well with the maximum signal-to-background ratios ranging from 100 to 200 for NAD+NADH and from 50 to 100 for NADP+NADPH detection. The assays are robust (Z′ value >0.7) and the inhibitor response curves generated using a known NAD biosynthetic pathway inhibitor FK866 correlate well with the reported data. More importantly, by multiplexing the dinucleotide detection assays with a fluorescent nonmetabolic cell viability assay, we show that dinucleotide levels can be decreased dramatically (>80%) by FK866 treatment before changes in cell viability are detected. The utility of the assays to identify modulators of intracellular nicotinamide adenine dinucleotide levels was further confirmed using an oncology active compound library, where novel dinucleotide regulating compounds were identified. For example, the histone deacetylase inhibitor entinostat was a potent inhibitor of cellular nicotinamide adenine dinucleotides, whereas the selective estrogen receptor modulator raloxifene unexpectedly caused a twofold increase in cellular nicotinamide adenine dinucleotide levels. PMID:25506801

  8. Increasing NAD synthesis in muscle via nicotinamide phosphoribosyltransferase is not sufficient to promote oxidative metabolism.

    PubMed

    Frederick, David W; Davis, James G; Dávila, Antonio; Agarwal, Beamon; Michan, Shaday; Puchowicz, Michelle A; Nakamaru-Ogiso, Eiko; Baur, Joseph A

    2015-01-16

    The NAD biosynthetic precursors nicotinamide mononucleotide and nicotinamide riboside are reported to confer resistance to metabolic defects induced by high fat feeding in part by promoting oxidative metabolism in skeletal muscle. Similar effects are obtained by germ line deletion of major NAD-consuming enzymes, suggesting that the bioavailability of NAD is limiting for maximal oxidative capacity. However, because of their systemic nature, the degree to which these interventions exert cell- or tissue-autonomous effects is unclear. Here, we report a tissue-specific approach to increase NAD biosynthesis only in muscle by overexpressing nicotinamide phosphoribosyltransferase, the rate-limiting enzyme in the salvage pathway that converts nicotinamide to NAD (mNAMPT mice). These mice display a ∼50% increase in skeletal muscle NAD levels, comparable with the effects of dietary NAD precursors, exercise regimens, or loss of poly(ADP-ribose) polymerases yet surprisingly do not exhibit changes in muscle mitochondrial biogenesis or mitochondrial function and are equally susceptible to the metabolic consequences of high fat feeding. We further report that chronic elevation of muscle NAD in vivo does not perturb the NAD/NADH redox ratio. These studies reveal for the first time the metabolic effects of tissue-specific increases in NAD synthesis and suggest that critical sites of action for supplemental NAD precursors reside outside of the heart and skeletal muscle. PMID:25411251

  9. Increasing NAD Synthesis in Muscle via Nicotinamide Phosphoribosyltransferase Is Not Sufficient to Promote Oxidative Metabolism*

    PubMed Central

    Frederick, David W.; Davis, James G.; Dávila, Antonio; Agarwal, Beamon; Michan, Shaday; Puchowicz, Michelle A.; Nakamaru-Ogiso, Eiko; Baur, Joseph A.

    2015-01-01

    The NAD biosynthetic precursors nicotinamide mononucleotide and nicotinamide riboside are reported to confer resistance to metabolic defects induced by high fat feeding in part by promoting oxidative metabolism in skeletal muscle. Similar effects are obtained by germ line deletion of major NAD-consuming enzymes, suggesting that the bioavailability of NAD is limiting for maximal oxidative capacity. However, because of their systemic nature, the degree to which these interventions exert cell- or tissue-autonomous effects is unclear. Here, we report a tissue-specific approach to increase NAD biosynthesis only in muscle by overexpressing nicotinamide phosphoribosyltransferase, the rate-limiting enzyme in the salvage pathway that converts nicotinamide to NAD (mNAMPT mice). These mice display a ∼50% increase in skeletal muscle NAD levels, comparable with the effects of dietary NAD precursors, exercise regimens, or loss of poly(ADP-ribose) polymerases yet surprisingly do not exhibit changes in muscle mitochondrial biogenesis or mitochondrial function and are equally susceptible to the metabolic consequences of high fat feeding. We further report that chronic elevation of muscle NAD in vivo does not perturb the NAD/NADH redox ratio. These studies reveal for the first time the metabolic effects of tissue-specific increases in NAD synthesis and suggest that critical sites of action for supplemental NAD precursors reside outside of the heart and skeletal muscle. PMID:25411251

  10. Flavonoid apigenin is an inhibitor of the NAD+ ase CD38: implications for cellular NAD+ metabolism, protein acetylation, and treatment of metabolic syndrome.

    PubMed

    Escande, Carlos; Nin, Veronica; Price, Nathan L; Capellini, Verena; Gomes, Ana P; Barbosa, Maria Thereza; O'Neil, Luke; White, Thomas A; Sinclair, David A; Chini, Eduardo N

    2013-04-01

    Metabolic syndrome is a growing health problem worldwide. It is therefore imperative to develop new strategies to treat this pathology. In the past years, the manipulation of NAD(+) metabolism has emerged as a plausible strategy to ameliorate metabolic syndrome. In particular, an increase in cellular NAD(+) levels has beneficial effects, likely because of the activation of sirtuins. Previously, we reported that CD38 is the primary NAD(+)ase in mammals. Moreover, CD38 knockout mice have higher NAD(+) levels and are protected against obesity and metabolic syndrome. Here, we show that CD38 regulates global protein acetylation through changes in NAD(+) levels and sirtuin activity. In addition, we characterize two CD38 inhibitors: quercetin and apigenin. We show that pharmacological inhibition of CD38 results in higher intracellular NAD(+) levels and that treatment of cell cultures with apigenin decreases global acetylation as well as the acetylation of p53 and RelA-p65. Finally, apigenin administration to obese mice increases NAD(+) levels, decreases global protein acetylation, and improves several aspects of glucose and lipid homeostasis. Our results show that CD38 is a novel pharmacological target to treat metabolic diseases via NAD(+)-dependent pathways. PMID:23172919

  11. Benzoylurea Chitin Synthesis Inhibitors.

    PubMed

    Sun, Ranfeng; Liu, Chunjuan; Zhang, Hao; Wang, Qingmin

    2015-08-12

    Benzoylurea chitin synthesis inhibitors are widely used in integrated pest management (IPM) and insecticide resistance management (IRM) programs due to their low toxicity to mammals and predatory insects. In the past decades, a large number of benzoylurea derivatives have been synthesized, and 15 benzoylurea chitin synthesis inhibitors have been commercialized. This review focuses on the history of commercial benzolyphenylureas (BPUs), synthetic methods, structure-activity relationships (SAR), action mechanism research, environmental behaviors, and ecotoxicology. Furthermore, their disadvantages of high risk to aquatic invertebrates and crustaceans are pointed out. Finally, we propose that the para-substituents at anilide of benzoylphenylureas should be the functional groups, and bipartite model BPU analogues are discussed in an attempt to provide new insight for future development of BPUs. PMID:26168369

  12. NAMPT-Mediated Salvage Synthesis of NAD+ Controls Morphofunctional Changes of Macrophages

    PubMed Central

    Venter, Gerda; Oerlemans, Frank T. J. J.; Willemse, Marieke; Wijers, Mietske; Fransen, Jack A. M.; Wieringa, Bé

    2014-01-01

    Functional morphodynamic behavior of differentiated macrophages is strongly controlled by actin cytoskeleton rearrangements, a process in which also metabolic cofactors ATP and NAD(H) (i.e. NAD+ and NADH) and NADP(H) (i.e. NADP+ and NADPH) play an essential role. Whereas the link to intracellular ATP availability has been studied extensively, much less is known about the relationship between actin cytoskeleton dynamics and intracellular redox state and NAD+-supply. Here, we focus on the role of nicotinamide phosphoribosyltransferase (NAMPT), found in extracellular form as a cytokine and growth factor, and in intracellular form as one of the key enzymes for the production of NAD+ in macrophages. Inhibition of NAD+ salvage synthesis by the NAMPT-specific drug FK866 caused a decrease in cytosolic NAD+ levels in RAW 264.7 and Maf-DKO macrophages and led to significant downregulation of the glycolytic flux without directly affecting cell viability, proliferation, ATP production capacity or mitochondrial respiratory activity. Concomitant with these differential metabolic changes, the capacity for phagocytic ingestion of particles and also substrate adhesion of macrophages were altered. Depletion of cytoplasmic NAD+ induced cell-morphological changes and impaired early adhesion in phagocytosis of zymosan particles as well as spreading performance. Restoration of NAD+ levels by NAD+, NMN, or NADP+ supplementation reversed the inhibitory effects of FK866. We conclude that direct coupling to local, actin-based, cytoskeletal dynamics is an important aspect of NAD+’s cytosolic role in the regulation of morphofunctional characteristics of macrophages. PMID:24824795

  13. Synthesis of Lysine Methyltransferase Inhibitors

    NASA Astrophysics Data System (ADS)

    Ye, Tao; Hui, Chunngai

    2015-07-01

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

  14. N-arylazido-. beta. -alanyl-NAD sup + , a new NAD sup + photoaffinity analogue. Synthesis and labeling of mitochondrial NADH dehydrogenase

    SciTech Connect

    Deng, P.S.K.; Chen, S. ); Hatefi, Y. )

    1990-01-30

    N-Arylaziod-{beta}-alanyl-NAD{sup +}(N3{prime}-0-(3-(N-(4-azido-2-nitrophenyl)amino)propionyl)NAD{sup +}) has been prepared by alkaline phosphatase treatment of arylaziod-{beta}-alanyl-NADP{sup +} (N3{prime}-O-(3-(N-(4-azido-2-nitrophenyl)amino)propionyl)NADP{sup +}). This NAD{sup +} analogue was found to be a potent competitive inhibitor with respect to NADH for the purified bovine heart mitochondrial NADH dehydrogenase. The enzyme was irreversibly inhibited as well as covalently labeled by this analogue upon photoirradiation. A stoichiometry of 1.15 mol of N-arylazido-{beta}-alanyl-NAD{sup +} bound/mol of enzyme, at 100% inactivation, was determined from incorporation studies using tritium-labeled analogue. Among the three subunits, 0.85 mol of the analogue was bound to the M{sub r} = 51,000 subunit, and each of the two smaller subunits contained 0.15 mol of the analogue when the dehydrogenase was completely inhibited upon photolysis. Both the irreversible inactivation and the covalent incorporation could be prevented by the presence of NADH during photolysis. These results indicate that N-arylaziod-{beta}-alanyl-NAD{sup +} is an active-site-directed photoaffinity label for the mitochondrial NADH dehydrogenase, and are further evidence that the M{sub r} = 51,000 subunit contain the NADH binding site. Results are also presented to show that N-arylazido-{beta}-alanyl-NAD{sup +} binds the dehydrogenase in a more effective manner than A-arylazido-{beta}-alanyl-NAD{sup +}.

  15. Synthesis of lysine methyltransferase inhibitors

    PubMed Central

    Hui, Chunngai; Ye, Tao

    2015-01-01

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

  16. [The synthesis of specific enzyme inhibitors].

    PubMed

    Iakovleva, G M

    1987-04-01

    The review deals with directed synthesis of specific enzyme inhibitors. They are classified within the framework of the mechanistic approach, namely, stable analogues of substrates, which form enzyme complexes mimicking the Michaelis complex or those which influence the chemical stages of enzyme catalysis; conformational inhibitors; substrate analogues participating in enzyme reactions and producing modified products; suicide inhibitors; stage inhibitors (inhibitors influencing certain stages of enzyme reaction); transition state analogues; multisubstrate analogues and collected substrates. Types of chemical modification used in synthesis of the specific inhibitors are discussed. Some possibilities of the quantity structure-activity relationship methods, computer modelling and molecular graphics in designing the optimal structure of inhibitors are mentioned. PMID:3300658

  17. Structure-Activity Relationship Studies and Biological Characterization of Human NAD+-dependent 15-Hydroxyprostaglandin Dehydrogenase Inhibitors

    PubMed Central

    Duveau, Damien Y.; Yasgar, Adam; Wang, Yuhong; Hu, Xin; Kouznetsova, Jennifer; Brimacombe, Kyle R.; Jadhav, Ajit; Simeonov, Anton; Thomas, Craig J.; Maloney, David J.

    2014-01-01

    The structure-activity relationship (SAR) study of two chemotypes identified as inhibitors of the human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (HPGD, 15-PGDH) was conducted. Top compounds from both series displayed potent inhibition (IC50 <50 nM), demonstrate excellent selectivity towards HPGD and potently induce PGE2 production in A549 lung cancer and LNCaP prostate cancer cells. PMID:24360556

  18. A [32P]-NAD+-based method to identify and quantitate long residence time enoyl-ACP reductase inhibitors

    PubMed Central

    Yu, Weixuan; Neckles, Carla; Chang, Andrew; Bommineni, Gopal Reddy; Spagnuolo, Lauren; Zhang, Zhuo; Liu, Nina; Lai, Christina; Truglio, James; Tonge, Peter J.

    2015-01-01

    The classical methods for quantifying drug-target residence time (tR) use loss or regain of enzyme activity in progress curve kinetic assays. However, such methods become imprecise at very long residence times, mitigating the use of alternative strategies. Using the NAD(P)H-dependent FabI enoyl-ACP reductase as a model system, we developed a Penefsky column-based method for direct measurement of tR, where the off-rate of the drug was determined with radiolabeled [adenylate-32P] NAD(P+) cofactor. Twenty-three FabI inhibitors were analyzed and a mathematical model was used to estimate limits to the tR values of each inhibitor based on percent drug-target complex recovery following gel filtration. In general, this method showed good agreement with the classical steady state kinetic methods for compounds with tR values of 10-100 min. In addition, we were able to identify seven long tR inhibitors (100-1500 min) and to accurately determine their tR values. The method was then used to measure tR as a function of temperature, an analysis not previously possible using the standard kinetic approach due to decreased NAD(P)H stability at elevated temperatures. In general, a 4-fold difference in tR was observed when the temperature was increased from 25 °C to 37 °C . PMID:25684450

  19. A Nampt inhibitor FK866 mimics vitamin B3 deficiency by causing senescence of human fibroblastic Hs68 cells via attenuation of NAD(+)-SIRT1 signaling.

    PubMed

    Song, Tuzz-Ying; Yeh, Shu-Lan; Hu, Miao-Lin; Chen, Mei-Yau; Yang, Nae-Cherng

    2015-12-01

    Vitamin B3 (niacin) deficiency can cause pellagra with symptoms of dermatitis, diarrhea and dementia. However, it is unclear whether the vitamin B3 deficiency causes human aging. FK866 (a Nampt inhibitor) can reduce intracellular NAD(+) level and induce senescence of human Hs68 cells. However, the mechanisms underlying FK866-induced senescence of Hs68 cells are unclear. In this study, we used FK866 to mimic the effects of vitamin B3 deficiency to reduce the NAD(+) level and investigated the mechanisms of FK866-induced senescence of Hs68 cells. We hypothesized that FK866 induced the senescence of Hs68 cells via an attenuation of NAD(+)-silent information regulator T1 (SIRT1) signaling. We found that FK866 induced cell senescence and diminished cellular NAD(+) levels and SIRT1 activity (detected by acetylation of p53), and these effects were dramatically antagonized by co-treatment with nicotinic acid, nicotinamide, or NAD(+). In contrast, the protein expression of SIRT1, AMP-activated protein kinase, mammalian target of rapamycin, and nicotinamide phosphoribosyltransferase (Nampt) was not affected by FK866. In addition, the role of GSH in the FK866-induced cells senescence may be limited, as N-acetylcysteine did not antagonize FK866-induced cell senescence. These results suggest that FK866 induces cell senescence via attenuation of NAD(+)-SIRT1 signaling. The effects of vitamin B3 deficiency on human aging warrant further investigation. PMID:26330291

  20. The Small Molecule GMX1778 Is a Potent Inhibitor of NAD+ Biosynthesis: Strategy for Enhanced Therapy in Nicotinic Acid Phosphoribosyltransferase 1-Deficient Tumors▿

    PubMed Central

    Watson, Mark; Roulston, Anne; Bélec, Laurent; Billot, Xavier; Marcellus, Richard; Bédard, Dominique; Bernier, Cynthia; Branchaud, Stéphane; Chan, Helen; Dairi, Kenza; Gilbert, Karine; Goulet, Daniel; Gratton, Michel-Olivier; Isakau, Henady; Jang, Anne; Khadir, Abdelkrim; Koch, Elizabeth; Lavoie, Manon; Lawless, Michael; Nguyen, Mai; Paquette, Denis; Turcotte, Émilie; Berger, Alvin; Mitchell, Matthew; Shore, Gordon C.; Beauparlant, Pierre

    2009-01-01

    GMX1777 is a prodrug of the small molecule GMX1778, currently in phase I clinical trials for the treatment of cancer. We describe findings indicating that GMX1778 is a potent and specific inhibitor of the NAD+ biosynthesis enzyme nicotinamide phosphoribosyltransferase (NAMPT). Cancer cells have a very high rate of NAD+ turnover, which makes NAD+ modulation an attractive target for anticancer therapy. Selective inhibition by GMX1778 of NAMPT blocks the production of NAD+ and results in tumor cell death. Furthermore, GMX1778 is phosphoribosylated by NAMPT, which increases its cellular retention. The cytotoxicity of GMX1778 can be bypassed with exogenous nicotinic acid (NA), which permits NAD+ repletion via NA phosphoribosyltransferase 1 (NAPRT1). The cytotoxicity of GMX1778 in cells with NAPRT1 deficiency, however, cannot be rescued by NA. Analyses of NAPRT1 mRNA and protein levels in cell lines and primary tumor tissue indicate that high frequencies of glioblastomas, neuroblastomas, and sarcomas are deficient in NAPRT1 and not susceptible to rescue with NA. As a result, the therapeutic index of GMX1777 can be widended in the treatment animals bearing NAPRT1-deficient tumors by coadministration with NA. This provides the rationale for a novel therapeutic approach for the use of GMX1777 in the treatment of human cancers. PMID:19703994

  1. Chlorolissoclimides: New inhibitors of eukaryotic protein synthesis

    PubMed Central

    Robert, Francis; Gao, Hong Qing; Donia, Marwa; Merrick, William C.; Hamann, Mark T.; Pelletier, Jerry

    2006-01-01

    Lissoclimides are cytotoxic compounds produced by shell-less molluscs through chemical secretions to deter predators. Chlorinated lissoclimides were identified as the active component of a marine extract from Pleurobranchus forskalii found during a high-throughput screening campaign to characterize new protein synthesis inhibitors. It was demonstrated that these compounds inhibit protein synthesis in vitro, in extracts prepared from mammalian and plant cells, as well as in vivo against mammalian cells. Our results suggest that they block translation elongation by inhibiting translocation, leading to an accumulation of ribosomes on mRNA. These data provide a rationale for the cytotoxic nature of this class of small molecule natural products. PMID:16540697

  2. ES936 stimulates DNA synthesis in HeLa cells independently on NAD(P)H:quinone oxidoreductase 1 inhibition, through a mechanism involving p38 MAPK.

    PubMed

    González-Aragón, David; Alcaín, Francisco J; Ariza, Julia; Jódar, Laura; Barbarroja, Nuria; López-Pedrera, Chary; Villalba, José M

    2010-07-30

    The indolequinone ES936 (5-methoxy-1,2-dimethyl-3-[(4-nitrophenol)methyl]-indole-4,7-dione) is a potent mechanism-based inhibitor of NAD(P)H:quinone oxidoreductase 1 (NQO1). Here, we report that ES936 significantly stimulated thymidine incorporation in sparse cultures of human adenocarcinoma HeLa cells, but was without effect in dense cultures. Stimulation of DNA synthesis was not related with a DNA repair response because an increase in thymidine incorporation was not observed in cells treated with 2,5 bis-[1-aziridyl]-1,4 benzoquinone, a well-established antitumor quinone that causes DNA damage. Conversely, it was related with an increase of cell growth. NQO1 inhibition was not involved in ES936 stimulation of DNA synthesis, because the same response was observed in cells where NQO1 expression had been knocked down by small interfering RNA. Stimulation of DNA synthesis was reverted by treatment with ambroxol, a SOD mimetic, and by pyruvate, an efficient peroxide scavenger, supporting the involvement of alterations in cellular redox state. Pharmacological inhibition of p38 with either SB203580 or PD169316 completely abolished ES936-stimulated DNA synthesis, indicating the requirement of p38 activity. This is the first report that demonstrates the existence of an ES936-sensitive system which is separate from NQO1, modulating the redox state and cell growth in HeLa cells through a p38-dependent mechanism. Our results show that the effect ES936 exerts on DNA synthesis may be either positive or negative depending on the cellular context and growth conditions. PMID:20433816

  3. Design, synthesis and biological characterization of novel inhibitors of CD38

    PubMed Central

    Dong, Min; Si, Yuan-Qi; Sun, Shuang-Yong; Pu, Xiao-Ping; Yang, Zhen-Jun; Zhang, Liang-Ren; Zhang, Li-He; Leung, Fung Ping; Lam, Connie Mo Ching.; Kwong, Anna Ka Yee; Yue, Jianbo; Zhou, Yeyun; Kriksunov, Irina A.; Hao, Quan; Lee, Hon Cheung

    2012-01-01

    Human CD38 is a novel multi-functional protein that acts not only as an antigen for B-lymphocyte activation, but also an enzyme catalyzing the synthesis of a Ca2+ messenger molecule, cyclic ADP-ribose, from NAD+. It is well established that this novel Ca2+ signaling enzyme is responsible for regulating a wide range of physiological functions. Based on the crystal structure of the CD38/NAD+ complex, we synthesized a series of simplified N-substituted nicotinamide derivatives (Compound 1–14). A number of these compounds exhibited moderate inhibition of the NAD+ utilizing activity of CD38, with Compound 4 showing the higher potency. The crystal structure of CD38/ Compound 4 complex and computer simulation of Compound 7 docking to CD38 show a significant role of the nicotinamide moiety and the distal aromatic group of the compounds for substrate recognition by the active site of CD38. Biologically, we showed that both Compounds 4 and 7 effectively relaxed the agonist-induced contraction of muscle preparations form rats and guinea pigs. This study is a rational design of inhibitors for CD38 that exhibit important physiological effects, and can serve as a model for future drug development. PMID:21431168

  4. Inhibition of de novo NAD(+) synthesis by oncogenic URI causes liver tumorigenesis through DNA damage.

    PubMed

    Tummala, Krishna S; Gomes, Ana L; Yilmaz, Mahmut; Graña, Osvaldo; Bakiri, Latifa; Ruppen, Isabel; Ximénez-Embún, Pilar; Sheshappanavar, Vinayata; Rodriguez-Justo, Manuel; Pisano, David G; Wagner, Erwin F; Djouder, Nabil

    2014-12-01

    Molecular mechanisms responsible for hepatocellular carcinoma (HCC) remain largely unknown. Using genetically engineered mouse models, we show that hepatocyte-specific expression of unconventional prefoldin RPB5 interactor (URI) leads to a multistep process of HCC development, whereas its genetic reduction in hepatocytes protects against diethylnitrosamine (DEN)-induced HCC. URI inhibits aryl hydrocarbon (AhR)- and estrogen receptor (ER)-mediated transcription of enzymes implicated in L-tryptophan/kynurenine/nicotinamide adenine dinucleotide (NAD(+)) metabolism, thereby causing DNA damage at early stages of tumorigenesis. Restoring NAD(+) pools with nicotinamide riboside (NR) prevents DNA damage and tumor formation. Consistently, URI expression in human HCC is associated with poor survival and correlates negatively with L-tryptophan catabolism pathway. Our results suggest that boosting NAD(+) can be prophylactic or therapeutic in HCC. PMID:25453901

  5. [Enzymes related with NAD synthesis promote conversion of 1,4-butanediol to 4-hydroxybutyrate].

    PubMed

    Zhang, Xin; Chen, Guoqiang

    2011-12-01

    Besides medical application, 4-hydroxybutyrate (4-HB) is a precursor of P3HB4HB, a bioplastic showing excellent physical properties and degradability. Escherichia coli S17-1 (pZL-dhaT-aldD) can transform 1, 4-butanediol (1,4-BD) into 4HB with participation of cofactor NAD. To enhance productivity, nicotinic acid phosphoribosyltransferase (PncB) and nicotinamide adenine dinucleotide synthetase (NadE) were overexpressed to increase intracellular nicotinamide adenine dinucleotide concentration and promote reaction process. The shake flask fermentation result showed that the conversion rate increased by 13.03% with help of PncB-NadE, leading to 4.87 g/L 4HB from 10 g/L 1,4-BD, and productivity was increased by 40.91% to 1.86 g/g. These results demonstrated that expression of PncB and NadE is beneficial for conversion of 1,4-BD to 4HB. PMID:22506415

  6. Protein synthesis inhibitor from potato tuber

    SciTech Connect

    Romaen, R. )

    1989-04-01

    A protein fraction capable of inhibit in vitro protein synthesis was found in potato tubers in fresh and wounded tissue. Inhibitor activity from fresh tissue decays with wounding. Inhibition activity was detected absorbed to ribsomal fraction and cytosol of potato tuber tissue by a partially reconstituted in vitro system from potato tuber and wheat germ. Adsorbed ribosomal fraction was more suitable of purification. This fraction was washed from ribosomes with 0.3M KCl, concentrated with ammonium sulfate precipitation and purified through sephadex G100 and sephadex G-75 columns chromatography. After 61 fold purification adsorbed protein fraction can inhibit germination of maize, wheat and sesame seeds, as well as {sup 3}H-leucine incorporation into protein by imbibed maize embryos. Inhibition activity was lost by temperature, alkali and protease-K hydrolysis. Preliminar analysis could not show presence of reductor sugars. Physiological role of this inhibitor in relation to rest and active tissue remains to be studied.

  7. Tools for Characterizing Bacterial Protein Synthesis Inhibitors

    PubMed Central

    Orelle, Cédric; Carlson, Skylar; Kaushal, Bindiya; Almutairi, Mashal M.; Liu, Haipeng; Ochabowicz, Anna; Quan, Selwyn; Pham, Van Cuong; Squires, Catherine L.; Murphy, Brian T.

    2013-01-01

    Many antibiotics inhibit the growth of sensitive bacteria by interfering with ribosome function. However, discovery of new protein synthesis inhibitors is curbed by the lack of facile techniques capable of readily identifying antibiotic target sites and modes of action. Furthermore, the frequent rediscovery of known antibiotic scaffolds, especially in natural product extracts, is time-consuming and expensive and diverts resources that could be used toward the isolation of novel lead molecules. In order to avoid these pitfalls and improve the process of dereplication of chemically complex extracts, we designed a two-pronged approach for the characterization of inhibitors of protein synthesis (ChIPS) that is suitable for the rapid identification of the site and mode of action on the bacterial ribosome. First, we engineered antibiotic-hypersensitive Escherichia coli strains that contain only one rRNA operon. These strains are used for the rapid isolation of resistance mutants in which rRNA mutations identify the site of the antibiotic action. Second, we show that patterns of drug-induced ribosome stalling on mRNA, monitored by primer extension, can be used to elucidate the mode of antibiotic action. These analyses can be performed within a few days and provide a rapid and efficient approach for identifying the site and mode of action of translation inhibitors targeting the bacterial ribosome. Both techniques were validated using a bacterial strain whose culture extract, composed of unknown metabolites, exhibited protein synthesis inhibitory activity; we were able to rapidly detect the presence of the antibiotic chloramphenicol. PMID:24041905

  8. Hydroperoxidic inhibitor of horse liver alcohol dehydrogenase activity, tightly bound to the enzyme-NAD+ complex, characteristically degrades the coenzyme.

    PubMed

    Skurský, L; Rezác, M; Khan, A N; Zídek, L; Rocek, J

    1992-01-01

    The strong inhibition of horse liver alcohol dehydrogenase (HLAD) by p-methylbenzyl hydroperoxide (XyHP) is only transient, XyHP behaves also as a pseudo-substrate of the enzyme and in the presence of NAD+, is degraded by HLAD to (as yet unidentified) non-inhibiting products while the NAD+ is converted to a derivative similar to the "NADX", originally observed in an analogous reaction of HLAD with hydrogen peroxide. The apparent KM for XyHP is approximately 10(4) times smaller than that for H2O2. The catalytic constant kcat for HLAD degradation of XyHP is two orders of magnitude less than that for ethanol dehydrogenation. XyHP inhibits both directions of the alcohol-aldehyde interconversion with equal potency. The first step of the inhibition mechanism is a tight binding of XyHP to the binary HLAD-NAD+ complex. PMID:1284958

  9. Drosophila NMNAT Maintains Neural Integrity Independent of Its NAD Synthesis Activity

    PubMed Central

    Zhai, R. Grace; Cao, Yu; Hiesinger, P. Robin; Zhou, Yi; Mehta, Sunil Q; Schulze, Karen L; Verstreken, Patrik; Bellen, Hugo J

    2006-01-01

    Wallerian degeneration refers to a loss of the distal part of an axon after nerve injury. Wallerian degeneration slow (Wlds) mice overexpress a chimeric protein containing the NAD synthase NMNAT (nicotinamide mononucleotide adenylyltransferase 1) and exhibit a delay in axonal degeneration. Currently, conflicting evidence raises questions as to whether NMNAT is the protecting factor and whether its enzymatic activity is required for such a possible function. Importantly, the link between nmnat and axon degeneration is at present solely based on overexpression studies of enzymatically active protein. Here we use the visual system of Drosophila as a model system to address these issues. We have isolated the first nmnat mutations in a multicellular organism in a forward genetic screen for synapse malfunction in Drosophila. Loss of nmnat causes a rapid and severe neurodegeneration that can be attenuated by blocking neuronal activity. Furthermore, in vivo neuronal expression of mutated nmnat shows that enzymatically inactive NMNAT protein retains strong neuroprotective effects and rescues the degeneration phenotype caused by loss of nmnat. Our data indicate an NAD-independent requirement of NMNAT for maintaining neuronal integrity that can be exploited to protect neurons from neuronal activity-induced degeneration by overexpression of the protein. PMID:17132048

  10. EFFECT OF ANTIBIOTICS AND INHIBITORS ON M PROTEIN SYNTHESIS

    PubMed Central

    Brock, Thomas D.

    1963-01-01

    Brock, Thomas D. (Western Reserve University, Cleveland, Ohio). Effect of antibiotics and inhibitors on M protein synthesis. J. Bacteriol. 85:527–531. 1963.—This work extends the observations of Fox and Krampitz on M protein synthesis in nongrowing cells of streptococci. A survey of a large number of antibiotics and other potential inhibitors was made. Some substances bring about inhibition of fermentation and inhibit M protein synthesis because they deprive the cell of the energy needed for this process. A second group of substances inhibit growth at concentrations tenfold or more lower than they inhibit M protein synthesis. These are the antibiotics which inhibit synthesis of cell wall or other structures in growing cells, but do not affect protein synthesis. A third group of substances inhibit growth and M protein synthesis at the same concentration. These substances probably inhibit growth because they inhibit general protein synthesis, and are therefore specific inhibitors of protein synthesis. In this class are chloramphenicol, erythromycin, and the tetracyclines. Several other antibiotics of previously unknown mode of action are in this class. A fourth group of substances had no effect on M protein synthesis. No substances were found which inhibited M protein synthesis at a lower concentration than that which inhibited growth. M protein synthesis in nongrowing cells may be a useful model system for obtaining a detailed understanding of protein synthesis. PMID:14042928

  11. Inhibition of the intrinsic NAD+ glycohydrolase activity of CD38 by carbocyclic NAD analogues.

    PubMed

    Wall, K A; Klis, M; Kornet, J; Coyle, D; Amé, J C; Jacobson, M K; Slama, J T

    1998-11-01

    Carba-NAD and pseudocarba-NAD are carbocyclic analogues of NAD+ in which a 2,3-dihydroxycyclopentane methanol replaces the beta-d-ribonucleotide ring of the nicotinamide riboside moiety of NAD+ [Slama and Simmons (1988) Biochemistry 27, 183-193]. These carbocyclic NAD+ analogues, related to each other as diastereomers, have been tested as inhibitors of the intrinsic NAD+ glycohydrolase activity of human CD38, dog spleen NAD+ glycohydrolase, mouse CD38 and Aplysia californica cADP-ribose synthetase. Pseudocarba-NAD, the carbocyclic dinucleotide in which l-2,3-dihydroxycyclopentane methanol replaces the d-ribose of the nicotinamide riboside moiety of NAD+, was found to be the more potent inhibitor. Pseudocarba-NAD was shown to inhibit the intrinsic NAD+ glycohydrolase activity of human CD38 competitively, with Ki=148 microM determined for the recombinant extracellular protein domain and Ki=180 microM determined for the native protein expressed as a cell-surface enzyme on cultured Jurkat cells. Pseudocarba-NAD was shown to be a non-competitive inhibitor of the purified dog spleen NAD+ glycohydrolase, with Kis=47 miroM and Kii=198 microM. Neither pseudocarba-NAD nor carba-NAD inhibited mouse CD38 or Aplysia californica cADP-ribose synthetase significantly at concentrations up to 1 mM. The results underscore significant species differences in the sensitivity of these enzymes to inhibition, and indicate that pseudocarba-NAD will be useful as an inhibitor of the enzymic activity of human but not mouse CD38 in studies using cultured cells. PMID:9794804

  12. Inhibition of the intrinsic NAD+ glycohydrolase activity of CD38 by carbocyclic NAD analogues.

    PubMed Central

    Wall, K A; Klis, M; Kornet, J; Coyle, D; Amé, J C; Jacobson, M K; Slama, J T

    1998-01-01

    Carba-NAD and pseudocarba-NAD are carbocyclic analogues of NAD+ in which a 2,3-dihydroxycyclopentane methanol replaces the beta-d-ribonucleotide ring of the nicotinamide riboside moiety of NAD+ [Slama and Simmons (1988) Biochemistry 27, 183-193]. These carbocyclic NAD+ analogues, related to each other as diastereomers, have been tested as inhibitors of the intrinsic NAD+ glycohydrolase activity of human CD38, dog spleen NAD+ glycohydrolase, mouse CD38 and Aplysia californica cADP-ribose synthetase. Pseudocarba-NAD, the carbocyclic dinucleotide in which l-2,3-dihydroxycyclopentane methanol replaces the d-ribose of the nicotinamide riboside moiety of NAD+, was found to be the more potent inhibitor. Pseudocarba-NAD was shown to inhibit the intrinsic NAD+ glycohydrolase activity of human CD38 competitively, with Ki=148 microM determined for the recombinant extracellular protein domain and Ki=180 microM determined for the native protein expressed as a cell-surface enzyme on cultured Jurkat cells. Pseudocarba-NAD was shown to be a non-competitive inhibitor of the purified dog spleen NAD+ glycohydrolase, with Kis=47 miroM and Kii=198 microM. Neither pseudocarba-NAD nor carba-NAD inhibited mouse CD38 or Aplysia californica cADP-ribose synthetase significantly at concentrations up to 1 mM. The results underscore significant species differences in the sensitivity of these enzymes to inhibition, and indicate that pseudocarba-NAD will be useful as an inhibitor of the enzymic activity of human but not mouse CD38 in studies using cultured cells. PMID:9794804

  13. NAD(P)H:Quinone Oxidoreductase-1 Expression Sensitizes Malignant Melanoma Cells to the HSP90 Inhibitor 17-AAG.

    PubMed

    Kasai, Shuya; Arakawa, Nobuyuki; Okubo, Ayaka; Shigeeda, Wataru; Yasuhira, Shinji; Masuda, Tomoyuki; Akasaka, Toshihide; Shibazaki, Masahiko; Maesawa, Chihaya

    2016-01-01

    The KEAP1-NRF2 pathway regulates cellular redox homeostasis by transcriptional induction of genes associated with antioxidant synthesis and detoxification in response to oxidative stress. Previously, we reported that KEAP1 mutation elicits constitutive NRF2 activation and resistance to cisplatin (CDDP) and dacarbazine (DTIC) in human melanomas. The present study was conducted to clarify whether an HSP90 inhibitor, 17-AAG, efficiently eliminates melanoma with KEAP1 mutation, as the NRF2 target gene, NQO1, is a key enzyme in 17-AAG bioactivation. In melanoma and non-small cell lung carcinoma cell lines with or without KEAP1 mutations, NQO1 expression and 17-AAG sensitivity are inversely correlated. NQO1 is highly expressed in normal melanocytes and in several melanoma cell lines despite the presence of wild-type KEAP1, and the NQO1 expression is dependent on NRF2 activation. Because either CDDP or DTIC produces reactive oxygen species that activate NRF2, we determined whether these agents would sensitize NQO1-low melanoma cells to 17-AAG. Synergistic cytotoxicity of the 17-AAG and CDDP combination was detected in four out of five NQO1-low cell lines, but not in the cell line with KEAP1 mutation. These data indicate that 17-AAG could be a potential chemotherapeutic agent for melanoma with KEAP1 mutation or NQO1 expression. PMID:27045471

  14. NAD(P)H:Quinone Oxidoreductase-1 Expression Sensitizes Malignant Melanoma Cells to the HSP90 Inhibitor 17-AAG

    PubMed Central

    Kasai, Shuya; Arakawa, Nobuyuki; Okubo, Ayaka; Shigeeda, Wataru; Yasuhira, Shinji; Masuda, Tomoyuki; Akasaka, Toshihide; Shibazaki, Masahiko; Maesawa, Chihaya

    2016-01-01

    The KEAP1-NRF2 pathway regulates cellular redox homeostasis by transcriptional induction of genes associated with antioxidant synthesis and detoxification in response to oxidative stress. Previously, we reported that KEAP1 mutation elicits constitutive NRF2 activation and resistance to cisplatin (CDDP) and dacarbazine (DTIC) in human melanomas. The present study was conducted to clarify whether an HSP90 inhibitor, 17-AAG, efficiently eliminates melanoma with KEAP1 mutation, as the NRF2 target gene, NQO1, is a key enzyme in 17-AAG bioactivation. In melanoma and non-small cell lung carcinoma cell lines with or without KEAP1 mutations, NQO1 expression and 17-AAG sensitivity are inversely correlated. NQO1 is highly expressed in normal melanocytes and in several melanoma cell lines despite the presence of wild-type KEAP1, and the NQO1 expression is dependent on NRF2 activation. Because either CDDP or DTIC produces reactive oxygen species that activate NRF2, we determined whether these agents would sensitize NQO1-low melanoma cells to 17-AAG. Synergistic cytotoxicity of the 17-AAG and CDDP combination was detected in four out of five NQO1-low cell lines, but not in the cell line with KEAP1 mutation. These data indicate that 17-AAG could be a potential chemotherapeutic agent for melanoma with KEAP1 mutation or NQO1 expression. PMID:27045471

  15. Synthesis of new sulfonamides as lipoxygenase inhibitors.

    PubMed

    Mustafa, Ghulam; Khan, Islam Ullah; Ashraf, Muhammad; Afzal, Iftikhar; Shahzad, Sohail Anjum; Shafiq, Muhammad

    2012-04-15

    The present study describes a convenient method for the synthesis of new lipoxygenase inhibitors, 4-(toluene-4-sulfonylamino)-benzoic acids from p-amino benzoic acid. Reaction of p-amino benzoic acid with p-toluenesulfonyl chloride provided thirteen N- and O-alkylation products 4a-4m in moderate to good yields. Lipoxygenase inhibition of newly formed sulfonamide derivatives was investigated and some of these compounds 4m, 4g, 4e, 4f and 4j showed good lipoxygenase inhibitory activities with IC(50) values ranged between 15.8 ± 0.57 and 91.7 ± 0.61 μmol whilst all other compounds exhibited mild anti-lipoxygenase activities with IC(50) values ranged between 139.2 ± 0.75 and 232.1 ± 0.78 μmol. N-alkylated products were more active against the enzyme than O-alkylated or both N- and O-alkylated ones. All synthesized sulfonamides were recrystallized in chloroform to give these title compounds which were characterized using FTIR, (1)H NMR, (13)C NMR, elemental analysis and single crystal X-ray diffraction techniques. PMID:22436389

  16. Complexes of Bacterial Nicotinate Mononucleotide Adenylyltransferase with Inhibitors: Implication for Structure-Based Drug Design and Improvement

    SciTech Connect

    Huang, Nian; Kolhatkar, Rohit; Eyobo, Yvonne; Sorci, Leonardo; Rodionova, Irina; Osterman, Andrei L.; MacKerell, Jr., Alexander D.; Zhang, Hong

    2010-12-07

    Bacterial nicotinate mononucleotide adenylyltransferase encoded by the essential gene nadD plays a central role in the synthesis of the redox cofactor NAD{sup +}. The NadD enzyme is conserved in the majority of bacterial species and has been recognized as a novel target for developing new and potentially broad-spectrum antibacterial therapeutics. Here we report the crystal structures of Bacillus anthracis NadD in complex with three NadD inhibitors, including two analogues synthesized in the present study. These structures revealed a common binding site shared by different classes of NadD inhibitors and explored the chemical environment surrounding this site. The structural data obtained here also showed that the subtle changes in ligand structure can lead to significant changes in the binding mode, information that will be useful for future structure-based optimization and design of high affinity inhibitors.

  17. Synthesis and In Vitro Evaluation of Aspartate Transcarbamoylase Inhibitors

    PubMed Central

    Coudray, Laëtitia; Pennebaker, Anne F.; Montchamp, Jean-Luc

    2009-01-01

    The design, synthesis, and evaluation of a series of novel inhibitors of aspartate transcarbamoylase (ATCase) are reported. Several submicromolar phosphorus-containing inhibitors are described, but all-carboxylate compounds are inactive. Compounds were synthesized to probe the postulated cyclic transition-state of the enzyme-catalyzed reaction. In addition, the associated role of the protonation state at the phosphorus acid moiety was evaluated using phosphinic and carboxylic acids. Although none of the synthesized inhibitors is more potent than N-phosphonacetyl-L-aspartate (PALA), the compounds provide useful mechanistic information, as well as the basis for the design of future inhibitors and/or prodrugs. PMID:19828320

  18. Microbial NAD metabolism: lessons from comparative genomics.

    PubMed

    Gazzaniga, Francesca; Stebbins, Rebecca; Chang, Sheila Z; McPeek, Mark A; Brenner, Charles

    2009-09-01

    NAD is a coenzyme for redox reactions and a substrate of NAD-consuming enzymes, including ADP-ribose transferases, Sir2-related protein lysine deacetylases, and bacterial DNA ligases. Microorganisms that synthesize NAD from as few as one to as many as five of the six identified biosynthetic precursors have been identified. De novo NAD synthesis from aspartate or tryptophan is neither universal nor strictly aerobic. Salvage NAD synthesis from nicotinamide, nicotinic acid, nicotinamide riboside, and nicotinic acid riboside occurs via modules of different genes. Nicotinamide salvage genes nadV and pncA, found in distinct bacteria, appear to have spread throughout the tree of life via horizontal gene transfer. Biochemical, genetic, and genomic analyses have advanced to the point at which the precursors and pathways utilized by a microorganism can be predicted. Challenges remain in dissecting regulation of pathways. PMID:19721089

  19. SimC7 Is a Novel NAD(P)H-Dependent Ketoreductase Essential for the Antibiotic Activity of the DNA Gyrase Inhibitor Simocyclinone

    PubMed Central

    Schäfer, Martin; Le, Tung B.K.; Hearnshaw, Stephen J.; Maxwell, Anthony; Challis, Gregory L.; Wilkinson, Barrie; Buttner, Mark J.

    2015-01-01

    Simocyclinone D8 (SD8) is a potent DNA gyrase inhibitor produced by Streptomyces antibioticus Tü6040. The simocyclinone (sim) biosynthetic gene cluster has been sequenced and a hypothetical biosynthetic pathway has been proposed. The tetraene linker in SD8 was suggested to be the product of a modular type I polyketide synthase working in trans with two monofunctional enzymes. One of these monofunctional enzymes, SimC7, was proposed to supply a dehydratase activity missing from two modules of the polyketide synthase. In this study, we report the function of SimC7. We isolated the entire ~ 72-kb sim cluster on a single phage artificial chromosome clone and produced simocyclinone heterologously in a Streptomyces coelicolor strain engineered for improved antibiotic production. Deletion of simC7 resulted in the production of a novel simocyclinone, 7-oxo-SD8, which unexpectedly carried a normal tetraene linker but was altered in the angucyclinone moiety. We demonstrate that SimC7 is an NAD(P)H-dependent ketoreductase that catalyzes the conversion of 7-oxo-SD8 into SD8. 7-oxo-SD8 was essentially inactive as a DNA gyrase inhibitor, and the reduction of the keto group by SimC7 was shown to be crucial for high-affinity binding to the enzyme. Thus, SimC7 is an angucyclinone ketoreductase that is essential for the biological activity of simocyclinone. PMID:25861759

  20. SimC7 Is a Novel NAD(P)H-Dependent Ketoreductase Essential for the Antibiotic Activity of the DNA Gyrase Inhibitor Simocyclinone.

    PubMed

    Schäfer, Martin; Le, Tung B K; Hearnshaw, Stephen J; Maxwell, Anthony; Challis, Gregory L; Wilkinson, Barrie; Buttner, Mark J

    2015-06-19

    Simocyclinone D8 (SD8) is a potent DNA gyrase inhibitor produced by Streptomyces antibioticus Tü6040. The simocyclinone (sim) biosynthetic gene cluster has been sequenced and a hypothetical biosynthetic pathway has been proposed. The tetraene linker in SD8 was suggested to be the product of a modular type I polyketide synthase working in trans with two monofunctional enzymes. One of these monofunctional enzymes, SimC7, was proposed to supply a dehydratase activity missing from two modules of the polyketide synthase. In this study, we report the function of SimC7. We isolated the entire ~72-kb sim cluster on a single phage artificial chromosome clone and produced simocyclinone heterologously in a Streptomyces coelicolor strain engineered for improved antibiotic production. Deletion of simC7 resulted in the production of a novel simocyclinone, 7-oxo-SD8, which unexpectedly carried a normal tetraene linker but was altered in the angucyclinone moiety. We demonstrate that SimC7 is an NAD(P)H-dependent ketoreductase that catalyzes the conversion of 7-oxo-SD8 into SD8. 7-oxo-SD8 was essentially inactive as a DNA gyrase inhibitor, and the reduction of the keto group by SimC7 was shown to be crucial for high-affinity binding to the enzyme. Thus, SimC7 is an angucyclinone ketoreductase that is essential for the biological activity of simocyclinone. PMID:25861759

  1. High-Affinity Inhibitors of Human NAD+-Dependent 15-Hydroxyprostaglandin Dehydrogenase: Mechanisms of Inhibition and Structure-Activity Relationships

    PubMed Central

    Niesen, Frank H.; Schultz, Lena; Jadhav, Ajit; Bhatia, Chitra; Guo, Kunde; Maloney, David J.; Pilka, Ewa S.; Wang, Minghua; Oppermann, Udo; Heightman, Tom D.; Simeonov, Anton

    2010-01-01

    Background 15-hydroxyprostaglandin dehydrogenase (15-PGDH, EC 1.1.1.141) is the key enzyme for the inactivation of prostaglandins, regulating processes such as inflammation or proliferation. The anabolic pathways of prostaglandins, especially with respect to regulation of the cyclooxygenase (COX) enzymes have been studied in detail; however, little is known about downstream events including functional interaction of prostaglandin-processing and -metabolizing enzymes. High-affinity probes for 15-PGDH will, therefore, represent important tools for further studies. Principal Findings To identify novel high-affinity inhibitors of 15-PGDH we performed a quantitative high-throughput screen (qHTS) by testing >160 thousand compounds in a concentration-response format and identified compounds that act as noncompetitive inhibitors as well as a competitive inhibitor, with nanomolar affinity. Both types of inhibitors caused strong thermal stabilization of the enzyme, with cofactor dependencies correlating with their mechanism of action. We solved the structure of human 15-PGDH and explored the binding modes of the inhibitors to the enzyme in silico. We found binding modes that are consistent with the observed mechanisms of action. Conclusions Low cross-reactivity in screens of over 320 targets, including three other human dehydrogenases/reductases, suggest selectivity of the present inhibitors for 15-PGDH. The high potencies and different mechanisms of action of these chemotypes make them a useful set of complementary chemical probes for functional studies of prostaglandin-signaling pathways. Enhanced version This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S2. PMID:21072165

  2. Synthesis of amino heterocycle aspartyl protease inhibitors.

    PubMed

    Chambers, Rachel K; Khan, Tanweer A; Olsen, David B; Sleebs, Brad E

    2016-06-14

    Aspartyl proteases are important pharmacological targets. Historically aspartyl proteases have been commonly targeted with transition state derived peptidomimetics. The strategy to develop aspartyl protease inhibitors has undertaken a dramatic paradigm shift in the last 10 years. The pharmaceutical industry in 2005 disclosed several scaffolds or "head groups" that prompted the field to move beyond peptidomimetic derived inhibitors. Since the discovery of the first amino heterocycle aspartyl protease inhibitor, the amino hydantoin, industry and academia have positioned themselves for a foothold on the new molecular space, designing a variety of related "head groups". Both the design and synthetic efforts involved in constructing these scaffolds are varied and complex. Here we highlight the synthetic strategies used to access these amino heterocycle scaffolds. PMID:27143279

  3. Hypolipidemic drugs are inhibitors of phosphatidylcholine synthesis.

    PubMed Central

    Parthasarathy, S; Kritchevsky, D; Baumann, W J

    1982-01-01

    Clofibric acid (CPIB) and several other systemic hypolipidemic drugs are shown to block phosphatidylcholine synthesis by inhibiting cholinephosphotransferase (ChoPTase; CDPcholine:1,2-diacylglycerol cholinephosphotransferase, EC 2.7.8.2) and particularly lysolecithin acyltransferase (LLAcylTase; acyl-CoA:1-acylglycero-3-phosphocholine O-acyltransferase, EC 2.3.1.23) of rat liver microsomes. Whereas millimolar drug concentrations are required to affect de novo lecithin synthesis catalyzed by ChoPTase, reacylation of lysolecithin by LLAcylTase is inhibited at micromolar levels. Increasing effectiveness in ChoPTase inhibition is observed in the series CPIB, SaH-42-348, tibric acid, S-321328, WY-14643, S-8527, and DH-990, with IC50 ranging from 22 mM (CPIB) to 0.3 mM (DH-990). LLAcylTase inhibition by the hypolipidemic drugs follows the same general pattern, but IC50 concentrations range from 9 mM (CPIB) to 40 microM (DH-990). The agents inhibit ChoPTase (Ki, 25-0.25 mM) and LLAcylTase (Ki, 10-0.025 mM) noncompetitively. The data suggest that inhibition of phosphatidylcholine synthesis, particularly by the LLAcylTase pathway, may be related to a drug's effectiveness in decreasing serum triglyceride and cholesterol levels by blocking lipoprotein synthesis. PMID:6294663

  4. Studies on bacterial cell wall inhibitors. VI. Screening method for the specific inhibitors of peptidoglycan synthesis.

    PubMed

    Omura, S; Tanaka, H; Oiwa, R; Nagai, T; Koyama, Y; Takahashi, Y

    1979-10-01

    A screening method was established for selecting new specific inhibitors of bacterial cell wall peptidoglycan synthesis. In the primary test, culture broths of soil isolates were selected based on relative microbial activity. A culture, to be retained, must be active against Bacillus subtilis and lack activities against Acholeplasma laidawii. In the secondary test, inhibitors of bacterial cell wall synthesis were identified by their ability to prevent the incorporation of meso-[3H]diaminopimelic acid but not to prevent the incorporation of L-[4C]leucine into the acid-insoluble macromolecular fraction of growing cells of Bacillus sp. ATCC 21206 (Dpm-). As the tertiary test, inhibitors with molecular weights under 1,000 were selected by passage through a Diaflo UM-2 membrane. By this screening procedure, six known antibiotics and one new one were picked out from ten thousand soil isolates. PMID:528376

  5. NAD(+)- dependent deacetylase SIRT3 regulates mitochondrial protein synthesis by deacetylation of the ribosomal protein MRPL10

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A member of the sirtuin family of NAD (+)-dependent deacetylases, SIRT3, is located in mammalian mitochondria and is important for regulation of mitochondrial metabolism, cell survival, and longevity. In this study, MRPL10 (mitochondrial ribosomal protein L10) was identified as the major acetylated ...

  6. Eukaryotic protein synthesis inhibitors identified by comparison of cytotoxicity profiles

    PubMed Central

    CHAN, JENNY; KHAN, SHAKILA N.; HARVEY, ISABELLE; MERRICK, WILLIAM; PELLETIER, JERRY

    2004-01-01

    The National Cancer Institute (NCI) Human Tumor Cell Line Anti-Cancer Drug Screen has evaluated the cytotoxicity profiles of a large number of synthetic compounds, natural products, and plant extracts on 60 different cell lines. The data for each compound/extract can be assessed for similarity of cytotoxicity pattern, relative to a given test compound, using an algorithm called COMPARE. In applying a chemical biology approach to better understand the mechanism of eukaryotic protein synthesis, we used these resources to search for novel inhibitors of translation. The cytotoxicity profiles of 31 known protein synthesis inhibitors were used to identify compounds from the NCI database with similar activity profiles. Using this approach, two natural products, phyllanthoside and nagilactone C, were identified and characterized as novel protein synthesis inhibitors. Both compounds are specific for the eukaryotic translation apparatus, function in vivo and in vitro, and interfere with translation elongation. Our results demonstrate the feasibility of utilizing cytotoxicity profiles to identify new inhibitors of translation. PMID:14970397

  7. Synthesis and evaluation of bibenzyl glycosides as potent tyrosinase inhibitors.

    PubMed

    Tajima, Reiko; Oozeki, Hiromi; Muraoka, Seiichi; Tanaka, Saori; Motegi, Yukari; Nihei, Hiroyuki; Yamada, Yoichi; Masuoka, Noriyoshi; Nihei, Ken-ichi

    2011-04-01

    Bibenzyl glycosides 1-6 were synthesized from 2,4-dihydoxybenzaldehyde and xylose, glucose, cellobiose or maltose. The key steps in the synthesis were the Wittig reaction and trichloroacetimidate glycosylation. Tests for tyrosinase inhibitory activity showed that all were significantly active, indicating that they are unique hydrophilic tyrosinase inhibitors. Bibenzyl xyloside 2 is a particularly potent inhibitor (IC(50) = 0.43 μM, 17 times higher than that of kojic acid). These results suggest that the hydrophilic cavity of tyrosinase might accommodate the bulky carbohydrate on the bibenzyl scaffold. PMID:21334791

  8. Design and synthesis of boronic acid inhibitors of endothelial lipase.

    PubMed

    O'Connell, Daniel P; LeBlanc, Daniel F; Cromley, Debra; Billheimer, Jeffrey; Rader, Daniel J; Bachovchin, William W

    2012-02-01

    Endothelial lipase (EL) and lipoprotein lipase (LPL) are homologous lipases that act on plasma lipoproteins. EL is predominantly a phospholipase and appears to be a key regulator of plasma HDL-C. LPL is mainly a triglyceride lipase regulating (V)LDL levels. The existing biological data indicate that inhibitors selective for EL over LPL should have anti-atherogenic activity, mainly through increasing plasma HDL-C levels. We report here the synthesis of alkyl, aryl, or acyl-substituted phenylboronic acids that inhibit EL. Many of the inhibitors evaluated proved to be nearly equally potent against both EL and LPL, but several exhibited moderate to good selectivity for EL. PMID:22225633

  9. Synthesis of the Pitstop family of clathrin inhibitors.

    PubMed

    Robertson, Mark J; Deane, Fiona M; Stahlschmidt, Wiebke; von Kleist, Lisa; Haucke, Volker; Robinson, Phillip J; McCluskey, Adam

    2014-07-01

    This protocol describes the synthesis of two classes of clathrin inhibitors, Pitstop 1 and Pitstop 2, along with two inactive analogs that can be used as negative controls (Pitstop inactive controls, Pitnot-2 and Pitnot-2-100). Pitstop-induced inhibition of clathrin TD function acutely interferes with clathrin-mediated endocytosis (CME), synaptic vesicle recycling and cellular entry of HIV, whereas clathrin-independent internalization pathways and secretory traffic proceed unperturbed; these reagents can, therefore, be used to investigate clathrin function, and they have potential pharmacological applications. Pitstop 1 is synthesized in two steps: sulfonation of 1,8-naphthalic anhydride and subsequent reaction with 4-amino(methyl)aniline. Pitnot-1 results from the reaction of 4-amino(methyl)aniline with commercially available 4-sulfo-1,8-naphthalic anhydride potassium salt. Reaction of 1-naphthalene sulfonyl chloride with pseudothiohydantoin followed by condensation with 4-bromobenzaldehyde yields Pitstop 2. The synthesis of the inactive control commences with the condensation of 4-bromobenzaldehyde with the rhodanine core. Thioketone methylation and displacement with 1-napthylamine affords the target compound. Although Pitstop 1-series compounds are not cell permeable, they can be used in biochemical assays or be introduced into cells via microinjection. The Pitstop 2-series compounds are cell permeable. The synthesis of these compounds does not require specialist equipment and can be completed in 3-4 d. Microwave irradiation can be used to reduce the synthesis time. The synthesis of the Pitstop 2 family is easily adaptable to enable the synthesis of related compounds such as Pitstop 2-100 and Pitnot-2-100. The procedures are also simple, efficient and amenable to scale-up, enabling cost-effective in-house synthesis for users of these inhibitor classes. PMID:24922269

  10. The antiproliferative activity of the heat shock protein 90 inhibitor IPI-504 is not dependent on NAD(P)H:quinone oxidoreductase 1 activity in vivo.

    PubMed

    Douglas, Mark; Lim, Alice R; Porter, James R; West, Kip; Pink, Melissa M; Ge, Jie; Wylie, Andrew A; Tibbits, Thomas T; Biggs, Kurtis; Curtis, Michael; Palombella, Vito J; Adams, Julian; Fritz, Christian C; Normant, Emmanuel

    2009-12-01

    IPI-504, a water-soluble ansamycin analogue currently being investigated in clinical trials, is a potent inhibitor of the protein chaperone heat shock protein 90 (Hsp90). Inhibition of Hsp90 by IPI-504 triggers the degradation of important oncogenic client proteins. In cells, the free base of IPI-504 hydroquinone exists in a dynamic redox equilibrium with its corresponding quinone (17-AAG); the hydroquinone form binding 50 times more tightly to Hsp90. It has been proposed recently that the NAD(P)H:quinone oxidoreductase NQO1 can produce the active hydroquinone and could be essential for the activity of IPI-504. Here, we have devised a method to directly measure the intracellular ratio of hydroquinone to quinone (HQ/Q) and have applied this measurement to correlate NQO1 enzyme abundance with HQ/Q ratio and cellular activity of IPI-504 in 30 cancer cell lines. Interestingly, the intracellular HQ/Q ratio was correlated with NQO1 levels only in a subset of cell lines and overall was poorly correlated with the growth inhibitory activity of IPI-504. Although artificial overexpression of NQO1 is able to increase the level of hydroquinone and cell sensitivity to IPI-504, it has little effect on the activity of 17-amino-17-demethoxy-geldanamycin, the major active metabolite of IPI-504. This finding could provide an explanation for the biological activity of IPI-504 in xenograft models of cell lines that are not sensitive to IPI-504 in vitro. Our results suggest that NQO1 activity is not a determinant of IPI-504 activity in vivo and, therefore, unlikely to become an important resistance mechanism to IPI-504 in the clinic. PMID:19952119

  11. New peptide deformylase inhibitors design, synthesis and pharmacokinetic assessment.

    PubMed

    Lv, Fengping; Chen, Chen; Tang, Yang; Wei, Jianhai; Zhu, Tong; Hu, Wenhao

    2016-08-01

    The docking approach for the screening of designed small molecule ligands, led to the identification of a critical arginine residue in peptide deformylase for spiro cyclopropyl PDF inhibitor's extra hydrophobic binding, providing us a useful tool for searching more efficient PDF inhibitors to fight for horrifying antibiotics resistance. Further synthetic modification was undertaken to optimize the potency of amide compounds. To lower metabolic susceptibility and in turn reduce unwanted metabolic toxicity that was observed clinically, while retaining desired antibacterial activity, the use of azoles as amide bioisosteres had also been investigated. After the completion of chemical synthesis, all the compounds were evaluated through in vitro antibacterial activity assay, some of which were further subject to in vivo rat pharmacokinetic assessment. Those findings in this letter showed that spiro cyclopropyl proline N-formyl hydroxylamines, and especially the bioisosteric azoles, can represent a promising class of PDF inhibitors. PMID:27293070

  12. Thrombin or Ca(++)-ionophore-mediated fall in endothelial ATP levels independent of poly(ADP-Ribose) polymerase activity and NAD levels--comparison with the effects of hydrogen peroxide.

    PubMed

    Halldórsson, Haraldur; Thors, Brynhildur; Thorgeirsson, Gudmundur

    2015-01-01

    To test the hypothesis that a fall in cellular ATP following stimulation of endothelial cells with thrombin is secondary to a decrease in NAD levels caused by poly(ADP-Ribose)polymerase (PARP), we measured the levels of NAD and ATP in endothelial cells after treatment with thrombin, the Ca(++)-ionophore A23187, or hydrogen peroxide (H2O2), and compared the effects of inhibitors of PARP, NAD synthesis, and ADP-ribose breakdown on these responses. Neither thrombin nor A23187 caused a reduction in endothelial NAD levels and A23187 affected ATP levels independently of NAD levels or PARP activity. H2O2 induced lowering of NAD caused modest lowering of ATP but marked additional ATP-lowering, independent of PARP and NAD, was also demonstrated. We conclude that in endothelial cells ATP levels are largely independent of NAD and PARP, which do not play a role in thrombin or Ca(++)-ionophore-mediated lowering of ATP. H2O2 caused ATP lowering through a similar mechanism as thrombin and A23187 but, additionally, caused a further ATP lowering through its intense stimulation of PARP and marked lowering of NAD. PMID:25774718

  13. NAD metabolism in HPRT-deficient mice

    PubMed Central

    Jacomelli, Gabriella; Di Marcello, Federica; Notarantonio, Laura; Sestini, Silvia; Cerboni, Barbara; Bertelli, Matteo; Pompucci, Giuseppe; Jinnah, Hyder A.

    2016-01-01

    The activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT) is virtually absent in Lesch-Nyhan disease (LND), an X-linked genetic disorder characterized by uric acid accumulation and neurodevelopmental dysfunction. The biochemical basis for the neurological and behavioral abnormalities have not yet been completely explained. Prior studies of cells from affected patients have shown abnormalities of NAD metabolism. In the current studies, NAD metabolism was evaluated in HPRT gene knock-out mice. NAD content and the activities of the enzymes required for synthesis and breakdown of this coenzyme were investigated in blood, brain and liver of HPRT− and control mice. NAD concentration and enzyme activities were found to be significantly increased in liver, but not in brain or blood of the HPRT− mice. These results demonstrate that changes in NAD metabolism occur in response to HPRT deficiency depending on both species and tissue type. PMID:19319672

  14. Prostaglandin synthesis inhibitors block alcohol-induced fetal hypoplasia.

    PubMed

    Pennington, S; Allen, Z; Runion, J; Farmer, P; Rowland, L; Kalmus, G

    1985-01-01

    Alcohol-induced growth retardation is a fetal effect consistently associated with maternal ethanol consumption. In humans, those infants whose mothers consume even a limited amount of ethanol during pregnancy have a significant incidence of growth inhibition. The molecular mechanism responsible for this growth deficiency is unknown, and prevention depends on maternal abstinence during pregnancy. The data reported here suggest that ethanol-mediated increases in tissue prostaglandin (PG) E levels (PGE1 plus PGE2) are correlated with the growth retardation. Further, simultaneous administration of PG synthesis inhibitors with the alcohol blocks the rise in tissue PG levels and protects against the alcohol-induced hypoplasia. PMID:3904508

  15. Exploring NAD+ metabolism in host-pathogen interactions.

    PubMed

    Mesquita, Inês; Varela, Patrícia; Belinha, Ana; Gaifem, Joana; Laforge, Mireille; Vergnes, Baptiste; Estaquier, Jérôme; Silvestre, Ricardo

    2016-03-01

    Nicotinamide adenine dinucleotide (NAD(+)) is a vital molecule found in all living cells. NAD(+) intracellular levels are dictated by its synthesis, using the de novo and/or salvage pathway, and through its catabolic use as co-enzyme or co-substrate. The regulation of NAD(+) metabolism has proven to be an adequate drug target for several diseases, including cancer, neurodegenerative or inflammatory diseases. Increasing interest has been given to NAD(+) metabolism during innate and adaptive immune responses suggesting that its modulation could also be relevant during host-pathogen interactions. While the maintenance of NAD(+) homeostatic levels assures an adequate environment for host cell survival and proliferation, fluctuations in NAD(+) or biosynthetic precursors bioavailability have been described during host-pathogen interactions, which will interfere with pathogen persistence or clearance. Here, we review the double-edged sword of NAD(+) metabolism during host-pathogen interactions emphasizing its potential for treatment of infectious diseases. PMID:26718485

  16. Design and synthesis of novel selective anaplastic lymphoma kinase inhibitors.

    PubMed

    Michellys, Pierre-Yves; Chen, Bei; Jiang, Tao; Jin, Yunho; Lu, Wenshuo; Marsilje, Thomas H; Pei, Wei; Uno, Tetsuo; Zhu, Xuefeng; Wu, Baogen; Nguyen, Truc Ngoc; Bursulaya, Badry; Lee, Christian; Li, Nanxin; Kim, Sungjoon; Tuntland, Tove; Liu, Bo; Sun, Frank; Steffy, Auzon; Hood, Tami

    2016-02-01

    Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase belonging to the insulin receptor superfamily. Expression of ALK in normal human tissues is only found in a subset of neural cells, however it is involved in the genesis of several cancers through genetic aberrations involving translocation of the kinase domain with multiple fusion partners (e.g., NPM-ALK in anaplastic large cell lymphoma ALCL or EML4-ALK in non-small cell lung cancer) or activating mutations in the full-length receptor resulting in ligand-independent constitutive activation (e.g., neuroblastoma). Here we are reporting the discovery of novel and selective anaplastic lymphoma kinase inhibitors from specific modifications of the 2,4-diaminopyridine core present in TAE684 and LDK378. Synthesis, structure activity relationships (SAR), absorption, distribution, metabolism, and excretion (ADME) profile, and in vivo efficacy in a mouse xenograft model of anaplastic large cell lymphoma are described. PMID:26750252

  17. Enhanced Degradation of Dihydrofolate Reductase through Inhibition of NAD Kinase by Nicotinamide Analogs

    PubMed Central

    Hsieh, Yi-Ching; Tedeschi, Philip; AdeBisi Lawal, Rialnat; Banerjee, Debabrata; Scotto, Kathleen; Kerrigan, John E.; Lee, Kuo-Chieh; Johnson-Farley, Nadine; Bertino, Joseph R.

    2013-01-01

    Dihydrofolate reductase (DHFR), because of its essential role in DNA synthesis, has been targeted for the treatment of a wide variety of human diseases, including cancer, autoimmune diseases, and infectious diseases. Methotrexate (MTX), a tight binding inhibitor of DHFR, is one of the most widely used drugs in cancer treatment and is especially effective in the treatment of acute lymphocytic leukemia, non-Hodgkin’s lymphoma, and osteosarcoma. Limitations to its use in cancer include natural resistance and acquired resistance due to decreased cellular uptake and decreased retention due to impaired polyglutamylate formation and toxicity at higher doses. Here, we describe a novel mechanism to induce DHFR degradation through cofactor depletion in neoplastic cells by inhibition of NAD kinase, the only enzyme responsible for generating NADP, which is rapidly converted to NADPH by dehydrogenases/reductases. We identified an inhibitor of NAD kinase, thionicotinamide adenine dinucleotide phosphate (NADPS), which led to accelerated degradation of DHFR and to inhibition of cancer cell growth. Of importance, combination treatment of NADPS with MTX displayed significant synergy in a metastatic colon cancer cell line and was effective in a MTX-transport resistant leukemic cell line. We suggest that NAD kinase is a valid target for further inhibitor development for cancer treatment. PMID:23197646

  18. Boosting NAD+ for the prevention and treatment of liver cancer

    PubMed Central

    Djouder, Nabil

    2015-01-01

    Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide yet has limited therapeutic options. We recently demonstrated that inhibition of de novo nicotinamide adenine dinucleotide (NAD+) synthesis is responsible for DNA damage, thereby initiating hepatocarcinogenesis. We propose that boosting NAD+ levels might be used as a prophylactic or therapeutic approach in HCC. PMID:27308492

  19. Boosting NAD(+) for the prevention and treatment of liver cancer.

    PubMed

    Djouder, Nabil

    2015-01-01

    Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide yet has limited therapeutic options. We recently demonstrated that inhibition of de novo nicotinamide adenine dinucleotide (NAD(+)) synthesis is responsible for DNA damage, thereby initiating hepatocarcinogenesis. We propose that boosting NAD(+) levels might be used as a prophylactic or therapeutic approach in HCC. PMID:27308492

  20. 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

  1. 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. PMID:25394180

  2. Synthesis and biological evaluation of neutrophilic inflammation inhibitors.

    PubMed

    Bruno, Olga; Brullo, Chiara; Arduino, Nicoletta; Schenone, Silvia; Ranise, Angelo; Bondavalli, Francesco; Ottonello, Luciano; Dapino, Patrizia; Dallegri, Franco

    2004-03-01

    In several non-infectious human diseases, such as ulcerous colitis, rheumatoid arthritis, chronic obstructive pulmonary disease (COPD), the extravasal recruitment of neutrophils plays a crucial role in the development of tissue damage, which, when persistent, can lead to the irreversible organ dysfunction. The neutrophil activation is controlled by a number of intracellular pathways, particularly by a cAMP-dependent protein kinase A (PKA) which also acts on phosphodiesterase IV (PDE4) gene stimulating the synthesis of this enzyme, able to transform cAMP to inactive AMP. PDE4 inhibitors enhance intracellular cAMP and decrease inflammatory cell activation. Several 3-cyclopentyloxy-4-methoxybenzaldehyde and 3-cyclopentyloxy-4-methoxybenzoic acid derivatives were synthesized and studied by us to evaluate their ability to inhibit the superoxide anion production in human neutrophils. These compounds were found able to inhibit the neutrophil activation and some of them increased the cAMP level on tumor necrosis factor-alpha-stimulated neutrophils. Moreover, they also inhibited selectively the human PDE4 enzyme, although they are less potent than the reference compound Rolipram. We report here synthesis, biological studies and some SAR considerations concerning the above mentioned compounds. PMID:14987986

  3. NAD(+) biosynthesis and salvage--a phylogenetic perspective.

    PubMed

    Gossmann, Toni I; Ziegler, Mathias; Puntervoll, Pål; de Figueiredo, Luis F; Schuster, Stefan; Heiland, Ines

    2012-09-01

    NAD is best known as an electron carrier and a cosubstrate of various redox reactions. However, over the past 20 years, NAD(+) has been shown to be a key signaling molecule that mediates post-translational protein modifications and serves as precursor of ADP-ribose-containing messenger molecules, which are involved in calcium mobilization. In contrast to its role as a redox carrier, NAD(+)-dependent signaling processes involve the release of nicotinamide (Nam) and require constant replenishment of cellular NAD(+) pools. So far, very little is known about the evolution of NAD(P) synthesis in eukaryotes. In the present study, genes involved in NAD(P) metabolism in 45 species were identified and analyzed with regard to similarities and differences in NAD(P) synthesis. The results show that the Preiss-Handler pathway and NAD(+) kinase are present in all organisms investigated, and thus seem to be ancestral routes. Additionally, two pathways exist that convert Nam to NAD(+); we identified several species that have apparently functional copies of both biosynthetic routes, which have been thought to be mutually exclusive. Furthermore, our findings suggest the parallel phylogenetic appearance of Nam N-methyltransferase, Nam phosphoribosyl transferase, and poly-ADP-ribosyltransferases. PMID:22404877

  4. Total synthesis of the squalene synthase inhibitor zaragozic acid C.

    PubMed

    Nakamura, Seiichi

    2005-01-01

    Zaragozic acids and squalestatins were documented by Merck, Glaxo, and Tokyo Noko University/Mitsubishi Kasei Corporation as part of a program aimed at identifying novel inhibitors of squalene synthase, as well as farnesyl transferase. These natural products have attracted considerable attention from numerous synthetic chemists because of their therapeutic potential and novel architecture. This review highlights our total syntheses of zaragozic acid C by two convergent strategies. The key steps in our first-generation synthesis involve 1) simultaneous creation of the C4 and C5 quaternary stereocenters through the Sn(OTf)2-promoted aldol coupling reaction between the alpha-keto ester and silyl ketene thioacetal derived from L- and D-tartaric acids, respectively; and 2) construction of the bicyclic core structure via acid-catalyzed internal ketalization under kinetically controlled conditions. The second-generation strategy relies on a tandem carbonyl ylide formation/1,3-dipolar cycloaddition approach and features elongation of the C1 alkyl side chain through an olefin cross-metathesis as well as high convergency and flexibility. PMID:15635219

  5. Salicylic Acid Inhibits Synthesis of Proteinase Inhibitors in Tomato Leaves Induced by Systemin and Jasmonic Acid.

    PubMed Central

    Doares, S. H.; Narvaez-Vasquez, J.; Conconi, A.; Ryan, C. A.

    1995-01-01

    Salicylic acid (SA) and acetylsalicylic acid (ASA), previously shown to inhibit proteinase inhibitor synthesis induced by wounding, oligouronides (H.M. Doherty, R.R. Selvendran, D.J. Bowles [1988] Physiol Mol Plant Pathol 33: 377-384), and linolenic acid (H. Pena-Cortes, T. Albrecht, S. Prat, E.W. Weiler, L. Willmitzer [1993] Planta 191: 123-128), are shown here to be potent inhibitors of systemin-induced and jasmonic acid (JA)-induced synthesis of proteinase inhibitor mRNAs and proteins. The inhibition by SA and ASA of proteinase inhibitor synthesis induced by systemin and JA, as well as by wounding and oligosaccharide elicitors, provides further evidence that both oligosaccharide and polypeptide inducer molecules utilize the octadecanoid pathway to signal the activation of proteinase inhibitor genes. Tomato (Lycopersicon esculentum) leaves were pulse labeled with [35S]methionine, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the inhibitory effects of SA are shown to be specific for the synthesis of a small number of JA-inducible proteins that includes the proteinase inhibitors. Previous results have shown that SA inhibits the conversion of 13S-hydroperoxy linolenic acid to 12-oxo-phytodienoic acid, thereby inhibiting the signaling pathway by blocking synthesis of JA. Here we report that the inhibition of synthesis of proteinase inhibitor proteins and mRNAs by SA in both light and darkness also occurs at a step in the signal transduction pathway, after JA synthesis but preceding transcription of the inhibitor genes. PMID:12228577

  6. Practical Synthesis of PC190723, An Inhibitor of the Bacterial Cell Division Protein FtsZ

    PubMed Central

    Sorto, Nohemy A.; Olmstead, Marilyn M.; Shaw, Jared T.

    2010-01-01

    A high-yielding and practical synthesis of the bacterial cell division inhibitor PC190723 is described. The synthesis is completed in a longest linear sequence of five steps from commercially available starting materials and can be readily executed on a multi-gram scale. PMID:21033691

  7. Synthesis and evaluation of transthyretin amyloidosis inhibitors containing carborane pharmacophores

    PubMed Central

    Julius, Richard L.; Farha, Omar K.; Chiang, Janet; Perry, L. Jeanne; Hawthorne, M. Frederick

    2007-01-01

    Carboranes represent a potentially rich but underutilized class of inorganic and catabolism-inert pharmacophores. The regioselectivity and ease of derivatization of carboranes allows for facile syntheses of a wide variety of novel structures. The steric bulk, rigidity, and ease of B- and C-derivatization and lack of π-interactions associated with hydrophobic carboranes may be exploited to enhance the selectivity of previously identified bioactive molecules. Transthyretin (TTR) is a thyroxine-transport protein found in the blood that has been implicated in a variety of amyloid related diseases. Previous investigations have identified a variety of nonsteroidal antiinflammatory drugs (NSAIDs) and structurally related derivatives that imbue kinetic stabilization to TTR, thus inhibiting its dissociative fragmentation and subsequent aggregation to form putative toxic amyloid fibrils. However, the cyclooxygenase (COX) activity associated with these pharmaceuticals may limit their potential as long-term therapeutic agents for TTR amyloid diseases. Here, we report the synthesis and evaluation of carborane-containing analogs of the promising NSAID pharmaceuticals previously identified. The replacement of a phenyl ring in the NSAIDs with a carborane moiety greatly decreases their COX activity with the retention of similar efficacy as an inhibitor of TTR dissociation. The most promising of these compounds, 1-carboxylic acid-7-[3-fluorophenyl]-1,7-dicarba-closo-dodecaborane, showed effectively no COX-1 or COX-2 inhibition at a concentration more than an order of magnitude larger than the concentration at which TTR dissociation is nearly completely inhibited. This specificity is indicative of the potential for the exploitation of the unique properties of carboranes as potent and selective pharmacophores. PMID:17360344

  8. Separation of cordycepin from Cordyceps militaris fermentation supernatant using preparative HPLC and evaluation of its antibacterial activity as an NAD+-dependent DNA ligase inhibitor

    PubMed Central

    Zhou, Xiaofeng; Cai, Guoqiang; He, Yi; Tong, Guotong

    2016-01-01

    Cordycepin exhibits various bio-activities, including anticancer, antibacterial, antiviral and immune regulation activities, and is a significant focus of research. However, the preparation of high-purity cordycepin remains challenging. Also, the molecular target with which cordycepin interacts to cause an antibacterial effect remains unknown. In the present study, cordycepin was prepared by preparative high-performance liquid chromatography (prep-HPLC) and the purity obtained was 99.6%, indicating that this technique may be useful for the large-scale isolation of cordycepin in the future. The results of computational molecular docking analysis indicated that the interaction energy between cordycepin and NAD+-dependent DNA ligase (LigA) was lower than that between cordycepin and other common antibacterial targets. The highly pure cordycepin obtained by prep-HPLC demonstrated inhibitory activity against LigA from various bacteria in vitro. In conclusion, cordycepin may be useful as a broad-spectrum antibiotic targeting LigA in various bacteria. PMID:27588098

  9. Intentional synthesis of corrosion inhibitors based on secondary products of sugar cane processing

    SciTech Connect

    Ledovskykh, V.M.

    1988-07-01

    Secondary products of sugar cane processing (mosto, wax, furfurol) were studied as starting raw materials for creating inhibitors for different purposes and temporary means of protecting metals from corrosion. In order to protect metals in different corrosive media the following inhibitors have been developed: an inhibitor for acid solutions (pickling metals, acid washing of the equipment) based on high-tonnage water-soluble waste mosto and combined synergistic inhibitors based on mixtures of it with cation- and anion-active surfactants, including nitrogen- and sulfur-containing substances obtained by intentional synthesis of another secondary product, furfurol; inhibitors for two-phase media (oil recovery and refining) of the carbonic acid amide and 2-alkylimidazoline classes from sugar cane wax; and inhibitors comprised of Li-, Na-, Ca-, and Al-plastic greases from sugar cane wax for atmospheric conditions.

  10. Possible role of NAD-dependent glyceraldehyde-3-phosphate dehydrogenase in growth promotion of Arabidopsis seedlings by low levels of selenium.

    PubMed

    Takeda, Toru; Fukui, Yuki

    2015-01-01

    We explored functional significance of selenium (Se) in Arabidopsis physiology. Se at very low concentrations in cultivation exerted a considerable positive effect on Arabidopsis growth with no indication of oxidative stress, whereas Se at higher concentrations significantly suppressed the growth and brought serious oxidative damage. Respiration, ATP levels, and the activity of NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (NAD-GAPDH) were enhanced in Arabidopsis grown in the medium containing 1.0 μM Se. Addition of an inhibitor of glutathione (GSH) synthesis to the medium abolished both of the Se-dependent growth promotion and NAD-GAPDH up-regulation. Assay of NAD-GAPDH purified from seedlings subjected to Se interventions raised the possibility of a direct connection between the activity of this enzyme and Arabidopsis growth. These results reveal that trace amounts of Se accelerate Arabidopsis growth, and suggest that this pro-growth effect of Se arises enhancing mitochondrial performance in a GSH-dependent manner, in which NAD-GAPDH may serve as a key regulator. PMID:25988618

  11. Enantioselective Synthesis of Dioxatriquinane Structural Motifs for HIV-1 Protease Inhibitors Using a Cascade Radical Cyclization†

    PubMed Central

    Ghosh, Arun K.; Xu, Chun-Xiao; Osswald, Heather L.

    2015-01-01

    Synthesis of novel HIV-1 protease inhibitors incorporating dioxatriquinane-derived P2-ligands is described. The tricyclic ligand alcohol contains five contiguous chiral centers. The ligand alcohols were prepared in optically active form by an enzymatic asymmetrization of mesodiacetate, cascade radical cyclization, and Lewis acid catalyzed reduction as the key steps. Inhibitors with dioxatriquinane-derived P2-ligands exhibited low nanomolar HIV-1 protease activity. PMID:26185337

  12. Design and synthesis of hepatoselective, pyrrole-based HMG-CoA reductase inhibitors.

    PubMed

    Pfefferkorn, Jeffrey A; Song, Yuntao; Sun, Kuai-Lin; Miller, Steven R; Trivedi, Bharat K; Choi, Chulho; Sorenson, Roderick J; Bratton, Larry D; Unangst, Paul C; Larsen, Scott D; Poel, Toni-Jo; Cheng, Xue-Min; Lee, Chitase; Erasga, Noe; Auerbach, Bruce; Askew, Valerie; Dillon, Lisa; Hanselman, Jeffrey C; Lin, Zhiwu; Lu, Gina; Robertson, Andrew; Olsen, Karl; Mertz, Thomas; Sekerke, Catherine; Pavlovsky, Alexander; Harris, Melissa S; Bainbridge, Graeme; Caspers, Nicole; Chen, Huifen; Eberstadt, Matthias

    2007-08-15

    This manuscript describes the design and synthesis of a series of pyrrole-based inhibitors of HMG-CoA reductase for the treatment of hypercholesterolemia. Analogs were optimized using structure-based design and physical property considerations resulting in the identification of 44, a hepatoselective HMG-CoA reductase inhibitor with excellent acute and chronic efficacy in a pre-clinical animal models. PMID:17574412

  13. Design and Synthesis of Potent, Selective Inhibitors of Matriptase

    PubMed Central

    2012-01-01

    Matriptase is a member of the type II transmembrane serine protease family. Several studies have reported deregulated matriptase expression in several types of epithelial cancers, suggesting that matriptase constitutes a potential target for cancer therapy. We report herein a new series of slow, tight-binding inhibitors of matriptase, which mimic the P1–P4 substrate recognition sequence of the enzyme. Preliminary structure–activity relationships indicate that this benzothiazole-containing RQAR-peptidomimetic is a very potent inhibitor and possesses a good selectivity for matriptase versus other serine proteases. A molecular model was generated to elucidate the key contacts between inhibitor 1 and matriptase. PMID:24900505

  14. Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays

    PubMed Central

    Aleshin, Vasily A.; Artiukhov, Artem V.; Oppermann, Henry; Kazantsev, Alexey V.; Lukashev, Nikolay V.; Bunik, Victoria I.

    2015-01-01

    Cellular NAD(P)H-dependent oxidoreductase activity with artificial dyes (NAD(P)H-OR) is an indicator of viability, as the cellular redox state is important for biosynthesis and antioxidant defense. However, high NAD(P)H due to impaired mitochondrial oxidation, known as reductive stress, should increase NAD(P)H-OR yet perturb viability. To better understand this complex behavior, we assayed NAD(P)H-OR with resazurin (Alamar Blue) in glioblastoma cell lines U87 and T98G, treated with inhibitors of central metabolism, oxythiamin, and phosphonate analogs of 2-oxo acids. Targeting the thiamin diphosphate (ThDP)-dependent enzymes, the inhibitors are known to decrease the NAD(P)H production in the pentose phosphate shuttle and/or upon mitochondrial oxidation of 2-oxo acids. Nevertheless, the inhibitors elevated NAD(P)H-OR with resazurin in a time- and concentration-dependent manner, suggesting impaired NAD(P)H oxidation rather than increased viability. In particular, inhibition of the ThDP-dependent enzymes affects metabolism of malate, which mediates mitochondrial oxidation of cytosolic NAD(P)H. We showed that oxythiamin not only inhibited mitochondrial 2-oxo acid dehydrogenases, but also induced cell-specific changes in glutamate and malate dehydrogenases and/or malic enzyme. As a result, inhibition of the 2-oxo acid dehydrogenases compromises mitochondrial metabolism, with the dysregulated electron fluxes leading to increases in cellular NAD(P)H-OR. Perturbed mitochondrial oxidation of NAD(P)H may thus complicate the NAD(P)H-based viability assay. PMID:26308058

  15. Design and synthesis of potent, isoxazole-containing renin inhibitors.

    PubMed

    Fournier, Pierre-André; Arbour, Mélissa; Cauchon, Elizabeth; Chen, Austin; Chefson, Amandine; Ducharme, Yves; Falgueyret, Jean-Pierre; Gagné, Sébastien; Grimm, Erich; Han, Yongxin; Houle, Robert; Lacombe, Patrick; Lévesque, Jean-François; MacDonald, Dwight; Mackay, Bruce; McKay, Dan; Percival, M David; Ramtohul, Yeeman; St-Jacques, René; Toulmond, Sylvie

    2012-04-15

    The design and optimization of a novel isoxazole S(1) linker for renin inhibitor is described herein. This effort culminated in the identification of compound 18, an orally bioavailable, sub-nanomolar renin inhibitor even in the presence of human plasma. When compound 18 was found to inhibit CYP3A4 in a time dependent manner, two strategies were pursued that successfully delivered equipotent compounds with minimal TDI potential. PMID:22450130

  16. Synthesis of potent inhibitors of anthrax toxin based on poly-L-glutamic acid.

    PubMed

    Joshi, Amit; Saraph, Arundhati; Poon, Vincent; Mogridge, Jeremy; Kane, Ravi S

    2006-01-01

    We report the synthesis of biodegradable polyvalent inhibitors of anthrax toxin based on poly-L-glutamic acid (PLGA). These biocompatible polyvalent inhibitors are at least 4 orders of magnitude more potent than the corresponding monovalent peptides in vitro and are comparable in potency to polyacrylamide-based inhibitors of anthrax toxin assembly. We have elucidated the influence of peptide density on inhibitory potency and demonstrated that these inhibitory potencies are limited by kinetics, with even higher activities seen when the inhibitors are preincubated with the heptameric receptor-binding subunit of anthrax toxin prior to exposure to cells. These polyvalent inhibitors are also effective at neutralizing anthrax toxin in vivo and represent attractive leads for designing biocompatible anthrax therapeutics. PMID:16984137

  17. The high-resolution crystal structure of periplasmic Haemophilus influenzae NAD nucleotidase reveals a novel enzymatic function of human CD73 related to NAD metabolism.

    PubMed

    Garavaglia, Silvia; Bruzzone, Santina; Cassani, Camilla; Canella, Laura; Allegrone, Gianna; Sturla, Laura; Mannino, Elena; Millo, Enrico; De Flora, Antonio; Rizzi, Menico

    2012-01-01

    Haemophilus influenzae is a major pathogen of the respiratory tract in humans that has developed the capability to exploit host NAD(P) for its nicotinamide dinucleotide requirement. This strategy is organized around a periplasmic enzyme termed NadN (NAD nucleotidase), which plays a central role by degrading NAD into adenosine and NR (nicotinamide riboside), the latter being subsequently internalized by a specific permease. We performed a biochemical and structural investigation on H. influenzae NadN which determined that the enzyme is a Zn2+-dependent 5'-nucleotidase also endowed with NAD(P) pyrophosphatase activity. A 1.3 Å resolution structural analysis revealed a remarkable conformational change that occurs during catalysis between the open and closed forms of the enzyme. NadN showed a broad substrate specificity, recognizing either mono- or di-nucleotide nicotinamides and different adenosine phosphates with a maximal activity on 5'-adenosine monophosphate. Sequence and structural analysis of H. influenzae NadN led us to discover that human CD73 is capable of processing both NAD and NMN, therefore disclosing a possible novel function of human CD73 in systemic NAD metabolism. Our data may prove to be useful for inhibitor design and disclosed unanticipated fascinating evolutionary relationships. PMID:21933152

  18. Design and synthesis of novel, conformationally restricted HMG-CoA reductase inhibitors.

    PubMed

    Pfefferkorn, Jeffrey A; Choi, Chulho; Song, Yuntao; Trivedi, Bharat K; Larsen, Scott D; Askew, Valerie; Dillon, Lisa; Hanselman, Jeffrey C; Lin, Zhiwu; Lu, Gina; Robertson, Andrew; Sekerke, Catherine; Auerbach, Bruce; Pavlovsky, Alexander; Harris, Melissa S; Bainbridge, Graeme; Caspers, Nicole

    2007-08-15

    Using structure-based design, a novel series of conformationally restricted, pyrrole-based inhibitors of HMG-CoA reductase were discovered. Leading analogs demonstrated potent inhibition of cholesterol synthesis in both in vitro and in vivo models and may be useful for the treatment of hypercholesterolemia and related lipid disorders. PMID:17574411

  19. [The clinical evaluation of the hypocholesterolemic effects of an inhibitor of cholesterol synthesis: mevalonic acid].

    PubMed

    Del Nero, E; Aloe, N; Augeri, C; Avola, F; Carta, G; Cavagnaro, A; De Grandi, R; Gianfreda, M; Magro, G P; Mazzarello, G P

    1992-07-01

    Twenty eight patients with heterozygous familial hypercholesterolemia were treated with mevalonic acid (an inhibitor of cholesterol synthesis) for 45 days. Patients received a daily dose of 750 to 1500 mg mevalonic acid depending on plasma cholesterol levels. Results showed a significant reduction in cholesterol values whereas no significant difference was observed in HDL cholesterol and triglyceride levels. PMID:1505176

  20. Bait matrix for delivery of chitin synthesis inhibitors to the formosan subterranean termite (Isoptera: Rhinotermitidae).

    PubMed

    Rojas, M G; Morales-Ramos, J A

    2001-04-01

    The efficacy of three chitin synthesis inhibitors, diflubenzuron, hexaflumuron, and chlorfluazuron, incorporated into a novel bait matrix to kill the Formosan subterranean termite, Coptotermes formosanus Shiraki, was evaluated in the laboratory. The bait matrix was significantly preferred by C. formosanus over southern yellow pine wood in a two-choice feeding test. Bait formulations containing 250 ppm of the three chitin synthesis inhibitors were presented to termite nests with 2,500 individuals (80% workers and 20% soldiers) in the presence of alternative food sources consisting of cardboard and southern yellow pine, Pinus taeda L., wood. None of the bait formulations were significantly repellent or feeding deterrent to the termite workers evidenced by the lack of full consumption of alternative food sources. All nests presented with the bait formulations died within 9 wk, whereas the control nests (bait with no chitin synthesis inhibitors) remained alive 6 mo after the end of the study. No significant differences in consumption were observed among the chitin synthesis inhibitor treatments. Importance of this study for the improvement of current bait technology is discussed. PMID:11332846

  1. Design, Synthesis, and Biological Evaluation of PKD Inhibitors

    PubMed Central

    George, Kara M.; Frantz, Marie-Céline; Bravo-Altamirano, Karla; LaValle, Courtney R.; Tandon, Manuj; Leimgruber, Stephanie; Sharlow, Elizabeth R.; Lazo, John S.; Wang, Q. Jane; Wipf, Peter

    2011-01-01

    Protein kinase D (PKD) belongs to a family of serine/threonine kinases that play an important role in basic cellular processes and are implicated in the pathogenesis of several diseases. Progress in our understanding of the biological functions of PKD has been limited due to the lack of a PKD-specific inhibitor. The benzoxoloazepinolone CID755673 was recently reported as the first potent and kinase-selective inhibitor for this enzyme. For structure-activity analysis purposes, a series of analogs was prepared and their in vitro inhibitory potency evaluated. PMID:22267986

  2. Design, synthesis and biological evaluation of potent FAAH inhibitors.

    PubMed

    Tuo, Wei; Leleu-Chavain, Natascha; Barczyk, Amélie; Renault, Nicolas; Lemaire, Lucas; Chavatte, Philippe; Millet, Régis

    2016-06-01

    A new series of 3-carboxamido-5-aryl-isoxazoles was designed, synthesized and evaluated for their biological activity. Different pharmacomodulations have been explored and the lipophilicity of these compounds was assessed. Investigation of the in vitro biological activity led to the identification of 5 compounds as potent FAAH inhibitors, their good FAAH inhibition capacity is probably correlated with their suitable lipophilicity. Specifically, compound 25 showed similar inhibition potency against FAAH in comparison with URB597, one of the most potent FAAH inhibitor known to date. PMID:27117424

  3. Enzymology of mammalian NAD metabolism in health and disease.

    PubMed

    Magni, Giulio; Orsomando, Giuseppe; Raffelli, Nadia; Ruggieri, Silverio

    2008-01-01

    Mounting evidence attests to the paramount importance of the non-redox NAD functions. Indeed, NAD homeostasis is related to the free radicals-mediated production of reactive oxygen species responsible for irreversible cellular damage in infectious disease, diabetes, inflammatory syndromes, neurodegeneration and cancer. Because the cellular redox status depends on both the absolute concentration of pyridine dinucleotides and their respective ratios of oxidized and reduced forms (i.e., NAD/NADH and NADP/NADPH), it is conceivable that an altered regulation of the synthesis and degradation of NAD impairs the cell redox state and likely contributes to the mechanisms underlying the pathogenesis of the above mentioned diseases. Taking into account the recent appearance in the literature of comprehensive reviews covering different aspects of the significance of NAD metabolism, with particular attention to the enzymes involved in NAD cleavage, this monograph includes the most recent results on NAD biosynthesis in mammals and humans. Due to recent findings on nicotinamide riboside as a nutrient, its inclusion under "niacins" is proposed. Here, the enzymes involved in the de novo and reutilization pathways are overviewed. PMID:18508649

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

    PubMed Central

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

    2015-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-05-01

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

  6. Protein synthesis inhibitors prevent both spontaneous and hormone-dependent maturation of isolated mouse oocytes

    SciTech Connect

    Downs, S.M. )

    1990-11-01

    The present study was carried out to examine the role of protein synthesis in mouse oocyte maturation in vitro. In the first part of this study, the effects of cycloheximide (CX) were tested on spontaneous meiotic maturation when oocytes were cultured in inhibitor-free medium. CX reversibly suppressed maturation of oocytes as long as maturation was either initially prevented by the phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthine (IBMX), or delayed by follicle-stimulating hormone (FSH). In the second part of this study, the actions of protein synthesis inhibitors were tested on hormone-induced maturation. CEO were maintained in meiotic arrest for 21-22 h with hypoxanthine, and germinal vesicle breakdown (GVB) was induced with follicle-stimulating hormone (FSH). Three different protein synthesis inhibitors (CX, emetine (EM), and puromycin (PUR)) each prevented the stimulatory action of FSH on GVB in a dose-dependent fashion. This was accompanied by a dose-dependent suppression of 3H-leucine incorporation by oocyte-cumulus cell complexes. The action of these inhibitors on FSH- and epidermal growth factor (EGF)-induced GVB was next compared. All three drugs lowered the frequency of GVB in the FSH-treated groups, below even that of the controls (drug + hypoxanthine); the drugs maintained meiotic arrest at the control frequencies in the EGF-treated groups. Puromycin aminonucleoside, an analog of PUR with no inhibitory action on protein synthesis, had no effect. The three inhibitors also suppressed the stimulatory action of FSH on oocyte maturation when meiotic arrest was maintained with the cAMP analog, dbcAMP.

  7. Isolation and partial characterization of a protein synthesis inhibitor from brine shrimp embryos.

    PubMed

    Warner, A H; Shridhar, V; Finamore, F J

    1977-09-01

    Encysted embryos of the brine shrimp, Artemia salina, contain an inhibitor of protein synthesis that appears to be important in translational control. In cyst homogenates, the inhibitor appears to be partitioned almost equally between the cytosol and ribosome fractions and it has been purified from both fractions to near homogeneity. In a cell-free protein-synthesizing system derived from Artemia cysts, with poly(U) as messenger, the protein inhibits polyphenylalanine synthesis proportional to inhibitor concentration up to about 75% inhibition, and the primary site of action appears to be at the elongation step. The inhibitor activity is not altered by 50-150 mM KCl in the reaction mixture, but it is slightly more effective at 5 mM MgCl2 than at 10 mM MgCl2. The inhibitor is a heat-labile protein of 130000 molecular weight and is devoid of hydrolase activity. Our data indicate that the inhibitor is not elongation factor EF-1 or EF-2, but we are studying the possibility that it may be a modified form of elongation factor EF-2. PMID:907902

  8. NMNAT expression and its relation to NAD metabolism.

    PubMed

    Jayaram, H N; Kusumanchi, P; Yalowitz, J A

    2011-01-01

    Nicotinamide mononucleotide adenylyltransferease (NMNAT), a rate-limiting enzyme present in all organisms, reversibly catalyzes the important step in the biosynthesis of NAD from ATP and NMN. NAD and NADP are used reversibly in anabolic and catabolic reactions. NAD is necessary for cell survival in oxidative stress and DNA damage. Based on their localization, three different NMNAT's have been recognized, NMNAT-1 (homohexamer) in the nucleus (chromosome 1 p32-35), NMNAT-2 (homodimer) in the cytoplasm (chromosome 1q25) and NMNAT-3 (homotetramer) in the mitochondria. NMNAT also catalyzes the metabolic conversion of potent antitumor prodrugs like tiazofurin and benzamide riboside to their active forms which are analogs of NAD. NAD synthase-NMNAT acts as a chaperone to protect against neurodegeneration, injury-induced axonal degeneration and also correlates with DNA synthesis during cell cycle. Since its activity is rather low in tumor cells it can be exploited as a source for therapeutic targeting. Steps involved in NAD synthesis are being utilized as targets for chemoprevention, radiosensitization and therapy of wide range of diseases, such as cancer, multiple sclerosis, neurodegeneration and Huntington's disease. PMID:21517776

  9. SARM1 activation triggers axon degeneration locally via NAD+ destruction

    PubMed Central

    Gerdts, Josiah; Brace, E.J.; Sasaki, Yo; DiAntonio, Aaron

    2015-01-01

    Axon degeneration is an intrinsic self-destruction program that underlies axon loss during injury and disease. Sterile alpha and TIR motif containing 1 (SARM1) protein is an essential mediator of axon degeneration. We report that SARM1 initiates a local destruction program involving rapid breakdown of NAD+ after injury. We used an engineered protease-sensitized SARM1 to demonstrate that SARM1 activity is required after axon injury to induce axon degeneration. Dimerization of the Toll-Interleukin Receptor (TIR) domain of SARM1 alone was sufficient to induce locally-mediated axon degeneration. Formation of the SARM1 TIR dimer triggered rapid breakdown of NAD+, whereas SARM1-induced axon destruction could be counteracted by increased NAD+ synthesis. SARM1-induced depletion of NAD+ may explain the potent axon protection in Wallerian Degeneration slow (Wlds) mutant mice. PMID:25908823

  10. Synthesis and Biochemical Evaluation of Thiochromanone Thiosemicarbazone Analogues as Inhibitors of Cathepsin L

    PubMed Central

    2012-01-01

    A series of 36 thiosemicarbazone analogues containing the thiochromanone molecular scaffold functionalized primarily at the C-6 position were prepared by chemical synthesis and evaluated as inhibitors of cathepsins L and B. The most promising inhibitors from this group are selective for cathepsin L and demonstrate IC50 values in the low nanomolar range. In nearly all cases, the thiochromanone sulfide analogues show superior inhibition of cathepsin L as compared to their corresponding thiochromanone sulfone derivatives. Without exception, the compounds evaluated were inactive (IC50 > 10000 nM) against cathepsin B. The most potent inhibitor (IC50 = 46 nM) of cathepsin L proved to be the 6,7-difluoro analogue 4. This small library of compounds significantly expands the structure–activity relationship known for small molecule, nonpeptidic inhibitors of cathepsin L. PMID:24900494

  11. Carborane-containing urea-based inhibitors of glutamate carboxypeptidase II: Synthesis and structural characterization.

    PubMed

    Youn, Sihyun; Kim, Kyung Im; Ptacek, Jakub; Ok, Kiwon; Novakova, Zora; Kim, YunHye; Koo, JaeHyung; Barinka, Cyril; Byun, Youngjoo

    2015-11-15

    Glutamate carboxypeptidase II (GCPII) is a zinc metalloprotease on the surface of astrocytes which cleaves N-acetylaspartylglutamate to release N-acetylaspartate and glutamate. GCPII inhibitors can decrease glutamate concentration and play a protective role against apoptosis or degradation of brain neurons. Herein, we report the synthesis and structural analysis of novel carborane-based GCPII inhibitors. We determined the X-ray crystal structure of GCPII in complex with a carborane-containing inhibitor at 1.79Å resolution. The X-ray analysis revealed that the bulky closo-carborane cluster is located in the spacious entrance funnel region of GCPII, indicating that the carborane cluster can be further structurally modified to identify promising lead structures of novel GCPII inhibitors. PMID:26459214

  12. A Novel Selective Prostaglandin E2 Synthesis Inhibitor Relieves Pyrexia and Chronic Inflammation in Rats.

    PubMed

    Sugita, Ryusuke; Kuwabara, Harumi; Sugimoto, Kotaro; Kubota, Kazufumi; Imamura, Yuichiro; Kiho, Toshihiro; Tengeiji, Atsushi; Kawakami, Katsuhiro; Shimada, Kohei

    2016-04-01

    Prostaglandin E2 (PGE2) is a terminal prostaglandin in the cyclooxygenase (COX) pathway. Inhibition of PGE2 production may relieve inflammatory symptoms such as fever, arthritis, and inflammatory pain. We report here the profile of a novel selective PGE2 synthesis inhibitor, compound A [N-[(1S,3S)-3-carbamoylcyclohexyl]-1-(6-methyl-3-phenylquinolin-2-yl)piperidine-4-carboxamide], in animal models of pyrexia and inflammation. The compound selectively suppressed the synthesis of PGE2 in human alveolar adenocarcinoma cell line A549 cells and rat macrophages. In the lipopolysaccharide-induced pyrexia model, this compound selectively reduced PGE2 production in cerebrospinal fluid and showed an anti-pyretic effect. In the adjuvant-induced arthritis model, compound A therapeutically decreased foot swelling in the established arthritis. Our data demonstrates that selective suppression of PGE2 synthesis shows anti-pyretic and anti-inflammatory effects, suggesting that selective PGE2 synthesis inhibitors can be applied as an alternative treatment to nonsteroidal, anti-inflammatory drugs (NSAIDs) or COX-2-selective inhibitors. PMID:26923147

  13. Synthesis and Biological Evaluation of Resveratrol Derivatives as Melanogenesis Inhibitors.

    PubMed

    Liu, Qing; Kim, CheongTaek; Jo, Yang Hee; Kim, Seon Beom; Hwang, Bang Yeon; Lee, Mi Kyeong

    2015-01-01

    Resveratrol (1), a naturally occurring stilbene compound, has been suggested as a potential whitening agent with strong inhibitory activity on melanin synthesis. However, the use of resveratrol in cosmetics has been limited due to its chemical instability and poor bioavailability. Therefore, resveratrol derivatives were prepared to improve bioavailability and anti-melanogenesis activity. Nine resveratrol derivatives including five alkyl ether derivatives with C₂H₅, C₄H₉, C₅H11, C₆H13, and C₈H17 (2a-2e) and four ester derivatives with CH₃, CH=C(CH₃)₂, CH(C₂H₅)C₄H₉, C₇H15 (3a-3d) were newly synthesized and their effect on melanin synthesis were assessed. All the synthetic derivatives efficiently reduced the melanin content in α-MSH stimulated B16F10 melanoma cells. Further investigation showed that the inhibitory effect of 2a on melanin synthesis was achieved not by the inhibition of tyrosinase activity but by the inhibition of melanogenic enzyme expressions such as tyrosinase and tyrosinase-related protein (TRP)-1. Our synthetic resveratrol derivatives have more lipophilic properties than resveratrol by the addition of alkyl or acyl chains to free hydroxyl moiety of resveratrol; thus, they are expected to show better bioavailability in skin application. Therefore, we suggest that our synthetic resveratrol derivatives might be promising candidates for better practical application to skin-whitening cosmetics. PMID:26393543

  14. Design, synthesis and preliminary biological evaluation of indoline-2,3-dione derivatives as novel HDAC inhibitors.

    PubMed

    Jin, Kang; Li, Shanshan; Li, Xiaoguang; Zhang, Jian; Xu, Wenfang; Li, Xuechen

    2015-08-01

    Histone deacetylases (HDACs) are zinc-dependent or NAD(+) dependent enzymes and play a critical role in the process of tumor development. Herein a series of indoline-2,3-dione derivatives have been designed and synthesized as potential HDACs inhibitors. The preliminary biological evaluation showed that most compounds synthesized have exhibited moderate Hela cell nuclear extract inhibitory activities, among which compound 25a (IC50=10.13 nM) has shown the best efficacy. The anti-proliferative activities of some of these compounds were also discussed. PMID:26100440

  15. Emerging therapeutic roles for NAD(+) metabolism in mitochondrial and age-related disorders.

    PubMed

    Srivastava, Sarika

    2016-12-01

    Nicotinamide adenine dinucleotide (NAD(+)) is a central metabolic cofactor in eukaryotic cells that plays a critical role in regulating cellular metabolism and energy homeostasis. NAD(+) in its reduced form (i.e. NADH) serves as the primary electron donor in mitochondrial respiratory chain, which involves adenosine triphosphate production by oxidative phosphorylation. The NAD(+)/NADH ratio also regulates the activity of various metabolic pathway enzymes such as those involved in glycolysis, Kreb's cycle, and fatty acid oxidation. Intracellular NAD(+) is synthesized de novo from L-tryptophan, although its main source of synthesis is through salvage pathways from dietary niacin as precursors. NAD(+) is utilized by various proteins including sirtuins, poly ADP-ribose polymerases (PARPs) and cyclic ADP-ribose synthases. The NAD(+) pool is thus set by a critical balance between NAD(+) biosynthetic and NAD(+) consuming pathways. Raising cellular NAD(+) content by inducing its biosynthesis or inhibiting the activity of PARP and cADP-ribose synthases via genetic or pharmacological means lead to sirtuins activation. Sirtuins modulate distinct metabolic, energetic and stress response pathways, and through their activation, NAD(+) directly links the cellular redox state with signaling and transcriptional events. NAD(+) levels decline with mitochondrial dysfunction and reduced NAD(+)/NADH ratio is implicated in mitochondrial disorders, various age-related pathologies as well as during aging. Here, I will provide an overview of the current knowledge on NAD(+) metabolism including its biosynthesis, utilization, compartmentalization and role in the regulation of metabolic homoeostasis. I will further discuss how augmenting intracellular NAD(+) content increases oxidative metabolism to prevent bioenergetic and functional decline in multiple models of mitochondrial diseases and age-related disorders, and how this knowledge could be translated to the clinic for human relevance. PMID

  16. Mitochondria-localized NAD biosynthesis by nicotinamide mononucleotide adenylyltransferase in Jerusalem artichoke (Helianthus tuberosus L.) heterotrophic tissues.

    PubMed

    Di Martino, Catello; Pallotta, Maria Luigia

    2011-10-01

    Current studies in plants suggest that the content of the coenzyme NAD is variable and potentially important in determining cell fate. In cases that implicate NAD consumption, re-synthesis must occur to maintain dinucleotide pools. Despite information on the pathways involved in NAD synthesis in plants, the existence of a mitochondrial nicotinamide mononucleotide adenylyltransferase (NMNAT) activity which catalyses NAD synthesis from nicotinamide mononucleotide (NMN) and ATP has not been reported. To verify the latter assumed pathway, experiments with purified and bioenergetically active mitochondria prepared from tubers of Jerusalem artichoke (Helianthus tuberosus L.) were performed. To determine whether NAD biosynthesis might occur, NMN was added to Jerusalem artichoke mitochondria (JAM) and NAD biosynthesis was tested by means of HPLC and spectroscopically. Our results indicate that JAM contain a specific NMNAT inhibited by Na-pyrophosphate, AMP and ADP-ribose. The dependence of NAD synthesis rate on NMN concentration shows saturation kinetics with K (m) and V (max) values of 82 ± 1.05 μM and 4.20 ± 0.20 nmol min(-1) mg(-1) protein, respectively. The enzyme's pH and temperature dependence were also investigated. Fractionation studies revealed that mitochondrial NMNAT activity was present in the soluble matrix fraction. The NAD pool needed constant replenishment that might be modulated by environmental inputs. Thus, the mitochondrion in heterotrophic plant tissues ensures NAD biosynthesis by NMNAT activity and helps to orchestrate NAD metabolic network in implementing the survival strategy of cells. PMID:21598001

  17. Synthesis of benzopentathiepin analogs and their evaluation as inhibitors of the phosphatase STEP

    PubMed Central

    Baguley, Tyler D.; Nairn, Angus C.; Lombroso, Paul J.; Ellman, Jonathan A.

    2015-01-01

    Striatal-enriched protein tyrosine phosphatase (STEP) is a brain specific protein tyrosine phosphatase that has been implicated in many neurodegenerative diseases, such as Alzheimer’s disease. We recently reported the benzopentathiepin TC-2153 as a potent inhibitor of STEP in vitro, cells and animals. Herein, we report the synthesis and evaluation of TC-2153 analogs in order to define what structural features are important for inhibition and to identify positions tolerant of substitution for further study. The trifluoromethyl substitution is beneficial for inhibitor potency, and the amine is tolerant of acylation, and thus provides a convenient handle for introducing additional functionality such as reporter groups. PMID:25666825

  18. Arylsulfonamide inhibitors of aggrecanases as potential therapeutic agents for osteoarthritis: synthesis and biological evaluation.

    PubMed

    Nuti, Elisa; Santamaria, Salvatore; Casalini, Francesca; Yamamoto, Kazuhiro; Marinelli, Luciana; La Pietra, Valeria; Novellino, Ettore; Orlandini, Elisabetta; Nencetti, Susanna; Marini, Anna Maria; Salerno, Silvia; Taliani, Sabrina; Da Settimo, Federico; Nagase, Hideaki; Rossello, Armando

    2013-04-01

    Aggrecanases, in particular aggrecanase-2 (ADAMTS-5), are considered the principal proteases responsible for aggrecan degradation in osteoarthritis. For this reason, considerable effort has been put on the discovery and development of aggrecanase inhibitors able to slow down or halt the progression of osteoarthritis. We report herein the synthesis and biological evaluation of a series of arylsulfonamido-based hydroxamates as aggrecanase inhibitors. Compound 18 was found to have a nanomolar activity for ADAMTS-5, ADAMTS-4 and MMP-13 and high selectivity over MMP-1 and MMP-14. Furthermore, this compound proved to be effective in blocking ex vivo cartilage degradation without having effect on cell cytotoxicity. PMID:23376997

  19. Synthesis and metabolism of inhibitors of ribonucleotide reductase

    SciTech Connect

    Smith, F.T.

    1985-01-01

    In an effort to prepare more effective inhibitors of ribo-nucleotide reductase a series of 2-substituted-4,6-dihydroxypyrimidines was prepared via the appropriately substituted benzamidine. None of the compounds exhibited in vivo activity against L1210 leukemia. No further testing was performed. In order to investigate the metabolism of 3,4-dihydroxybenzohydroxamic acid, a known inhibitor of ribonucleotide reductase, radiolabeled 3,4-dihydroxybenzohydroxamic acid was synthesized by a modification of the procedure of Pichat and Tostain. /sup 14/C-3,4-Dihydroxybenzoic acid was converted to the methyl ester and subsequently reacted with hydroxylamine to give the hydroxamic acid. /sup 14/C-3,4-Dihydroxybenzohydroxamic acid was given i.p. to Sprague-Dawley rats. Excretion occurred mainly (72%) via the urine. HPLC coupled with GC/MS analyses showed that the compound was excreted mainly unchanged. The compound was metabolized to 3,4-dihydroxybenzamide, 4-methoxy-3-hydroxybenzohydroxamic acid, and 4-hydroxy-3-methoxybenzohydroxamic acid. HPLC analysis also showed the lack of formation of any glucuronide or sulfate conjugates through either the hydroxamic acid or catechol functionalities.

  20. Synthesis and Biological Evaluation of Botulinum Neurotoxin A Protease Inhibitors

    PubMed Central

    Li, Bing; Pai, Ramdas; Cardinale, Steven C.; Butler, Michelle M.; Peet, Norton P.; Moir, Donald T.; Bavari, Sina; Bowlin, Terry L.

    2010-01-01

    NSC 240898 was previously identified as a botulinum neurotoxin A light chain (BoNT/A LC) endopeptidase inhibitor by screening the National Cancer Institute Open Repository diversity set. Two types of analogs have been synthesized and shown to inhibit BoNT/A LC in a FRET-based enzyme assay, with confirmation in an HPLC-based assay. These two series of compounds have also been evaluated for inhibition of anthrax lethal factor (LF), an unrelated metalloprotease, to examine enzyme specificity of the BoNT/A LC inhibition. The most potent inhibitor against BoNT/A LC in these two series is compound 12 (IC50 = 2.5 µM, FRET assay), which is 4.4-fold more potent than the lead structure, and 11.2-fold more selective for BoNT/A LC versus the anthrax LF metalloproteinase. Structure-activity relationship studies have revealed structural features important to potency and enzyme specificity. PMID:20155918

  1. The synthesis of 19-norandrostenedione from dehydroepiandrosterone in equine placenta is inhibited by aromatase inhibitors 4-hydroxyandrostenedione and fadrozole.

    PubMed

    Moslemi, S; Silberzahn, P; Gaillard, J L

    1995-12-01

    19-Norandrostenedione was synthesized in vitro from dehydroepiandrosterone by explants of equine full-term placenta. The synthesis of 19-norandrostenedione was inhibited by two specific aromatase inhibitors, 4-hydroxyandrostenedione and fadrozole. PMID:8590376

  2. Large-scale asymmetric synthesis of a cathepsin S inhibitor.

    PubMed

    Lorenz, Jon C; Busacca, Carl A; Feng, XuWu; Grinberg, Nelu; Haddad, Nizar; Johnson, Joe; Kapadia, Suresh; Lee, Heewon; Saha, Anjan; Sarvestani, Max; Spinelli, Earl M; Varsolona, Rich; Wei, Xudong; Zeng, Xingzhong; Senanayake, Chris H

    2010-02-19

    A potent reversible inhibitor of the cysteine protease cathepsin-S was prepared on large scale using a convergent synthetic route, free of chromatography and cryogenics. Late-stage peptide coupling of a chiral urea acid fragment with a functionalized aminonitrile was employed to prepare the target, using 2-hydroxypyridine as a robust, nonexplosive replacement for HOBT. The two key intermediates were prepared using a modified Strecker reaction for the aminonitrile and a phosphonation-olefination-rhodium-catalyzed asymmetric hydrogenation sequence for the urea. A palladium-catalyzed vinyl transfer coupled with a Claisen reaction was used to produce the aldehyde required for the side chain. Key scale up issues, safety calorimetry, and optimization of all steps for multikilogram production are discussed. PMID:20102230

  3. [Current conservative treatment of renal colic: value of prostaglandin synthesis inhibitors].

    PubMed

    Zwergel, U; Felgner, J; Rombach, H; Zwergel, T

    1998-04-20

    Prostaglandin synthesis inhibitors and parasympatholytic drugs are often used as analgetics in the case of renal colic. This paper analyzes how and whether these drug effects are important for the analgetic therapy. In an animal and in a human model with acutely obstructed kidneys we found that intravenous application of Indometacine and dipyrone significantly reduces renal pelvic pressure. The parasympatholytic drug hyoscine butylbromide did not produce any change of upper urinary tract dynamics. Inhibitors of prostaglandin synthesis thus effect pressure reduction in the renal pelvis, which is necessary for analgetic therapy. In contrast, hyoscine butylbromide does not have any influence on the acute upper urinary tract obstruction; consequently its usefulness in the treatment of renal colic is rather doubtful. PMID:12799978

  4. Ovicidal activity of chitin synthesis inhibitors when fed to adult German cockroaches (Dictyoptera: Blattellidae).

    PubMed

    DeMark, J J; Bennett, G W

    1990-07-01

    Ovicidal activity was observed in four adult groups (virgin males; virgin females; newly gravid females; and inseminated, reproducing females) of the German cockroach, Blattella germanica (L.), fed the chitin synthesis inhibitors triflumuron, chlorfluazuron, hexafluron, and UC 84572 (structure not disclosed) at the LC50's and LC95's determined from fifth-stage nymphs. All compounds were active only when fed to reproducing females (including the feeding period in which the ootheca is developing). Hexafluron and triflumuron at the LC50 caused 100% inhibition of hatch in reproducing females. Chlorfluazuron and UC 84572 at the LC50 had similar ovicidal activity (45.8 and 50.0% hatch, respectively). Female German cockroaches fed the chitin synthesis inhibitors before mating and after the ootheca had protruded from the abdomen were not affected. Reproductive capabilities of males were not affected, and males did not effectively transfer the compounds to untreated females during mating. PMID:2388230

  5. Fidaxomicin Is an Inhibitor of the Initiation of Bacterial RNA Synthesis

    PubMed Central

    Artsimovitch, Irina; Seddon, Jaime; Sears, Pamela

    2012-01-01

    Fidaxomicin was recently approved for the treatment of Clostridium difficile infection. It inhibits transcription by bacterial RNA polymerase. Because transcription is a multistep process, experiments were conducted in which fidaxomicin was added at different stages of transcriptional initiation to identify the blocked step. DNA footprinting experiments were also conducted to further elucidate the stage inhibited. Fidaxomicin blocks initiation only if added before the formation of the “open promoter complex,” in which the template DNA strands have separated but RNA synthesis has not yet begun. Binding of fidaxomicin precludes the initial separation of DNA strands that is prerequisite to RNA synthesis. These studies show that it has a mechanism distinct from that of elongation inhibitors, such as streptolydigin, and from the transcription initiation inhibitors myxopyronin and the rifamycins. PMID:22752861

  6. Amino Compounds as Inhibitors of De Novo Synthesis of Chlorobenzenes.

    PubMed

    Wang, Si-Jia; He, Pin-Jing; Lu, Wen-Tao; Shao, Li-Ming; Zhang, Hua

    2016-01-01

    The inhibitory effects of four amino compounds on the formation of chlorobenzenes (CBzs) - dioxin precursors and indicators, and the inhibitory mechanisms were explored. The results show NH4H2PO4 can decrease the total yields of CBzs (1,2di-CBz, 1,3di-CBz, 1,4di-CBz, penta-CBz and hexa-CBz) by 98.1%±1.6% and 96.1%±0.7% under air and nitrogen flow. The inhibitory effects indicated by the total yields of CBzs follow the order NH4H2PO4 > NH4HF2 > (NH4)2SO4 > NH4Br under air flow and NH4H2PO4 ≈ (NH4)2SO4 ≈ NH4HF2 >NH4Br under nitrogen flow. The inhibition mechanism revealed by thermal analysis that CuCl2 was converted to CuPO3 by reacting with NH4H2PO4 below 200 °C, which can block the transfer of chlorine and formation of C-Cl bonds at 350 °C. The effects of the other three inhibitors were weaker because their reactions with CuCl2, which form other copper compounds, and the reaction of CuCl2 with carbon, which forms C-Cl bonds, were almost simultaneous and competitive. Oxygen influenced the yield of CBzs obviously, and the total yield of five CBzs sharply increased with oxygen. Because of their high efficiency, low environmental impact, low cost, and availability, amino compounds - especially NH4H2PO4 - can be utilized as inhibitors of CBzs during incineration. PMID:27034259

  7. Amino Compounds as Inhibitors of De Novo Synthesis of Chlorobenzenes

    NASA Astrophysics Data System (ADS)

    Wang, Si-Jia; He, Pin-Jing; Lu, Wen-Tao; Shao, Li-Ming; Zhang, Hua

    2016-04-01

    The inhibitory effects of four amino compounds on the formation of chlorobenzenes (CBzs) - dioxin precursors and indicators, and the inhibitory mechanisms were explored. The results show NH4H2PO4 can decrease the total yields of CBzs (1,2di-CBz, 1,3di-CBz, 1,4di-CBz, penta-CBz and hexa-CBz) by 98.1%±1.6% and 96.1%±0.7% under air and nitrogen flow. The inhibitory effects indicated by the total yields of CBzs follow the order NH4H2PO4 > NH4HF2 > (NH4)2SO4 > NH4Br under air flow and NH4H2PO4 ≈ (NH4)2SO4 ≈ NH4HF2 >NH4Br under nitrogen flow. The inhibition mechanism revealed by thermal analysis that CuCl2 was converted to CuPO3 by reacting with NH4H2PO4 below 200 °C, which can block the transfer of chlorine and formation of C–Cl bonds at 350 °C. The effects of the other three inhibitors were weaker because their reactions with CuCl2, which form other copper compounds, and the reaction of CuCl2 with carbon, which forms C–Cl bonds, were almost simultaneous and competitive. Oxygen influenced the yield of CBzs obviously, and the total yield of five CBzs sharply increased with oxygen. Because of their high efficiency, low environmental impact, low cost, and availability, amino compounds - especially NH4H2PO4 - can be utilized as inhibitors of CBzs during incineration.

  8. Amino Compounds as Inhibitors of De Novo Synthesis of Chlorobenzenes

    PubMed Central

    Wang, Si-Jia; He, Pin-Jing; Lu, Wen-Tao; Shao, Li-Ming; Zhang, Hua

    2016-01-01

    The inhibitory effects of four amino compounds on the formation of chlorobenzenes (CBzs) - dioxin precursors and indicators, and the inhibitory mechanisms were explored. The results show NH4H2PO4 can decrease the total yields of CBzs (1,2di-CBz, 1,3di-CBz, 1,4di-CBz, penta-CBz and hexa-CBz) by 98.1%±1.6% and 96.1%±0.7% under air and nitrogen flow. The inhibitory effects indicated by the total yields of CBzs follow the order NH4H2PO4 > NH4HF2 > (NH4)2SO4 > NH4Br under air flow and NH4H2PO4 ≈ (NH4)2SO4 ≈ NH4HF2 >NH4Br under nitrogen flow. The inhibition mechanism revealed by thermal analysis that CuCl2 was converted to CuPO3 by reacting with NH4H2PO4 below 200 °C, which can block the transfer of chlorine and formation of C–Cl bonds at 350 °C. The effects of the other three inhibitors were weaker because their reactions with CuCl2, which form other copper compounds, and the reaction of CuCl2 with carbon, which forms C–Cl bonds, were almost simultaneous and competitive. Oxygen influenced the yield of CBzs obviously, and the total yield of five CBzs sharply increased with oxygen. Because of their high efficiency, low environmental impact, low cost, and availability, amino compounds - especially NH4H2PO4 - can be utilized as inhibitors of CBzs during incineration. PMID:27034259

  9. BRCA1 as a nicotinamide adenine dinucleotide (NAD)-dependent metabolic switch in ovarian cancer

    PubMed Central

    Li, Da; Chen, Na-Na; Cao, Ji-Min; Sun, Wu-Ping; Zhou, Yi-Ming; Li, Chun-Yan; Wang, Xiu-Xia

    2014-01-01

    Both hereditary factors (e.g., BRCA1) and nicotinamide adenine dinucleotide (NAD)-dependent metabolic pathways are implicated in the initiation and progression of ovarian cancer. However, whether crosstalk exists between BRCA1 and NAD metabolism remains largely unknown. Here, we showed that: (i) BRCA1 inactivation events (mutation and promoter methylation) were accompanied by elevated levels of NAD; (ii) the knockdown or overexpression of BRCA1 was an effective way to induce an increase or decrease of nicotinamide phosphoribosyltransferase (Nampt)-related NAD synthesis, respectively; and (iii) BRCA1 expression patterns were inversely correlated with NAD levels in human ovarian cancer specimens. In addition, it is worth noting that: (i) NAD incubation induced increased levels of BRCA1 in a concentration-dependent manner; (ii) Nampt knockdown-mediated reduction in NAD levels was effective at inhibiting BRCA1 expression; and (iii) the overexpression of Nampt led to higher NAD levels and a subsequent increase in BRCA1 levels in primary ovarian cancer cells and A2780, HO-8910 and ES2 ovarian cancer cell lines. These results highlight a novel link between BRCA1 and NAD. Our findings imply that genetic (e.g., BRCA1 inactivation) and NAD-dependent metabolic pathways are jointly involved in the malignant progression of ovarian cancer. PMID:25486197

  10. The effect of chitin synthesis inhibitors on the development of Brugia malayi in Aedes aegypti.

    PubMed

    Mohapatra, R; Ranjit, M R; Dash, A P

    1996-09-01

    Two chitin synthesis inhibitors (CSIs) viz., triflumuron and hexaflumuron interfere++ with the development of Brugia malayi in Aedes aegypti (a black-eyed Liverpool strain). The development of B. malayi was slow in both the treated populations and the infection rate, infectivity rate and L3 load per mosquito decreased significantly (P < 0.001) in comparison with untreated controls. Hexaflumuron was found to be more inhibiting than triflumuron. PMID:8984113

  11. Synthesis and biological evaluation of analogues of the kinase inhibitor nilotinib as Abl and Kit inhibitors

    PubMed Central

    Duveau, Damien Y.; Hu, Xin; Walsh, Martin J.; Shukla, Suneet; Skoumbourdis, Amanda P.; Boxer, Matthew B.; Ambudkar, Suresh V.; Shen, Min; Thomas, Craig J.

    2013-01-01

    The importance of the trifluoromethyl group in the polypharmacological profile of nilotinib was investigated. Molecular editing of nilotinib led to the design, synthesis and biological evaluation of analogues where the trifluoromethyl group was replaced by a proton, fluorine and a methyl group. While these analogues were less active than nilotinib toward Abl, their activity toward Kit was comparable, with the monofluorinated analogue being the most active. Docking of nilotinib and of analogues 2a–c to the binding pocket of Abl and of Kit showed that the lack of shape complementarity in Kit is compensated by the stabilizing effect from its juxtamembrane region. PMID:23273517

  12. Induction of DNA synthesis in isolated nuclei by cytoplasmic factors: inhibition by protease inhibitors.

    PubMed Central

    Wong, R L; Gutowski, J K; Katz, M; Goldfarb, R H; Cohen, S

    1987-01-01

    Cytoplasmic extracts from spontaneously proliferating and mitogen-activated lymphoid cells contain a protein factor called ADR (activator of DNA replication) that induces DNA synthesis in isolated quiescent nuclei. ADR-containing preparations have proteolytic activity, as indicated by their ability to degrade fibrin in a plasminogen-independent and plasminogen-dependent manner. In addition, aprotinin, a nonspecific protease inhibitor, abrogates ADR-induced DNA synthesis in a dose-dependent fashion. Preincubation studies demonstrated that the effect of aprotinin is not due to its suppressive effects on the nuclei themselves. Other protease inhibitors such as leupeptin, p-aminobenzamidine, and N-alpha-tosyllysine chloromethyl ketone are also inhibitory, but soybean trypsin inhibitor is without effect. ADR activity can be removed from active extracts by adsorption with aprotinin-conjugated agarose beads and can be recovered by elution with an acetate buffer (pH 5). These findings are consistent with the interpretation that the initiation of DNA synthesis in resting nuclei may be protease dependent and, further, that the cytoplasmic stimulatory factor we have called ADR may be a protease itself. PMID:3540956

  13. A PHGDH inhibitor reveals coordination of serine synthesis and one-carbon unit fate.

    PubMed

    Pacold, Michael E; Brimacombe, Kyle R; Chan, Sze Ham; Rohde, Jason M; Lewis, Caroline A; Swier, Lotteke J Y M; Possemato, Richard; Chen, Walter W; Sullivan, Lucas B; Fiske, Brian P; Cho, Steve; Freinkman, Elizaveta; Birsoy, Kıvanç; Abu-Remaileh, Monther; Shaul, Yoav D; Liu, Chieh Min; Zhou, Minerva; Koh, Min Jung; Chung, Haeyoon; Davidson, Shawn M; Luengo, Alba; Wang, Amy Q; Xu, Xin; Yasgar, Adam; Liu, Li; Rai, Ganesha; Westover, Kenneth D; Vander Heiden, Matthew G; Shen, Min; Gray, Nathanael S; Boxer, Matthew B; Sabatini, David M

    2016-06-01

    Serine is both a proteinogenic amino acid and the source of one-carbon units essential for de novo purine and deoxythymidine synthesis. In the canonical pathway of glucose-derived serine synthesis, Homo sapiens phosphoglycerate dehydrogenase (PHGDH) catalyzes the first, rate-limiting step. Genetic loss of PHGDH is toxic toward PHGDH-overexpressing breast cancer cell lines even in the presence of exogenous serine. Here, we used a quantitative high-throughput screen to identify small-molecule PHGDH inhibitors. These compounds reduce the production of glucose-derived serine in cells and suppress the growth of PHGDH-dependent cancer cells in culture and in orthotopic xenograft tumors. Surprisingly, PHGDH inhibition reduced the incorporation into nucleotides of one-carbon units from glucose-derived and exogenous serine. We conclude that glycolytic serine synthesis coordinates the use of one-carbon units from endogenous and exogenous serine in nucleotide synthesis, and we suggest that one-carbon unit wasting thus may contribute to the efficacy of PHGDH inhibitors in vitro and in vivo. PMID:27110680

  14. Total synthesis of amiclenomycin, an inhibitor of biotin biosynthesis.

    PubMed

    Mann, Stéphane; Carillon, Sophie; Breyne, Olivier; Marquet, Andrée

    2002-01-18

    We describe the first synthesis of amiclenomycin, a natural product that has been found to inhibit biotin biosynthesis and, as a consequence, to exhibit antibiotic properties. Structure 1, with a trans relationship between the ring substituents. had previously been proposed for amiclenomycin on the basis of its 1H NMR spectrum. We have prepared the trans and cis isomers 1 and 2 by unequivocal routes and we conclude that the natural product is in fact the cis isomer 2. The properly substituted cyclohexadienyl rings were constructed first. A cycloaddition reaction between 1,2-di(phenylsulfonyl)ethylene and the N-allyloxycarbonyl diene 13, followed by reductive elimination of the phenylsulfinyl groups, gave the cis isomer 15. To obtain the trans isomer, the O-trimethylsilyl diene was used to give the cis hydroxylated Diels-Alder adduct 33, which was transformed into the corresponding trans amino derivative by means of a Mitsunobu reaction. The L-alpha-amino acid functionality was introduced by means of a Strecker reaction on the aldehydes 16 and 42, followed by enzymatic hydrolysis with immobilised pronase. PMID:11843156

  15. Boosting NAD to spare hearing.

    PubMed

    Brenner, Charles

    2014-12-01

    Ex vivo experiments have strangely shown that inhibition or stimulation of NAD metabolism can be neuroprotective. In this issue of Cell Metabolism, Brown et al. (2014) demonstrate that cochlear NAD is diminished by deafening noise but protected by nicotinamide riboside or WldS mutation. Hearing protection by nicotinamide riboside depends on Sirt3. PMID:25470539

  16. Inhibitor of DNA synthesis is present in normal chicken serum

    SciTech Connect

    Franklin, R.A.; Davila, D.R.; Westly, H.J.; Kelley, K.W.

    1986-03-05

    The authors have found that heat-inactivated serum (57/sup 0/C for 1 hour) from normal chickens reduces the proliferation of mitogen-stimulated chicken and murine splenocytes as well as some transformed mammalian lymphoblastoid cell lines. Greater than a 50% reduction in /sup 3/H-thymidine incorporation was observed when concanavalin A (Con A)-activated chicken splenocytes that were cultured in the presence of 10% autologous or heterologous serum were compared to mitogen-stimulated cells cultured in the absence of serum. Normal chicken serum (10%) also caused greater than 95% suppression of /sup 3/H-thymidine incorporation by bovine (EBL-1 and BL-3) and gibbon ape (MLA 144) transformed lymphoblastoid cell lines. The only cell line tested that was not inhibited by chicken serum was an IL-2-dependent, murine cell line. Chicken serum also inhibited both /sup 3/H-thymidine incorporation and IL-2 synthesis by Con A-activated murine splenocytes. Suppression was caused by actions other than cytotoxicity because viability of chicken splenocytes was unaffected by increasing levels of chicken serum. Furthermore, dialyzed serum retained its activity, which suggested that thymidine in the serum was not inhibiting uptake of radiolabeled thymidine. Suppressive activity was not due to adrenal glucocorticoids circulating in plasma because neither physiologic nor pharmacologic doses of corticosterone had inhibitory effects on mitogen-stimulated chicken splenocytes. These data demonstrate that an endogenous factor that is found in normal chicken serum inhibits proliferation of T-cells from chickens and mice as well as some transformed mammalian lymphoblastoid cell lines.

  17. Cholesterol synthesis inhibitors protect against platelet-activating factor-induced neuronal damage

    PubMed Central

    Bate, Clive; Rumbold, Louis; Williams, Alun

    2007-01-01

    Background Platelet-activating factor (PAF) is implicated in the neuronal damage that accompanies ischemia, prion disease and Alzheimer's disease (AD). Since some epidemiological studies demonstrate that statins, drugs that reduce cholesterol synthesis, have a beneficial effect on mild AD, we examined the effects of two cholesterol synthesis inhibitors on neuronal responses to PAF. Methods Primary cortical neurons were treated with cholesterol synthesis inhibitors (simvastatin or squalestatin) prior to incubation with different neurotoxins. The effects of these drugs on neuronal cholesterol levels and neuronal survival were measured. Immunoblots were used to determine the effects of simvastatin or squalestatin on the distribution of the PAF receptor and an enzyme linked immunoassay was used to quantify the amounts of PAF receptor. Results PAF killed primary neurons in a dose-dependent manner. Pre-treatment with simvastatin or squalestatin reduced neuronal cholesterol and increased the survival of PAF-treated neurons. Neuronal survival was increased 50% by 100 nM simvastatin, or 20 nM squalestatin. The addition of mevalonate restored cholesterol levels, and reversed the protective effect of simvastatin. Simvastatin or squalestatin did not affect the amounts of the PAF receptor but did cause it to disperse from within lipid rafts. Conclusion Treatment of neurons with cholesterol synthesis inhibitors including simvastatin and squalestatin protected neurons against PAF. Treatment caused a percentage of the PAF receptors to disperse from cholesterol-sensitive domains. These results raise the possibility that the effects of statins on neurodegenerative disease are, at least in part, due to desensitisation of neurons to PAF. PMID:17233902

  18. Neuronal NAD(P)H Oxidases Contribute to ROS Production and Mediate RGC Death after Ischemia

    PubMed Central

    Dvoriantchikova, Galina; Grant, Jeff; Santos, Andrea Rachelle C.; Hernandez, Eleut; Ivanov, Dmitry

    2012-01-01

    Purpose. To study the role of neuronal nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase–dependent reactive oxygen species (ROS) production in retinal ganglion cell (RGC) death after ischemia. Methods. Ischemic injury was induced by unilateral elevation of intraocular pressure via direct corneal cannulation. For in vitro experiments, RGCs isolated by immunopanning from retinas were exposed to oxygen and glucose deprivation (OGD). The expression levels of NAD(P)H oxidase subunits were evaluated by quantitative PCR, immunocytochemistry, and immunohistochemistry. The level of ROS generated was assayed by dihydroethidium. The NAD(P)H oxidase inhibitors were then tested to determine if inhibition of NAD(P)H oxidase altered the production of ROS within the RGCs and promoted cell survival. Results. It was reported that RGCs express catalytic Nox1, Nox2, Nox4, Duox1, as well as regulatory Ncf1/p47phox, Ncf2/p67phox, Cyba/p22phox, Noxo1, and Noxa1 subunits of NAD(P)H oxidases under normal conditions and after ischemia. However, whereas RGCs express only low levels of catalytic Nox2, Nox4, and Duox1, and regulatory Ncf1/p47, Ncf2/p67 subunits, they exhibit significantly higher levels of catalytic subunit Nox1 and the subunits required for optimal activity of Nox1. It was observed that the nonselective NAD(P)H oxidase inhibitors VAS-2870, AEBSF, and the Nox1 NAD(P)H oxidase–specific inhibitor ML-090 decreased the ROS burst stimulated by OGD, which was associated with a decreased level of RGC death. Conclusions. The findings suggest that NAD(P)H oxidase activity in RGCs renders them vulnerable to ischemic death. Importantly, high levels of Nox1 NAD(P)H oxidase subunits in RGCs suggest that this enzyme could be a major source of ROS in RGCs produced by NAD(P)H oxidases. PMID:22467573

  19. Novel Antibiotic-Free Plasmid Selection System Based on Complementation of Host Auxotrophy in the NAD De Novo Synthesis Pathway▿ †

    PubMed Central

    Dong, Wei-Ren; Xiang, Li-Xin; Shao, Jian-Zhong

    2010-01-01

    The use of antibiotic resistance genes in plasmids causes potential biosafety and clinical hazards, such as the possibility of horizontal spread of resistance genes or the rapid emergence of multidrug-resistant pathogens. This paper introduces a novel auxotrophy complementation system that allowed plasmids and host cells to be effectively selected and maintained without the use of antibiotics. An Escherichia coli strain carrying a defect in NAD de novo biosynthesis was constructed by knocking out the chromosomal quinolinic acid phosphoribosyltransferase (QAPRTase) gene. The resistance gene in the plasmids was replaced by the QAPRTase gene of E. coli or the mouse. As a result, only expression of the QAPRTase gene from plasmids can complement and rescue E. coli host cells in minimal medium. This is the first time that a vertebrate gene has been used to construct a nonantibiotic selection system, and it can be widely applied in DNA vaccine and gene therapy. As the QAPRTase gene is ubiquitous in species ranging from bacteria to mammals, the potential environmental biosafety problems caused by horizontal gene transfer can be eliminated. PMID:20118370

  20. LP99: Discovery and Synthesis of the First Selective BRD7/9 Bromodomain Inhibitor**

    PubMed Central

    Clark, Peter G K; Vieira, Lucas C C; Tallant, Cynthia; Fedorov, Oleg; Singleton, Dean C; Rogers, Catherine M; Monteiro, Octovia P; Bennett, James M; Baronio, Roberta; Müller, Susanne; Daniels, Danette L; Méndez, Jacqui; Knapp, Stefan; Brennan, Paul E; Dixon, Darren J

    2015-01-01

    The bromodomain-containing proteins BRD9 and BRD7 are part of the human SWI/SNF chromatin-remodeling complexes BAF and PBAF. To date, no selective inhibitor for BRD7/9 has been reported despite its potential value as a biological tool or as a lead for future therapeutics. The quinolone-fused lactam LP99 is now reported as the first potent and selective inhibitor of the BRD7 and BRD9 bromodomains. Development of LP99 from a fragment hit was expedited through balancing structure-based inhibitor design and biophysical characterization against tractable chemical synthesis: Complexity-building nitro-Mannich/lactamization cascade processes allowed for early structure–activity relationship studies whereas an enantioselective organocatalytic nitro-Mannich reaction enabled the synthesis of the lead scaffold in enantioenriched form and on scale. This epigenetic probe was shown to inhibit the association of BRD7 and BRD9 to acetylated histones in vitro and in cells. Moreover, LP99 was used to demonstrate that BRD7/9 plays a role in regulating pro-inflammatory cytokine secretion. PMID:25864491

  1. 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 ...

  2. Synthesis of the proteinase inhibitor LEKTI domain 6 by the fragment condensation method and regioselective disulfide bond formation.

    PubMed

    Vasileiou, Zoe; Barlos, Kostas K; Gatos, Dimitrios; Adermann, Knut; Deraison, Celine; Barlos, Kleomenis

    2010-01-01

    Proteinase inhibitors are of high pharmaceutical interest and are drug candidates for a variety of indications. Specific kallikrein inhibitors are important for their antitumor activity and their potential application to the treatment of skin diseases. In this study we describe the synthesis of domain 6 of the kallikrein inhibitor Lympho-Epithilial Kazal-Type Inhibitor (LEKTI) by the fragment condensation method and site-directed cystine bridge formation. To obtain the linear LEKTI precursor, the condensation was best performed in solution, coupling the protected fragment 1-22 to 23-68. This method yielded LEKTI domain 6 of high purity and equipotent to the recombinantly produced peptide. PMID:20069636

  3. Design and synthesis of a series of serine derivatives as small molecule inhibitors of the SARS coronavirus 3CL protease.

    PubMed

    Konno, Hiroyuki; Wakabayashi, Masaki; Takanuma, Daiki; Saito, Yota; Akaji, Kenichi

    2016-03-15

    Synthesis of serine derivatives having the essential functional groups for the inhibitor of SARS 3CL protease and evaluation of their inhibitory activities using SARS 3CL R188I mutant protease are described. The lead compounds, functionalized serine derivatives, were designed based on the tetrapeptide aldehyde and Bai's cinnamoly inhibitor, and additionally performed with simulation on GOLD softwear. Structure activity relationship studies of the candidate compounds were given reasonable inhibitors ent-3 and ent-7k against SARS 3CL R188I mutant protease. These inhibitors showed protease selectivity and no cytotoxicity. PMID:26879854

  4. Finding Potent Sirt Inhibitor in Coffee: Isolation, Confirmation and Synthesis of Javamide-II (N-Caffeoyltryptophan) as Sirt1/2 Inhibitor

    PubMed Central

    Park, Jae B.

    2016-01-01

    Recent studies suggest that Sirt inhibition may have beneficial effects on several human diseases such as neurodegenerative diseases and cancer. Coffee is one of most popular beverages with several positive health effects. Therefore, in this paper, potential Sirt inhibitors were screened using coffee extract. First, HPLC was utilized to fractionate coffee extract, then screened using a Sirt1/2 inhibition assay. The screening led to the isolation of a potent Sirt1/2 inhibitor, whose structure was determined as javamide-II (N-caffeoyltryptophan) by NMR. For confirmation, the amide was chemically synthesized and its capacity of inhibiting Sirt1/2 was also compared with the isolated amide. Javamide-II inhibited Sirt2 (IC50; 8.7μM) better than Sirt1(IC50; 34μM). Since javamide-II is a stronger inhibitor for Sirt2 than Sirt1. The kinetic study was performed against Sirt2. The amide exhibited noncompetitive Sirt2 inhibition against the NAD+ (Ki = 9.8 μM) and showed competitive inhibition against the peptide substrate (Ki = 5.3 μM). Also, a docking simulation showed stronger binding pose of javamide-II to Sirt2 than AGK2. In cellular levels, javamide-II was able to increase the acetylation of total lysine, cortactin and histone H3 in neuronal NG108-15 cells. In the same cells, the amide also increased the acetylation of lysine (K382) in p53, but not (K305). This study suggests that Javamide-II found in coffee may be a potent Sirt1/2 inhibitor, probably with potential use in some conditions of human diseases. PMID:26986569

  5. Hydrogenase synthesis in Bradyrhizobium japonicum Hupc mutants is altered in sensitivity to DNA gyrase inhibitors.

    PubMed Central

    Novak, P D; Maier, R J

    1989-01-01

    In the Hupc mutants of Bradyrhizobium japonicum SR, regulation of expression of hydrogenase is altered; the mutants synthesize hydrogenase constitutively in the presence of atmospheric levels of oxygen. The DNA gyrase inhibitors nalidixic acid, novobiocin, and coumermycin were used to inhibit growth of wild-type and mutant cells. For each inhibitor tested, growth of mutant and wild-type strains was equally sensitive. However, in contrast to the wild type, the Hupc mutants synthesized hydrogenase in the presence of high levels of any inhibitor. Cells were incubated with the drugs and simultaneously labeled with 14C-labeled amino acids, and hydrogenase was immunoprecipitated with antibody to the large subunit of the enzyme. Fluorograms of antibody blots then were scanned to determine the relative amount of hydrogenase (large subunit) synthesized in the presence or absence of the gyrase inhibitors. The amount of hydrogenase synthesized by the Hupc mutants in the presence of 300 micrograms of nalidixic acid per ml was near the level of enzyme synthesized in the absence of the inhibitor. No hydrogenase was detected in antibody blots of wild-type cultures which were derepressed for hydrogenase in the presence of 100 micrograms of coumermycin or novobiocin per ml. In contrast, hydrogenase was synthesized by the Hupc mutants in the presence of 100 micrograms of either drug per ml. The amount synthesized ranged from 5 to 32% and 20 to 49%, respectively, of that in the absence of those inhibitors, but nevertheless, hydrogenase synthesis was detected in all of the mutants examined.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:2547335

  6. Nicotinic acid, nicotinamide, and nicotinamide riboside: a molecular evaluation of NAD+ precursor vitamins in human nutrition.

    PubMed

    Bogan, Katrina L; Brenner, Charles

    2008-01-01

    Although baseline requirements for nicotinamide adenine dinucleotide (NAD+) synthesis can be met either with dietary tryptophan or with less than 20 mg of daily niacin, which consists of nicotinic acid and/or nicotinamide, there is growing evidence that substantially greater rates of NAD+ synthesis may be beneficial to protect against neurological degeneration, Candida glabrata infection, and possibly to enhance reverse cholesterol transport. The distinct and tissue-specific biosynthetic and/or ligand activities of tryptophan, nicotinic acid, nicotinamide, and the newly identified NAD+ precursor, nicotinamide riboside, reviewed herein, are responsible for vitamin-specific effects and side effects. Because current data suggest that nicotinamide riboside may be the only vitamin precursor that supports neuronal NAD+ synthesis, we present prospects for human nicotinamide riboside supplementation and propose areas for future research. PMID:18429699

  7. Synthesis and characterization of potent inhibitors of Trypanosoma cruzi dihydrofolate reductase

    SciTech Connect

    Schormann, Norbert; Velu, Sadanandan E.; Murugesan, Srinivasan; Senkovich, Olga; Walker, Kiera; Chenna, Bala C.; Shinkre, Bidhan; Desai, Amar; Chattopadhyay, Debasish

    2010-09-17

    Dihydrofolate reductase (DHFR) of the parasite Trypanosoma cruzi (T. cruzi) is a potential target for developing drugs to treat Chagas disease. We have undertaken a detailed structure-activity study of this enzyme. We report here synthesis and characterization of six potent inhibitors of the parasitic enzyme. Inhibitory activity of each compound was determined against T. cruzi and human DHFR. One of these compounds, ethyl 4-(5-[(2,4-diamino-6-quinazolinyl)methyl]amino-2-methoxyphenoxy)butanoate (6b) was co-crystallized with the bifunctional dihydrofolate reductase-thymidylate synthase enzyme of T. cruzi and the crystal structure of the ternary enzyme:cofactor:inhibitor complex was determined. Molecular docking was used to analyze the potential interactions of all inhibitors with T. cruzi DHFR and human DHFR. Inhibitory activities of these compounds are discussed in the light of enzyme-ligand interactions. Binding affinities of each inhibitor for the respective enzymes were calculated based on the experimental or docked binding mode. An estimated 60-70% of the total binding energy is contributed by the 2,4-diaminoquinazoline scaffold.

  8. New selective carbonic anhydrase IX inhibitors: synthesis and pharmacological evaluation of diarylpyrazole-benzenesulfonamides.

    PubMed

    Rogez-Florent, Tiphaine; Meignan, Samuel; Foulon, Catherine; Six, Perrine; Gros, Abigaëlle; Bal-Mahieu, Christine; Supuran, Claudiu T; Scozzafava, Andrea; Frédérick, Raphaël; Masereel, Bernard; Depreux, Patrick; Lansiaux, Amélie; Goossens, Jean-François; Gluszok, Sébastien; Goossens, Laurence

    2013-03-15

    Carbonic anhydrase (CA) IX expression is increased upon hypoxia and has been proposed as a therapeutic target since it has been associated with poor prognosis, tumor progression and pH regulation. We report the synthesis and the pharmacological evaluation of a new class of human carbonic anhydrase (hCA) inhibitors, 4-(5-aryl-2-hydroxymethyl-pyrazol-1-yl)-benzenesulfonamides. A molecular modeling study was conducted in order to simulate the binding mode of this new family of enzyme inhibitors within the active site of hCA IX. Pharmacological studies revealed high hCA IX inhibitory potency in the parameters nanomolar range. This study showed that the position of sulfonamide group in meta of the 1-phenylpyrazole increase a selectivity hCA IX versus hCA II of our compounds. An in vitro antiproliferative screening has been performed on the breast cancer MDA-MB-231 cell using doxorubicin as cytotoxic agent and in presence of selected CA IX inhibitor. The results shown that the cytotoxic efficiency of doxorubicin in an hypoxic environment, expressed in IC50 value, is restored at 20% level with 1μM CA IX inhibitor. PMID:23168081

  9. Synthesis and characterization of constrained peptidomimetic dipeptidyl peptidase IV inhibitors: amino-lactam boroalanines.

    PubMed

    Lai, Jack H; Wu, Wengen; Zhou, Yuhong; Maw, Hlaing H; Liu, Yuxin; Milo, Lawrence J; Poplawski, Sarah E; Henry, Gillian D; Sudmeier, James L; Sanford, David G; Bachovchin, William W

    2007-05-17

    We describe here the epimerization-free synthesis and characterization of a new class of conformationally constrained lactam aminoboronic acid inhibitors of dipeptidyl peptidase IV (DPP IV; E.C. 3.4.14.5). These compounds have the advantage that they cannot undergo the pH-dependent cyclization prevalent in most dipeptidyl boronic acids that attenuates their potency at physiological pH. For example, D-3-amino-1-[L-1-boronic-ethyl]-pyrrolidine-2-one (amino-D-lactam-L-boroAla), one of the best lactam inhibitors of DPP IV, is several orders of magnitude less potent than L-Ala-L-boroPro, as measured by Ki values (2.3 nM vs 30 pM, respectively). At physiological pH, however, it is actually more potent than L-Ala-L-boroPro, as measured by IC50 values (4.2 nM vs 1400 nM), owing to the absence of the potency-attenuating cyclization. In an interesting and at first sight surprising reversal of the relationship between stereochemistry and potency observed with the conformationally unrestrained Xaa-boroPro class of inhibitors, the L-L diastereomers of the lactams are orders of magnitude less effective than the D-L lactams. However, this interesting reversal and the unexpected potency of the D-L lactams as DPP IV inhibitors can be understood in structural terms, which is explained and discussed here. PMID:17458948

  10. [The action of catecholamine-synthesis inhibitors and of spiperone on sea urchin and mouse embryos].

    PubMed

    Markova, L N; Sakharova, N Iu; Bezuglov, V V

    2000-01-01

    We studied the effects of three inhibitors of catecholamine synthesis on the development of sea urchins Sphaerechinus granularis and Paracentrotus lividus. These drugs affected the early embryogenesis, which was expressed in inhibition of the cleavage divisions, appearance of abnormal embryos, and developmental arrest. The addition of arachidonic acid amide and dopamine to the incubation medium weakened the effects of the inhibitors. Spiperone induced developmental defects in preimplantation mouse embryos and sea urchin embryos. Arachidonic acid amide with dopamine exerted a protective effect against spiperone when introduced to sea urchin embryos at the blastula or late gastrula stages, rather than after fertilization. In murine embryos, this amide induced developmental defects and arrest itself and its effect was reversible. Possible mechanisms underlying the effects of these drugs are discussed. PMID:10732361

  11. Inhibitors of the Hydrolytic Enzyme Dimethylarginine Dimethylaminohydrolase (DDAH): Discovery, Synthesis and Development.

    PubMed

    Murphy, Rhys B; Tommasi, Sara; Lewis, Benjamin C; Mangoni, Arduino A

    2016-01-01

    Dimethylarginine dimethylaminohydrolase (DDAH) is a highly conserved hydrolytic enzyme found in numerous species, including bacteria, rodents, and humans. In humans, the DDAH-1 isoform is known to metabolize endogenous asymmetric dimethylarginine (ADMA) and monomethyl arginine (l-NMMA), with ADMA proposed to be a putative marker of cardiovascular disease. Current literature reports identify the DDAH family of enzymes as a potential therapeutic target in the regulation of nitric oxide (NO) production, mediated via its biochemical interaction with the nitric oxide synthase (NOS) family of enzymes. Increased DDAH expression and NO production have been linked to multiple pathological conditions, specifically, cancer, neurodegenerative disorders, and septic shock. As such, the discovery, chemical synthesis, and development of DDAH inhibitors as potential drug candidates represent a growing field of interest. This review article summarizes the current knowledge on DDAH inhibition and the derived pharmacokinetic parameters of the main DDAH inhibitors reported in the literature. Furthermore, current methods of development and chemical synthetic pathways are discussed. PMID:27187323

  12. Synthesis of Novel Tricyclic Chromenone-Based Inhibitors of IRE-1 RNase Activity

    PubMed Central

    2015-01-01

    Inositol-requiring enzyme 1 (IRE-1) is a kinase/RNase ER stress sensor that is activated in response to excessive accumulation of unfolded proteins, hypoxic conditions, calcium imbalance, and other stress stimuli. Activation of IRE-1 RNase function exerts a cytoprotective effect and has been implicated in the progression of cancer via increased expression of the transcription factor XBP-1s. Here, we describe the synthesis and biological evaluation of novel chromenone-based covalent inhibitors of IRE-1. Preparation of a family of 8-formyltetrahydrochromeno[3,4-c]pyridines was achieved via a Duff formylation that is attended by an unusual cyclization reaction. Biological evaluation in vitro and in whole cells led to the identification of 30 as a potent inhibitor of IRE-1 RNase activity and XBP-1s expression in wild type B cells and human mantle cell lymphoma cell lines. PMID:24749861

  13. Solanocapsine derivatives as potential inhibitors of acetylcholinesterase: Synthesis, molecular docking and biological studies.

    PubMed

    García, Manuela E; Borioni, José L; Cavallaro, Valeria; Puiatti, Marcelo; Pierini, Adriana B; Murray, Ana P; Peñéñory, Alicia B

    2015-12-01

    The investigation of natural products in medicinal chemistry is essential today. In this context, acetylcholinesterase (AChE) inhibitors comprise one type of the compounds most actively studied in the search for an effective treatment of symptoms of Alzheimer's disease. This work describes the isolation of a natural compound, solanocapsine, the preparation of its chemical derivatives, the evaluation of AChE inhibitory activity, and the structure-activity analysis of relevant cases. The influence of structural variations on the inhibitory potency was carefully investigated by modifying different reactive parts of the parent molecule. A theoretical study was also carried out into the binding mode of representative compounds to the enzyme through molecular modeling. The biological properties of the series were investigated. Through this study valuable information was obtained of steroidal alkaloid-type compounds as a starting point for the synthesis of AChE inhibitors. PMID:26362598

  14. Design and synthesis of a new class of cryptophycins based tubulin inhibitors.

    PubMed

    Kumar, Arvind; Kumar, Manjeet; Sharma, Simmi; Guru, Santosh Kumar; Bhushan, Shashi; Shah, Bhahwal Ali

    2015-03-26

    Tubulin binding compounds represent one of the most attractive targets for anticancer drug development. They broadly fall into two categories viz., tubulin polymerization inhibitors, which block microtubule growth and destabilize microtubules like vinca alkaloids and cryptophycins, and the others, which polymerize microtubules into hyperstable forms represented by family of taxanes. In this context, we aimed at design and synthesis of cryptophycins based macrocyclic depsipeptides, which are synthetically more accessible, however have the basic information to target tubulins and establish structure activity relationship (SAR). Thus, a new class of cryptophycins based marocyclic depsipeptides with a truncated epoxide chain were synthesized as potential tubulin inhibitors. The resultant lead analogues 15a and 16a exhibited good anti-cancer activity, induced apoptosis, caused block/delay in cell cycle as well as significantly reduced the expression of α- and β-tubulins. Molecular modelling studies show that 15a and 16a bind in the same domain as that of cryptophycins. PMID:25647428

  15. Bioorganometallic chemistry: biocatalytic oxidation reactions with biomimetic nad+/nadh co-factors and [cp*rh(bpy)h]+ for selective organic synthesis

    SciTech Connect

    Lutz, Jochen; Hollman, Frank; Ho, The Vinh; Schnyder, Adrian; Fish, Richard H.; Schmid, Andreas

    2004-03-09

    The biocatalytic, regioselective hydroxylation of 2-hydroxybiphenyl to the corresponding catechol was accomplished utilizing the monooxygenase 2-hydroxybiphenyl 3-monooxygenase (HbpA). The necessary natural nicotinamide adenine dinucleotide (NAD{sup +}) co-factor for this biocatalytic process was replaced by a biomimetic co-factor, N-benzylnicotinamide bromide, 1a. The interaction between the flavin (FAD) containing HbpA enzyme and the corresponding biomimetic NADH compound, N-benzyl-1,4-dihdronicotinamide, 1b, for hydride transfers, was shown to readily occur. The in situ recycling of the reduced NADH biomimic 1b from 1a was accomplished with [Cp*Rh(bpy)H](Cl); however, productive coupling of this regeneration reaction to the enzymatic hydroxylation reaction was not totally successful, due to a deactivation process concerning the HbpA enzyme peripheral groups; i.e., -SH or -NH{sub 2} possibly reacting with the precatalyst, [Cp*Rh(bpy)(H{sub 2}O)](Cl){sub 2}, and thus inhibiting the co-factor regeneration process. The deactivation mechanism was studied, and a promising strategy of derivatizing these peripheral -SH or -NH{sub 2} groups with a polymer containing epoxide was successful in circumventing the undesired interaction between HbpA and the precatalyst. This latter strategy allowed tandem co-factor regeneration using 1a or 2a, [Cp*Rh(bpy)(H2O)](Cl){sub 2}, and formate ion, in conjunction with the polymer bound, FAD containing HbpA enzyme to provide the catechol product.

  16. Phenotypic Screening Identifies Protein Synthesis Inhibitors as H-Ras-Nanocluster-Increasing Tumor Growth Inducers.

    PubMed

    Najumudeen, Arafath K; Posada, Itziar M D; Lectez, Benoit; Zhou, Yong; Landor, Sebastian K-J; Fallarero, Adyary; Vuorela, Pia; Hancock, John; Abankwa, Daniel

    2015-12-15

    Ras isoforms H-, N-, and K-ras are each mutated in specific cancer types at varying frequencies and have different activities in cell fate control. On the plasma membrane, Ras proteins are laterally segregated into isoform-specific nanoscale signaling hubs, termed nanoclusters. As Ras nanoclusters are required for Ras signaling, chemical modulators of nanoclusters represent ideal candidates for the specific modulation of Ras activity in cancer drug development. We therefore conducted a chemical screen with commercial and in-house natural product libraries using a cell-based H-ras-nanoclustering FRET assay. Next to established Ras inhibitors, such as a statin and farnesyl-transferase inhibitor, we surprisingly identified five protein synthesis inhibitors as positive regulators. Using commonly employed cycloheximide as a representative compound, we show that protein synthesis inhibition increased nanoclustering and effector recruitment specifically of active H-ras but not of K-ras. Consistent with these data, cycloheximide treatment activated both Erk and Akt kinases and specifically promoted H-rasG12V-induced, but not K-rasG12V-induced, PC12 cell differentiation. Intriguingly, cycloheximide increased the number of mammospheres, which are enriched for cancer stem cells. Depletion of H-ras in combination with cycloheximide significantly reduced mammosphere formation, suggesting an exquisite synthetic lethality. The potential of cycloheximide to promote tumor cell growth was also reflected in its ability to increase breast cancer cell tumors grown in ovo. These results illustrate the possibility of identifying Ras-isoform-specific modulators using nanocluster-directed screening. They also suggest an unexpected feedback from protein synthesis inhibition to Ras signaling, which might present a vulnerability in certain tumor cell types. PMID:26568031

  17. Synthesis and evaluation of novel benzylphthalazine derivatives as hedgehog signaling pathway inhibitors.

    PubMed

    Bao, Xiaolong; Peng, Yuanqiu; Lu, Xiuhong; Yang, Jun; Zhao, Weili; Tan, Wenfu; Dong, Xiaochun

    2016-07-01

    We report herein the design and synthesis of a series of novel benzylphthalazine derivatives as hedgehog signaling pathway inhibitors. Gli-luciferase assay demonstrated that changing piperazine ring of Anta XV to different four, five or six-membered heterocyclic building blocks afforded significant influences on Hh pathway inhibition. In particular, compound 10e with piperidin-4-amine moiety was found to possess 12-fold higher Hh inhibitory activities comparing to the lead compound in vitro. In vivo efficacy of 10e in a ptch(+/-)p53(-/-) mouse medulloblastoma allograft model also indicated encouraging results. PMID:27180012

  18. Enantioselective Synthesis and Profiling of Two Novel Diazabicyclooctanone β-Lactamase Inhibitors

    PubMed Central

    2014-01-01

    The enantioselective synthesis of two novel cyclopropane-fused diazabicyclooctanones is reported here. Starting from butadiene monoxide, the key enone intermediate 7 was prepared in six steps. Subsequent stereoselective introduction of the cyclopropane group and further transformation led to compounds 1a and 1b as their corresponding sodium salt. The great disparity regarding their hydrolytic stability was rationalized by the steric interaction between the cyclopropyl methylene and urea carbonyl. These two novel β-lactamase inhibitors were active against class A, C, and D enzymes. PMID:25313328

  19. Growth inhibition of human prostate cells in vitro by novel inhibitors of androgen synthesis.

    PubMed

    Klus, G T; Nakamura, J; Li, J S; Ling, Y Z; Son, C; Kemppainen, J A; Wilson, E M; Brodie, A M

    1996-11-01

    The long-standing strategy for the treatment of metastatic prostate cancer has been to reduce androgenic stimulation of tumor growth by removal of the testes, the primary site of testosterone synthesis. However, a low level of androgenic stimulation may continue, even after castration, by the conversion of adrenal androgens to 5alpha-dihydrotestosterone (DHT) in the prostate tumor cells. Two important enzymes of the androgen biosynthetic pathway are 17alpha-hydroxylase/C17,20-lyase, which regulates an early step in the synthesis of testosterone and other androgens in both the testes and adrenal glands, and 5alpha-reductase, which converts testosterone to the more potent androgen, DHT, in the prostate. We have identified new inhibitors of these enzymes that may be of use in achieving a more complete ablation of androgens in the treatment of metastatic prostate cancer. Three derivatives of androstene were shown to inhibit 17alpha-hydroxylase/C17,20-lyase with potencies 2-20-fold greater than that of ketoconazole, a previously established inhibitor of this enzyme. Derivatives of pregnane and pregnene displayed activities against 5alpha-reductase that were comparable to that of N-(1,1-dimethyl-ethyl)-3-oxo-4-aza-5alpha-androst-1-ene-17beta-car boxamide. All of the 5alpha-reductase inhibitors were able to at least partially inhibit the mitogenic effect of testosterone in either histocultures of human benign prostatic hypertrophic tissue or in cultures of the LNCaP human prostatic tumor cell line. For these compounds, it appears that this inhibition can be attributed to a reduction of DHT synthesis in these cultures, because no inhibitory effect was observed in DHT-treated cultures, and none of the compounds had a cytotoxic effect. Surprisingly, one of the inhibitors of 17alpha-hydroxylase/C17,20-lyase, 17beta-(4-imidazolyl)-5-pregnen-3beta-ol, was also able to inhibit the mitogenic effect of testosterone in both the histoculture and cell culture assays and had an effect

  20. Rational Design, Synthesis and Evaluation of Coumarin Derivatives as Protein-protein Interaction Inhibitors.

    PubMed

    De Luca, Laura; Agharbaoui, Fatima E; Gitto, Rosaria; Buemi, Maria Rosa; Christ, Frauke; Debyser, Zeger; Ferro, Stefania

    2016-09-01

    Herein we describe the design and synthesis of a new series of coumarin derivatives searching for novel HIV-1 integrase (IN) allosteric inhibitors. All new obtained compounds were tested in order to evaluate their ability to inhibit the interaction between the HIV-1 IN enzyme and the nuclear protein lens epithelium growth factor LEDGF/p75. A combined approach of docking and molecular dynamic simulations has been applied to clarify the activity of the new compounds. Specifically, the binding free energies by using the method of molecular mechanics-generalized Born surface area (MM-GBSA) was calculated, whereas hydrogen bond occupancies were monitored throughout simulations methods. PMID:27546050

  1. Synthesis of novel, peptidic kinase inhibitors with cytostatic/cytotoxic activity.

    PubMed

    Szymanski, Wiktor; Zwolinska, Magdalena; Klossowski, Szymon; Młynarczuk-Biały, Izabela; Biały, Lukasz; Issat, Tadeusz; Malejczyk, Jacek; Ostaszewski, Ryszard

    2014-03-01

    The utility of a novel, chemoenzymatic procedure for the stereocontrolled synthesis of small peptides is presented in the preparation and structure optimisation of dipeptides with cytostatic/cytotoxic activity. The method uses Passerini multicomponent reaction for the preparation of racemic scaffold which is then enantioselectively hydrolysed by hydrolytic enzymes. Products of these transformations are further functionalised towards title compounds. Both activity and selectivity towards tumor cells is optimised. Final compound is shown to be an inhibitor of the protein kinase signaling pathway. PMID:24507826

  2. A Synthesis of a Spirocyclic Macrocyclic Protease Inhibitor for the Treatment of Hepatitis C.

    PubMed

    Chung, Cheol K; Cleator, Ed; Dumas, Aaron M; Hicks, Jacqueline D; Humphrey, Guy R; Maligres, Peter E; Nolting, Andrew F; Rivera, Nelo; Ruck, Rebecca T; Shevlin, Michael

    2016-03-18

    The development of a convergent and highly stereoselective synthesis of an HCV NS3/4a protease inhibitor possessing a unique spirocyclic and macrocyclic architecture is described. A late-stage spirocyclization strategy both enabled rapid structure-activity relationship studies in the drug discovery phase and simultaneously served as the basis for the large scale drug candidate preparation for clinical use. Also reported is the discovery of a novel InCl3-catalyzed carbonyl reduction with household aluminum foil or zinc powder as the terminal reductant. PMID:26950496

  3. Synthesis and biological evaluation of C(5)-substituted derivatives of leukotriene biosynthesis inhibitor BRP-7.

    PubMed

    Levent, Serkan; Gerstmeier, Jana; Olgaç, Abdurrahman; Nikels, Felix; Garscha, Ulrike; Carotti, Andrea; Macchiarulo, Antonio; Werz, Oliver; Banoglu, Erden; Çalışkan, Burcu

    2016-10-21

    Pharmacological intervention with 5-lipoxygenase (5-LO) pathway leading to suppression of leukotriene (LT) biosynthesis is a clinically validated strategy for treatment of respiratory and cardiovascular diseases such as asthma and atherosclerosis. Here we describe the synthesis of a series of C(5)-substituted analogues of the previously described 5-LO-activating protein (FLAP) inhibitor BRP-7 (IC50 = 0.31 μM) to explore the effects of substitution at the C(5)-benzimidazole (BI) ring as a strategy to increase the potency against FLAP-mediated 5-LO product formation. Incorporation of polar substituents on the C(5) position of the BI core, exemplified by compound 11 with a C(5)-nitrile substituent, significantly enhances the potency for suppression of 5-LO product synthesis in human neutrophils (IC50 = 0.07 μM) and monocytes (IC50 = 0.026 μM). PMID:27423639

  4. Action of Protein Synthesis Inhibitors in Blocking Electrogenic H+ Efflux from Corn Roots 12

    PubMed Central

    Chastain, Chris J.; Lafayette, Peter R.; Hanson, John B.

    1981-01-01

    The block in the electrogenic H+ efflux produced by protein synthesis inhibitors in corn root tissue can be released or by-passed by addition of fusicoccin or nigericin. The inhibition also lowers cell potential, and the release repolarizes. Associated with the inhibition of H+ efflux is inhibition of K+ influx and the growth of the root tip; fusicoccin partially relieves these inhibitions, but nigericin does not. The inhibition of H+ efflux which arises from blocking the proton channel of the ATPase by oligomycin or N,N′-dicyclohexylcarbodiimide can also be partially relieved by fusicoccin, but not by nigericin; the inhibition produced by diethylstilbestrol is not relieved by fusicoccin. The results are discussed in terms of the presumed mode of action of fusicoccin on the plasmalemma ATPase. Inhibition of protein synthesis appears to inactivate the proton channel of the ATPase, possibly as the indirect result of disrupted metabolism. Fusicoccin reactivates or bypasses the blocked channel. PMID:16661763

  5. Structural and functional characterization of human NAD kinase.

    PubMed

    Lerner, F; Niere, M; Ludwig, A; Ziegler, M

    2001-10-19

    NADP is essential for biosynthetic pathways, energy, and signal transduction. Its synthesis is catalyzed by NAD kinase. Very little is known about the structure, function, and regulation of this enzyme from multicellular organisms. We identified a human NAD kinase cDNA and the corresponding gene using available database information. A cDNA was amplified from a human fibroblast cDNA library and functionally overexpressed in Escherichia coli. The obtained cDNA, slightly different from that deposited in the database, encodes a protein of 49 kDa. The gene is expressed in most human tissues, but not in skeletal muscle. Human NAD kinase differs considerably from that of prokaryotes by subunit molecular mass (49 kDa vs 30-35 kDa). The catalytically active homotetramer is highly selective for its substrates, NAD and ATP. It did not phosphorylate the nicotinic acid derivative of NAD (NAAD) suggesting that the potent calcium-mobilizing pyridine nucleotide NAADP is synthesized by an alternative route. PMID:11594753

  6. Action of Inhibitors of RNA and Protein Synthesis on Cell Enlargement 1

    PubMed Central

    Noodén, Larry D.; Thimann, Kenneth V.

    1966-01-01

    Further studies with inhibitors of protein synthesis are presented to support the conclusion, drawn from work with chloramphenicol, that protein synthesis is a critical limiting factor in auxin-induced cell expansion. The indoleacetic acid-induced elongation of oat coleoptile sections was strongly inhibited by dl-p-fluorophenylalanine, and the inhibition is antagonized by phenylalanine. Puromycin at 10−4 m very strongly inhibited the indoleacetic acid-induced growth of oat coleoptile and artichoke tuber sections and exerted a less powerful effect on pea stem sections. As found earlier with chloramphenicol, concentrations of puromycin effective in inhibiting the growth of coleoptile sections had quantitatively similar effects on protein synthesis, as measured by the incorporation of C14-leucine into protein of the coleoptile tissue. Several analogues of RNA bases were also tested, but while 8-azaguanine very strongly inhibited growth of artichoke tuber disks, 6-azauracil was the only one of this group clearly inhibitory to growth in coleoptile or pea stem sections. Actinomycin D actively inhibited both elongation and the incorporation of C14-leucine into protein in oat coleoptile sections. Inhibition of the 2 processes went closely parallel. Actinomycin D also powerfully inhibited growth of artichoke tuber disks. All the compounds effective in inhibiting growth generally inhibited the uptake of leucine as well. The possibility that auxin causes cell enlargement in plants by inducing the synthesis of a messenger RNA and of one or more new but unstable enzymes, is discussed. Possible but less favored alternative explanations are: A) that auxin induces synthesis of a wall protein, or B) that the continued synthesis of some other unstable protein (by a process independent of auxin) may be a prerequisite for cell enlargement. PMID:5904588

  7. Transinhibition of C1 inhibitor synthesis in type I hereditary angioneurotic edema.

    PubMed Central

    Kramer, J; Rosen, F S; Colten, H R; Rajczy, K; Strunk, R C

    1993-01-01

    To ascertain the mechanism for decreased synthesis of C1 inhibitor (C1 INH) in certain patients with the autosomal dominant disorder hereditary angioneurotic edema, we studied expression of C1 INH in fibroblasts in which the mutant and wild type mRNA and protein could be distinguished because of deletion of exon 7 (delta Ex7). In the HANE delta Ex7 cells, the amount of wild type mRNA (2.1 kb) was expressed at 52 +/- 2% (n = 5) of normal, whereas the mutant mRNA was 17 +/- 1% (n = 5) of normal. Rates of synthesis of both wild type and mutant proteins (11 +/- 3 and 3 +/- 1% of normal, respectively) were lower than predicted from the mRNA levels. There was no evidence of increased C1 INH protein catabolism. These data indicate that there are multiple levels of control of C1 INH synthesis in type I hereditary angioneurotic edema. Pretranslational regulation results in < 50% of the mutant truncated 1.9-kb mRNA. In addition, translational regulation results in decreased synthesis of both wild type and mutatn C1 INH proteins. These data suggest a transinhibition of wild type C1 INH translation by mutant mRNA and/or protein. Images PMID:8450054

  8. NAD metabolism in Vibrio cholerae.

    PubMed Central

    Foster, J W; Brestel, C

    1982-01-01

    Extracts of Vibrio cholerae were assayed for various enzymatic activities associated with pyridine nucleotide cycle metabolism. The activities measured include NAD glycohydrolase, nicotinamide deamidase, nicotinamide mononucleotide deamidase, and nicotinic acid phosphoribosyltransferase. The results obtained demonstrate the existence in V. cholerae of the five-membered pyridine nucleotide cycle and the potential for a four-membered pyridine nucleotide cycle. The data presented also suggest that most of the NAD glycohydrolase in V. cholerae extracts is not directly related to cholera toxin. PMID:6119307

  9. Regulation of synthesis and activity of NAD(+)-dependent 15-hydroxy-prostaglandin dehydrogenase (15-PGDH) by dexamethasone and phorbol ester in human erythroleukemia (HEL) cells

    SciTech Connect

    Xun, C.Q.; Ensor, C.M.; Tai, H.H. )

    1991-06-28

    Dexamethasone stimulated 15-PGDH activity in HEL cells in a time and concentration dependent manner. Increase in 15-PGDH activity by dexamethasone was found to be accompanied by an increase in enzyme synthesis as revealed by Western blot and (35S)methionine labeling studies. In addition to dexamethasone, other anti-inflammatory steroids also increased 15-PGDH activity in the order of their glucocorticoid activity. Among sex steroids only progesterone increased significantly 15-PGDH activity. 12-0-Tetradecanoylphorbol-13-acetate (TPA) also induced the synthesis of 15-PGDH but inhibited the enzyme activity. It appears that TPA caused a time dependent inactivation of 15-PGDH by a protein kinase C mediated mechanism.

  10. Protein synthesis inhibitors attenuate water flow in vasopressin-stimulated toad urinary bladder

    SciTech Connect

    Hoch, B.S.; Ast, M.B.; Fusco, M.J.; Jacoby, M.; Levine, S.D. )

    1988-01-01

    Vasopressin stimulates the introduction of aggregated particles, which may represent pathways for water flow, into the luminal membrane of toad urinary bladder. It is not known whether water transport pathways are degraded on removal from membrane or whether they are recycled. The authors examined the effect of the protein synthesis inhibitors cycloheximide and puromycin using repeated 30-min cycles of vasopressin followed by washout of vasopressin, all in the presence of an osmotic gradient, a protocol that maximizes aggregate turnover. High dose cycloheximide inhibited flow immediately. Low dose cycloheximide did not affect initial flow. In the absence of vasopressin, inhibition did not develop. Despite the inhibition of flow in vasopressin-treated tissues, the cAMP-dependent protein kinase ratio was elevated in cycloheximide-treated tissues, suggesting modulation at a distal site in the stimulatory cascade. ({sup 14}C)urea permeability was not inhibited by cycloheximide. Puromycin also inhibited water flow by the fourth challenge with vasopressin. The data suggest that protein synthesis inhibitors attenuate flow at a site that is distal to cAMP-dependent protein kinase. However, the reversal of inhibition in MIX-treated tissues suggests that the water pathway can be fully manifested given suitable stimulation. They conclude that either large stores of the transport system are available or that the transport system is extensively recycled on retrieval from the membrane.

  11. Synthesis and Evaluation of 5-Lipoxygenase Translocation Inhibitors from Acylnitroso Hetero-Diels-Alder Cycloadducts†

    PubMed Central

    Bolger, Joshua K.; Tian, Wen; Wolter, William R.; Cho, Wonhwa; Suckow, Mark A.

    2012-01-01

    Acylnitroso cycloadducts have proven to be valuable intermediates in the syntheses of a plethora of biologically active molecules. Recently, organometallic reagents were shown to open bicyclic acylnitroso cycloadducts and, more interestingly, the prospect of highly regioselective openings was raised. This transformation was employed in the synthesis of a compound with excellent inhibitory activity against 5-lipoxygenase ((±)-4a, IC50 51 nM), an important mediator of inflammation intimately involved in a number of disease states including asthma and cancer. Optimization of the copper-mediated organometallic ring opening reaction was accomplished allowing the further exploration of the biological activity. Synthesis of a number of derivatives with varying affinity for metal binding as well as pendant groups in a range of sizes was accomplished. Analogues were tested in a whole cell assay which revealed a subset of the compounds to be inhibitors of enzyme translocation, a mode of action not previously known and, potentially, extremely important for better understanding of the enzyme and inhibitor development. Additionally, the lead compound was tested in vivo in an established colon cancer model and showed very encouraging anti-tumorogenic properties. PMID:21365098

  12. Laboratory evaluation of five chitin synthesis inhibitors against the colorado potato beetle, Leptinotarsa decemlineata.

    PubMed

    Karimzadeh, R; Hejazi, M J; Rahimzadeh Khoei, F; Moghaddam, M

    2007-01-01

    Results of laboratory experiments are reported that tested the effects of five chitin synthesis inhibitors, diflubenzuron, cyromazine, lufenuron, hexaflumuron and triflumuron. on second instars of the Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Crysomelidae), originally collected from potato fields of Bostanabaad, a town 66 km southeast of Tabriz, Iran. In bioassays, the larvae were fed potato leaves dipped in aqueous solutions containing chitin synthesis inhibitors. The mortalities and abnormalities of the treated larvae were recorded 72 hours after treatments. LC(50) values were 58.6, 69.6, 27.3, 0.79 and 81.4 mg ai/ L for diflubenzuron, cyromazine, lufenuron, hexaflumuron and triflumuron, respectively. Compared with phosalone, which is one of the common insecticides used for controlling this pest in Iran, lufenuron and hexaflumuron seem to be much more potent, and if they perform equally well in the field, they would be suitable candidates to be considered as reduced risk insecticides in management programs for L. decemlineata due to much wider margin of safety for mammals and considerably fewer undesirable environmental side effects. PMID:20345285

  13. Laboratory Evaluation of Five Chitin Synthesis Inhibitors Against the Colorado Potato Beetle, Leptinotarsa decemlineata

    PubMed Central

    Karimzadeh, R.; Hejazi, M. J.; Rahimzadeh Khoei, F.; Moghaddam, M.

    2007-01-01

    Results of laboratory experiments are reported that tested the effects of five chitin synthesis inhibitors, diflubenzuron, cyromazine, lufenuron, hexaflumuron and triflumuron. on second instars of the Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Crysomelidae), originally collected from potato fields of Bostanabaad, a town 66 km southeast of Tabriz, Iran. In bioassays, the larvae were fed potato leaves dipped in aqueous solutions containing chitin synthesis inhibitors. The mortalities and abnormalities of the treated larvae were recorded 72 hours after treatments. LC50 values were 58.6, 69.6, 27.3, 0.79 and 81.4 mg ai/ L for diflubenzuron, cyromazine, lufenuron, hexaflumuron and triflumuron, respectively. Compared with phosalone, which is one of the common insecticides used for controlling this pest in Iran, lufenuron and hexaflumuron seem to be much more potent, and if they perform equally well in the field, they would be suitable candidates to be considered as reduced risk insecticides in management programs for L. decemlineata due to much wider margin of safety for mammals and considerably fewer undesirable environmental side effects. PMID:20345285

  14. Synthesis of 4-substituted nipecotic acid derivatives and their evaluation as potential GABA uptake inhibitors.

    PubMed

    Hellenbrand, Tim; Höfner, Georg; Wein, Thomas; Wanner, Klaus T

    2016-05-01

    In this study, we disclose the design and synthesis of novel 4-susbtituted nipecotic acid derivatives as inhibitors of the GABA transporter mGAT1. Based on molecular modeling studies the compounds are assumed to adopt a binding pose similar to that of the potent mGAT1 inhibitor nipecotic acid. As substitution in 4-position should not cause an energetically unfavorable orientation of nipecotic acid as it is the case for N-substituted derivatives this is expected to lead to highly potent binders. For the synthesis of novel 4-substituted nipecotic acid derivatives a linear synthetic strategy was employed. As a key step, palladium catalyzed cross coupling reactions were used to attach the required biaryl moieties to the ω-position of the alkenyl- or alkynyl spacers of varying length in the 4-position of the nipecotic acid scaffold. The resulting amino acids were characterized with respect to their binding affinities and inhibitory potencies at mGAT1. Though the biological activities found were generally insignificant to poor, two compounds, one of which possesses a reasonable binding affinity for mGAT1, rac-57, the other a notable inhibitory potency at mGAT4, rac-84, both displaying a slight subtype selectivity for the individual transporters, could be identified. PMID:27039250

  15. The Leishmania nicotinamidase is essential for NAD+ production and parasite proliferation.

    PubMed

    Gazanion, E; Garcia, D; Silvestre, R; Gérard, C; Guichou, J F; Labesse, G; Seveno, M; Cordeiro-Da-Silva, A; Ouaissi, A; Sereno, D; Vergnes, B

    2011-10-01

    NAD+ is a central cofactor that plays important roles in cellular metabolism and energy production in all living cells. Genomics-based reconstruction of NAD+ metabolism revealed that Leishmania protozoan parasites are NAD+ auxotrophs. Consequently, these parasites require assimilating NAD+ precursors (nicotinamide, nicotinic acid, nicotinamide riboside) from their host environment to synthesize NAD+ by a salvage pathway. Nicotinamidase is a key enzyme of this salvage pathway that catalyses conversion of nicotinamide (NAm) to nicotinic acid (Na), and that is absent in higher eukaryotes. We present here the biochemical and functional characterizations of the Leishmania infantum nicotinamidase (LiPNC1). Generation of Lipnc1 null mutants leads to a decrease in NAD+ content, associated with a metabolic shutdown-like phenotype with an extensive lag phase of growth. Both phenotypes could be rescued by an add-back construct or by addition of exogenous Na. In addition, Lipnc1 null mutants were unable to establish a sustained infection in a murine experimental model. Altogether, these results illustrate that NAD+ homeostasis is a fundamental component of Leishmania biology and virulence, and that NAm constitutes its main NAD+ source in the mammalian host. The crystal structure of LiPNC1 we solved allows now the design of rational inhibitors against this new promising therapeutic target. PMID:21819459

  16. In vivo protection against soman toxicity by known inhibitors of acetylcholine synthesis in vitro.

    PubMed

    Sterling, G H; Doukas, P H; Sheldon, R J; O'Neill, J J

    1988-02-01

    Soman inhibits the enzyme acetylcholinesterase, essentially irreversibly, producing an accumulation of acetylcholine (ACh) which is responsible for many of its toxic effects. Current approaches to treatment include: (1) atropine, a muscarinic receptor blocker; (2) pyridine-2-aldoxime methylchloride (2-PAM), an enzyme reactivator; and (3) carbamate protection of the enzyme. However, no fully satisfactory regimen has been found, primarily because of the rapid aging process. In this study, compounds known to inhibit ACh synthesis in vitro were evaluated in combination with atropine and 2-PAM so as to assess their potential utility in protection against soman toxicity in rats. Acetylsecohemicholinium (100 micrograms/kg, i.c.v.t., 30 min prior to soman), an inhibitor of high affinity choline uptake (HAChU) and cholineacetyltransferase (ChAT) activity in vitro, enhanced the protective effects of atropine and 2-PAM, reducing the mortality within the first 2 hr following soman. N-Hydroxyethylnaphthylvinylpyridine (NHENVP), a quaternary ChAT inhibitor (1.7 mumol/kg, i.m.), significantly reduced the overall percent mortality due to soman from 80% to 20%. The compound was most effective when administered 2-3 min prior to soman and was effective only by the intramuscular route. N-Allyl-3-quinuclidinol, a potent HAChU inhibitor (1 mumol/kg, i.m.) was the most effective quinuclidine analog evaluated, also reducing the percent mortality for a 24-hr period. Unlike NHENVP, it was most effective when given 30-60 min prior to soman. It is suggested from the data that compounds that disrupt presynaptic ACh synthesis in vitro may prove effective in treating organophosphate poisoning. The results demonstrate interesting differences among the compounds studied and provide insight for the design of protectants against soman toxicity. These findings further underscore the need to examine the structure activity and pharmacokinetic properties of these compounds, i.e. comparison of routes of

  17. Synthesis of novel fluorocarbocyclic nucleosides and nucleotides as potential inhibitors of human immunodeficiency virus

    SciTech Connect

    Hilpert, H.

    1989-01-01

    3[prime]-Azido-3[prime]-deoxythymidine (AZT) and 2[prime], 3[prime]-dideoxycytidine (DDC) are potent in vivo inhibitors of human immunodeficiency virus. Due to their short half-life in the body and undesired side-effects compounds with improved bioavailability were designed. A feature of these analogues was the replacement of the heterocyclic oxygen atom by an isosteric CHF-group thus stabilizing the labile glycosidic bond against metabolic breakdown. A versatile and short synthesis, starting from ketone, serves to construct the highly functionalized and protected key intermediates. These ([alpha]- and [beta]-fluoro epimeric) intermediates were elaborated to eight fluorocarbocyclic nucleoside analogues linked with a thymine base, an adenine base, and a guanine base. An attempt was made to prepare analogues of the potent HIV inhibitor carbovir c. The unexpected oxidation of the double bond of compound d, instead of the desired Baeyer-Villiger ring-expansion, meant that the synthetic scheme was redundant. A second total synthesis involves the preparation of the three fluorocarbocyclic phosphonates. These analogues possess additionally a P-C linkage which should markedly enhance the stability of the side chain. To perform enzyme inhibition tests, three analogues were chemically activated to the biologically active triphosphates. Inhibition tests on HIV associated reverse transcriptase confirmed the high activity of one of the AZT triphosphates. The fluorocarbocyclic counterpart was two orders of magnitude less active. A fluorocarbocyclic phosphonate was twice as active as the AZT triphosphate. Neither the eight nucleoside analogues nor the three phosphonates displayed significant activity against HIV infected cells. Crystallographic data of two fluorocarbocyclic nucleosides, two potent HIV inhibitors, and some 20 examples of 2[prime]-deoxyribonucleosides have been compared.

  18. Design, Synthesis, and Protein Crystallography of Biaryltriazoles as Potent Tautomerase Inhibitors of Macrophage Migration Inhibitory Factor

    PubMed Central

    Dziedzic, Pawel; Cisneros, José A.; Robertson, Michael J.; Hare, Alissa A.; Danford, Nadia E.; Baxter, Richard H. G.; Jorgensen, William L.

    2015-01-01

    Optimization is reported for biaryltriazoles as inhibitors of the tautomerase activity of human macrophage migration inhibitory factor (MIF), a proinflammatory cytokine associated with numerous inflammatory diseases and cancer. A combined approach was taken featuring organic synthesis, enzymatic assaying, crystallography, and modeling including free-energy perturbation (FEP) calculations. X-ray crystal structures for 3a and 3b bound to MIF are reported and provided a basis for the modeling efforts. The accommodation of the inhibitors in the binding site is striking with multiple hydrogen bonds and aryl–aryl interactions. Additional modeling encouraged pursuit of 5-phenoxyquinolinyl analogues, which led to the very potent compound 3s. Activity was further enhanced by addition of a fluorine atom adjacent to the phenolic hydroxyl group as in 3w, 3z, 3aa, and 3bb to strengthen a key hydrogen bond. It is also shown that physical properties of the compounds can be modulated by variation of solvent-exposed substituents. Several of the compounds are likely the most potent known MIF tautomerase inhibitors; the most active ones are more than 1000-fold more active than the well-studied (R)-ISO-1 and more than 200-fold more active than the chromen-4-one Orita-13. PMID:25697265

  19. In silico design, synthesis, and screening of novel deoxyhypusine synthase inhibitors targeting HIV-1 replication.

    PubMed

    Schroeder, Marcus; Kolodzik, Adrian; Pfaff, Katharina; Priyadarshini, Poornima; Krepstakies, Marcel; Hauber, Joachim; Rarey, Matthias; Meier, Chris

    2014-05-01

    The human enzyme deoxyhypusine synthase (DHS) is an important host cell factor that participates in the post-translational hypusine modification of eukaryotic initiation factor 5A (eIF-5A). Hypusine-modified eIF-5A plays a role in a number of diseases, including HIV infection/AIDS. Thus, DHS represents a novel and attractive drug target. So far, four crystal structures are available, and various substances have been tested for inhibition of human DHS. Among these inhibitors, N-1-guanyl-1,7-diaminoheptane (GC7) has been co-crystallized in the active site of DHS. However, despite its potency, GC7 is not selective enough to be used in drug applications. Therefore, new compounds that target DHS are needed. Herein we report the in silico design, chemical synthesis, and biological evaluation of new DHS inhibitors. One of these inhibitors showed dose-dependent inhibition of DHS in vitro, as well as suppression of HIV replication in cell cultures. Furthermore, the compound exhibited no cytotoxic effects at active concentrations. Thus, this designed compound demonstrated proof of principle and represents a promising starting point for the development of new drug candidates to specifically interfere with DHS activity. PMID:24616161

  20. The Design, Synthesis and Structure-Activity Relationship of Mixed Serotonin, Norepinephrine and Dopamine Uptake Inhibitors

    NASA Astrophysics Data System (ADS)

    Chen, Zhengming; Yang, Ji; Skolnick, Phil

    The evolution of antidepressants over the past four decades has involved the replacement of drugs with a multiplicity of effects (e.g., TCAs) by those with selective actions (i.e., SSRIs). This strategy was employed to reduce the adverse effects of TCAs, largely by eliminating interactions with certain neurotransmitters or receptors. Although these more selective compounds may be better tolerated by patients, selective drugs, specifically SSRIs, are not superior to older drugs in treating depressed patients as measured by response and remission rates. It may be an advantage to increase synaptic levels of both serotonin and norepinephrine, as in the case of dual uptake inhibitors like duloxetine and venlafaxine. An important recent development has been the emergence of the triple-uptake inhibitors (TUIs/SNDRIs), which inhibit the uptake of the three neurotransmitters most closely linked to depression: serotonin, norepinephrine, and dopamine. Preclinical studies and clinical trials indicate that a drug inhibiting the reuptake of all three of these neurotransmitters could produce more rapid onset of action and greater efficacy than traditional antidepressants. This review will detail the medicinal chemistry involved in the design, synthesis and discovery of mixed serotonin, norepinephrine and dopamine transporter uptake inhibitors.

  1. A review on ROCK-II inhibitors: From molecular modelling to synthesis.

    PubMed

    Shah, Surmil; Savjani, Jignasa

    2016-05-15

    Rho kinase enzyme expressed in different disease conditions and involved in mediating vasoconstriction and vascular remodeling in the pathogenesis. There are two isoforms of Rho kinases, namely ROCK I and ROCK II, responsible for different physiological function due to difference in distribution, but almost similar in structure. The Rho kinase 2 belongs to AGC family and is widely distributed in brain, heart and muscles. It is responsible for contraction of vascular smooth muscles by calcium sensitization. Its defective and unwanted expression can lead to many medical conditions like multiple sclerosis, myocardial ischemia, inflammatory responses, etc. Many Rho kinase 1 and 2 inhibitors have been designed for Rho/Rho kinase pathway by use of molecular modeling studies. Most of the designed compounds have been modeled based on ROCK 1 enzyme. This article is focused on Rho kinase 2 inhibitors as there are many ways to improvise by use of Computer aided drug designing as very less quantum of research work carried out. Herein, the article highlights different stages of designing like docking, SAR and synthesis of ROCK inhibitors and recent advances. It also highlights future prospective to improve the activity. PMID:27080184

  2. Design, synthesis, and biological evaluation of substrate-competitive inhibitors of C-terminal Binding Protein (CtBP).

    PubMed

    Korwar, Sudha; Morris, Benjamin L; Parikh, Hardik I; Coover, Robert A; Doughty, Tyler W; Love, Ian M; Hilbert, Brendan J; Royer, William E; Kellogg, Glen E; Grossman, Steven R; Ellis, Keith C

    2016-06-15

    C-terminal Binding Protein (CtBP) is a transcriptional co-regulator that downregulates the expression of many tumor-suppressor genes. Utilizing a crystal structure of CtBP with its substrate 4-methylthio-2-oxobutyric acid (MTOB) and NAD(+) as a guide, we have designed, synthesized, and tested a series of small molecule inhibitors of CtBP. From our first round of compounds, we identified 2-(hydroxyimino)-3-phenylpropanoic acid as a potent CtBP inhibitor (IC50=0.24μM). A structure-activity relationship study of this compound further identified the 4-chloro- (IC50=0.18μM) and 3-chloro- (IC50=0.17μM) analogues as additional potent CtBP inhibitors. Evaluation of the hydroxyimine analogues in a short-term cell growth/viability assay showed that the 4-chloro- and 3-chloro-analogues are 2-fold and 4-fold more potent, respectively, than the MTOB control. A functional cellular assay using a CtBP-specific transcriptional readout revealed that the 4-chloro- and 3-chloro-hydroxyimine analogues were able to block CtBP transcriptional repression activity. This data suggests that substrate-competitive inhibition of CtBP dehydrogenase activity is a potential mechanism to reactivate tumor-suppressor gene expression as a therapeutic strategy for cancer. PMID:27156192

  3. Design, synthesis, and biological evaluation of pyrazinones containing novel P1 needles as inhibitors of TF/VIIa.

    PubMed

    Trujillo, John I; Huang, Horng-Chih; Neumann, William L; Mahoney, Matthew W; Long, Scott; Huang, Wei; Garland, Danny J; Kusturin, Carrie; Abbas, Zaheer; South, Michael S; Reitz, David B

    2007-08-15

    Herein is described the design, synthesis, and enzymatic activity of a series of substituted pyrazinones as inhibitors of the TF/VIIa complex. These inhibitors were designed to explore replacement and variation of the P1 amidine described previously [J. Med. Chem.2003, 46, 4050]. The P1 needle replacements were selected based upon their reduced basicity compared to the parent phenyl amidine (pKa approximately 12). A contributing factor towards the oral bioavailability of a compound is the ionization state of the compound in the intestinal tract. The desired outcome of the study was to identify an orally bioavailable TF-VIIa inhibitor. PMID:17566736

  4. Androgen synthesis inhibitors in the treatment of castration-resistant prostate cancer

    PubMed Central

    Stein, Mark N; Patel, Neal; Bershadskiy, Alexander; Sokoloff, Alisa; Singer, Eric A

    2014-01-01

    Suppression of gonadal testosterone synthesis represents the standard first line therapy for treatment of metastatic prostate cancer. However, in the majority of patients who develop castration-resistant prostate cancer (CRPC), it is possible to detect persistent activation of the androgen receptor (AR) through androgens produced in the adrenal gland or within the tumor itself. Abiraterone acetate was developed as an irreversible inhibitor of the dual functional cytochrome P450 enzyme CYP17 with activity as a 17α-hydroxylase and 17,20-lyase. CYP17 is necessary for production of nongonadal androgens from cholesterol. Regulatory approval of abiraterone in 2011, based on a phase III trial showing a significant improvement in overall survival (OS) with abiraterone and prednisone versus prednisone, represented proof of principle that targeting AR is essential for improving outcomes in men with CRPC. Inhibition of 17α-hydroxylase by abiraterone results in accumulation of upstream mineralocorticoids due to loss of cortisol-mediated suppression of pituitary adrenocorticotropic hormone (ACTH), providing a rationale for development of CYP17 inhibitors with increased specificity for 17,20-lyase (orteronel, galeterone and VT-464) that can potentially be administered without exogenous corticosteroids. In this article, we review the development of abiraterone and other CYP17 inhibitors; recent studies with abiraterone that inform our understanding of clinical parameters such as drug effects on quality-of-life, potential early predictors of response, and optimal sequencing of abiraterone with respect to other agents; and results of translational studies providing insights into resistance mechanisms to CYP17 inhibitors leading to clinical trials with drug combinations designed to prolong abiraterone benefit or restore abiraterone activity. PMID:24759590

  5. Evaluation of NAD(P)-Dependent Dehydrogenase Activities in Neutrophilic Granulocytes by the Bioluminescent Method.

    PubMed

    Savchenko, A A

    2015-09-01

    Bioluminescent method for measurements of the neutrophilic NAD(P)-dependent dehydrogenases (lactate dehydrogenase, NAD-dependent malate dehydrogenase, NADP-dependent decarboxylating malate dehydrogenase, NAD-dependent isocitrate dehydrogenase, and glucose- 6-phosphate dehydrogenase) is developed. The sensitivity of the method allows minimization of the volume of biological material for measurements to 104 neutrophils per analysis. The method is tried in patients with diffuse purulent peritonitis. Low levels of NADPH synthesis enzymes and high levels of enzymes determining the substrate flow by the Krebs cycle found in these patients can lead to attenuation of functional activity of cells. PMID:26468025

  6. Alteration in substrate specificity of horse liver alcohol dehydrogenase by an acyclic nicotinamide analog of NAD(+).

    PubMed

    Malver, Olaf; Sebastian, Mina J; Oppenheimer, Norman J

    2014-11-01

    A new, acyclic NAD-analog, acycloNAD(+) has been synthesized where the nicotinamide ribosyl moiety has been replaced by the nicotinamide (2-hydroxyethoxy)methyl moiety. The chemical properties of this analog are comparable to those of β-NAD(+) with a redox potential of -324mV and a 341nm λmax for the reduced form. Both yeast alcohol dehydrogenase (YADH) and horse liver alcohol dehydrogenase (HLADH) catalyze the reduction of acycloNAD(+) by primary alcohols. With HLADH 1-butanol has the highest Vmax at 49% that of β-NAD(+). The primary deuterium kinetic isotope effect is greater than 3 indicating a significant contribution to the rate limiting step from cleavage of the carbon-hydrogen bond. The stereochemistry of the hydride transfer in the oxidation of stereospecifically deuterium labeled n-butanol is identical to that for the reaction with β-NAD(+). In contrast to the activity toward primary alcohols there is no detectable reduction of acycloNAD(+) by secondary alcohols with HLADH although these alcohols serve as competitive inhibitors. The net effect is that acycloNAD(+) has converted horse liver ADH from a broad spectrum alcohol dehydrogenase, capable of utilizing either primary or secondary alcohols, into an exclusively primary alcohol dehydrogenase. This is the first example of an NAD analog that alters the substrate specificity of a dehydrogenase and, like site-directed mutagenesis of proteins, establishes that modifications of the coenzyme distance from the active site can be used to alter enzyme function and substrate specificity. These and other results, including the activity with α-NADH, clearly demonstrate the promiscuity of the binding interactions between dehydrogenases and the riboside phosphate of the nicotinamide moiety, thus greatly expanding the possibilities for the design of analogs and inhibitors of specific dehydrogenases. PMID:25280628

  7. NAD+ and sirtuins in aging and disease.

    PubMed

    Imai, Shin-ichiro; Guarente, Leonard

    2014-08-01

    Nicotinamide adenine dinucleotide (NAD(+)) is a classical coenzyme mediating many redox reactions. NAD(+) also plays an important role in the regulation of NAD(+)-consuming enzymes, including sirtuins, poly-ADP-ribose polymerases (PARPs), and CD38/157 ectoenzymes. NAD(+) biosynthesis, particularly mediated by nicotinamide phosphoribosyltransferase (NAMPT), and SIRT1 function together to regulate metabolism and circadian rhythm. NAD(+) levels decline during the aging process and may be an Achilles' heel, causing defects in nuclear and mitochondrial functions and resulting in many age-associated pathologies. Restoring NAD(+) by supplementing NAD(+) intermediates can dramatically ameliorate these age-associated functional defects, counteracting many diseases of aging, including neurodegenerative diseases. Thus, the combination of sirtuin activation and NAD(+) intermediate supplementation may be an effective antiaging intervention, providing hope to aging societies worldwide. PMID:24786309

  8. Larvicidal Activity of Novaluron, a Chitin Synthesis Inhibitor, Against the Housefly, Musca domestica

    PubMed Central

    Cetin, Huseyin; Erler, Fedai; Yanikoglu, Atila

    2006-01-01

    A chitin synthesis inhibitor, novaluron, was evaluated under laboratory conditions for its larvicidal activity against a field population of the housefly, Musca domestica L. (Diptera: Muscidae), by feeding and dipping methods. The concentrations used were 1, 2.5, 5, 10 and 20 mg a.i./kg in both methods. The product caused >80% larval mortality at 10 and 20 mg a.i./kg. Of the two methods, feeding was more effective for larvicidal activity at doses above 2.5 mg a.i./kg. After 72 hours, the LC50 and LC90 values were 1.66 and 8.25 mg a.i./kg, respectively, with the feeding method; and 2.72 and 17.88 mg a.i./kg, respectively, using the dipping method. The results showed that the product provided good control of housefly larvae and would greatly reduce adult emergence.

  9. The first synthesis of substituted azepanes mimicking monosaccharides: a new class of potent glycosidase inhibitors.

    PubMed

    Li, Hongqing; Blériot, Yves; Chantereau, Caroline; Mallet, Jean-Maurice; Sollogoub, Matthieu; Zhang, Yongmin; Rodríguez-García, Eliazar; Vogel, Pierre; Jiménez-Barbero, Jesús; Sinaÿ, Pierre

    2004-05-21

    The synthesis of the first examples of seven-membered ring iminoalditols, molecules displaying an extra hydroxymethyl substituent on their seven-membered ring compared to the previously reported polyhydroxylated azepanes, has been achieved from d-arabinose in 10 steps using RCM of a protected N-allyl-aminohexenitol as a key step. While the (2R,3R,4R)-2-hydroxymethyl-3,4-dihydroxy-azepane 10, a seven-membered ring analogue of fagomine, is a weak inhibitor of glycosidases, the (2R,3R,4R,5S,6S)-2-hydroxymethyl-3,4,5,6-tetrahydroxy-azepane 9 selectively inhibits green coffee bean alpha-galactosidase in the low micromolar range (Ki = 2.2 muM) despite a D-gluco relative configuration. PMID:15136805

  10. Synthesis and biological evaluation of novel FK228 analogues as potential isoform selective HDAC inhibitors.

    PubMed

    Narita, Koichi; Matsuhara, Keisuke; Itoh, Jun; Akiyama, Yui; Dan, Singo; Yamori, Takao; Ito, Akihiro; Yoshida, Minoru; Katoh, Tadashi

    2016-10-01

    Novel C4- and C7-modified FK228 analogues were efficiently synthesized in a highly convergent and unified manner. This synthesis features the amide condensation of glycine-d-cysteine-containing segments with d-valine-containing segments for the direct assembly of the corresponding seco-acids, which are key precursors of macrolactones. The HDAC inhibition assay and cell-growth inhibition analysis of the synthesized analogues revealed novel aspects of their structure-activity relationship. This study demonstrated that simple modification at the C4 and C7 side chains in FK228 is effective for improving both HDAC inhibitory activity and isoform selectivity; moreover, potent and highly isoform-selective class I HDAC1 inhibitors were identified. PMID:27318982

  11. Design, synthesis and biological evaluation of 6-pyridylmethylaminopurines as CDK inhibitors.

    PubMed

    Wilson, Stuart C; Atrash, Butrus; Barlow, Clare; Eccles, Susan; Fischer, Peter M; Hayes, Angela; Kelland, Lloyd; Jackson, Wayne; Jarman, Michael; Mirza, Amin; Moreno, Javier; Nutley, Bernard P; Raynaud, Florence I; Sheldrake, Peter; Walton, Mike; Westwood, Robert; Whittaker, Steven; Workman, Paul; McDonald, Edward

    2011-11-15

    The cyclin-dependent kinase (CDK) inhibitor seliciclib (1, CYC202) is in phase II clinical development for the treatment of cancer. Here we describe the synthesis of novel purines with greater solubility, lower metabolic clearance, and enhanced potency versus CDKs. These compounds exhibit novel selectivity profiles versus CDK isoforms. Compound αSβR-21 inhibits CDK2/cyclin E with IC(50)=30 nM, CDK7-cyclin H with IC(50)=1.3 μM, and CDK9-cyclinT with IC(50)=0.11 μM; it (CCT68127) inhibits growth of HCT116 colon cancer cells in vitro with GI(50)=0.7 μM; and shows antitumour activity when dosed p.o. at 50mg/kg to mice bearing HCT116 solid human tumour xenografts. PMID:21982796

  12. Isolation and Synthesis of a Bacterially Produced Inhibitor of Rosette Development in Choanoflagellates.

    PubMed

    Cantley, Alexandra M; Woznica, Arielle; Beemelmanns, Christine; King, Nicole; Clardy, Jon

    2016-04-01

    The choanoflagellate Salpingoeca rosetta is a microbial marine eukaryote that can switch between unicellular and multicellular states. As one of the closest living relatives of animals, this organism has become a model for understanding how multicellularity evolved in the animal lineage. Previously our laboratories isolated and synthesized a bacterially produced sulfonolipid that induces S. rosetta to form multicellular "rosettes." In this study, we report the identification of a bacterially produced inhibitor of rosettes (IOR-1) as well as the total synthesis of this molecule and all of its stereoisomers. Our results confirm the previously noted specificity and potency of rosette-modulating molecules, expand our understanding of the complex chemical ecology between choanoflagellates and rosette-inducing bacteria, and provide a synthetic probe template for conducting further mechanistic studies on the emergence of multicellularity. PMID:26998963

  13. An inhibitor of chondroitin sulfate proteoglycan synthesis promotes central nervous system remyelination

    PubMed Central

    Keough, Michael B.; Rogers, James A.; Zhang, Ping; Jensen, Samuel K.; Stephenson, Erin L.; Chen, Tieyu; Hurlbert, Mitchel G.; Lau, Lorraine W.; Rawji, Khalil S.; Plemel, Jason R.; Koch, Marcus; Ling, Chang-Chun; Yong, V. Wee

    2016-01-01

    Remyelination is the generation of new myelin sheaths after injury facilitated by processes of differentiating oligodendrocyte precursor cells (OPCs). Although this repair phenomenon occurs in lesions of multiple sclerosis patients, many lesions fail to completely remyelinate. A number of factors have been identified that contribute to remyelination failure, including the upregulated chondroitin sulfate proteoglycans (CSPGs) that comprise part of the astrogliotic scar. We show that in vitro, OPCs have dramatically reduced process outgrowth in the presence of CSPGs, and a medication library that includes a number of recently reported OPC differentiation drugs failed to rescue this inhibitory phenotype on CSPGs. We introduce a novel CSPG synthesis inhibitor to reduce CSPG content and find rescued process outgrowth from OPCs in vitro and accelerated remyelination following focal demyelination in mice. Preventing CSPG deposition into the lesion microenvironment may be a useful strategy to promote repair in multiple sclerosis and other neurological disorders. PMID:27115988

  14. Laboratory Evaluation of Flurox, a Chitin Synthesis Inhibitor, on the Termite, Microcerotermes diversus

    PubMed Central

    Habibpour, Behzad

    2010-01-01

    Microcerotermes diversus (Silvestri) (Isoptera: Termitidae) is the most economically destructive termite in structures in southwest Iran. One sustainable control strategy that usually helps to reduce subterranean termite damage in buildings, is the use of insect growth regualtors in a suitable bait matrix that are safe to the user and the environment. In the laboratory assays described here, the delayed toxicity of Flurox, a chitin synthesis inhibitor, to M. diversus was evaluated under force-feeding and choice trials. Flurox induced worker and nymph mortality and incomplete ecdysis in nymphs of M. diversus under no-choice and two-choice feeding tests. These adverse effects may cause disruption of the caste balance in M. diversus, leading to the collapse of the colony. These assays determined concentrations of Flurox that can be used in bait formulations. PMID:20569123

  15. Isolation and Synthesis of a Bacterially Produced Inhibitor of Rosette Development in Choanoflagellates

    PubMed Central

    2016-01-01

    The choanoflagellate Salpingoeca rosetta is a microbial marine eukaryote that can switch between unicellular and multicellular states. As one of the closest living relatives of animals, this organism has become a model for understanding how multicellularity evolved in the animal lineage. Previously our laboratories isolated and synthesized a bacterially produced sulfonolipid that induces S. rosetta to form multicellular “rosettes.” In this study, we report the identification of a bacterially produced inhibitor of rosettes (IOR-1) as well as the total synthesis of this molecule and all of its stereoisomers. Our results confirm the previously noted specificity and potency of rosette-modulating molecules, expand our understanding of the complex chemical ecology between choanoflagellates and rosette-inducing bacteria, and provide a synthetic probe template for conducting further mechanistic studies on the emergence of multicellularity. PMID:26998963

  16. An inhibitor of chondroitin sulfate proteoglycan synthesis promotes central nervous system remyelination.

    PubMed

    Keough, Michael B; Rogers, James A; Zhang, Ping; Jensen, Samuel K; Stephenson, Erin L; Chen, Tieyu; Hurlbert, Mitchel G; Lau, Lorraine W; Rawji, Khalil S; Plemel, Jason R; Koch, Marcus; Ling, Chang-Chun; Yong, V Wee

    2016-01-01

    Remyelination is the generation of new myelin sheaths after injury facilitated by processes of differentiating oligodendrocyte precursor cells (OPCs). Although this repair phenomenon occurs in lesions of multiple sclerosis patients, many lesions fail to completely remyelinate. A number of factors have been identified that contribute to remyelination failure, including the upregulated chondroitin sulfate proteoglycans (CSPGs) that comprise part of the astrogliotic scar. We show that in vitro, OPCs have dramatically reduced process outgrowth in the presence of CSPGs, and a medication library that includes a number of recently reported OPC differentiation drugs failed to rescue this inhibitory phenotype on CSPGs. We introduce a novel CSPG synthesis inhibitor to reduce CSPG content and find rescued process outgrowth from OPCs in vitro and accelerated remyelination following focal demyelination in mice. Preventing CSPG deposition into the lesion microenvironment may be a useful strategy to promote repair in multiple sclerosis and other neurological disorders. PMID:27115988

  17. Anti-amyloidogenic effects of glycosphingolipid synthesis inhibitors occur independently of ganglioside alterations.

    PubMed

    Noel, Anastasia; Ingrand, Sabrina; Barrier, Laurence

    2016-09-01

    Evidence has suggested that ganglioside abnormalities may be linked to the proteolytic processing of amyloid precursor protein (APP) in Alzheimer's disease (AD) and that pharmacological inhibition of ganglioside synthesis may reduce amyloid β-peptide (Aβ) production. In this study, we assessed the usefulness of two well-established glycosphingolipid (GSL) synthesis inhibitors, the synthetic ceramide analog D-PDMP (1-phenyl 2-decanoylamino-3-morpholino-1-propanol) and the iminosugar N-butyldeoxynojirimycin (NB-DNJ or miglustat), as anti-amyloidogenic drugs in a human cellular model of AD. We found that both GSL inhibitors were able to markedly inhibit Aβ production, although affecting differently the APP cleavage. Surprisingly, the L-enantiomer of PDMP, which promotes ganglioside accumulation, acted similarly to D-PDMP to inhibit Aβ production. Concurrently, both D- and L-PDMP strongly and equally reduced the levels of long-chain ceramides. Altogether, our data suggested that the anti-amyloidogenic effects of PDMP agents are independent of the altered cellular ganglioside composition, but may result, at least in part, from their ability to reduce ceramide levels. Moreover, our current study established for the first time that NB-DNJ, a drug already used as a therapeutic for Gaucher disease (a lysosomal storage disorder), was also able to reduce Aβ production in our cellular model. Therefore, our study provides novel information regarding the possibilities to target amyloidogenic processing of APP through modulation of sphingolipid metabolism and emphasizes the potential of the iminosugar NB-DNJ as a disease modifying therapy for AD. PMID:27373967

  18. Structural Basis of Inhibition of the Human NAD+ -Dependent Deacetylase SIRT5 by Suramin

    SciTech Connect

    Schuetz,A.; Min, J.; Antoshenko, T.; Wang, C.; Allali-Hassani, A.; Dong, A.; Loppnau, P.; vedadi, M.; Bochkarev, A.; et al.

    2007-01-01

    Sirtuins are NAD+-dependent protein deacetylases and are emerging as molecular targets for the development of pharmaceuticals to treat human metabolic and neurological diseases and cancer. To date, several sirtuin inhibitors and activators have been identified, but the structural mechanisms of how these compounds modulate sirtuin activity have not yet been determined. We identified suramin as a compound that binds to human SIRT5 and showed that it inhibits SIRT5 NAD+-dependent deacetylase activity with an IC50 value of 22 {mu}M. To provide insights into how sirtuin function is altered by inhibitors, we determined two crystal structures of SIRT5, one in complex with ADP-ribose, the other bound to suramin. Our structural studies provide a view of a synthetic inhibitory compound in a sirtuin active site revealing that suramin binds into the NAD+, the product, and the substrate-binding site. Finally, our structures may enable the rational design of more potent inhibitors.

  19. NAD+-Glycohydrolase Promotes Intracellular Survival of Group A Streptococcus

    PubMed Central

    Sharma, Onkar; O’Seaghdha, Maghnus; Velarde, Jorge J.; Wessels, Michael R.

    2016-01-01

    A global increase in invasive infections due to group A Streptococcus (S. pyogenes or GAS) has been observed since the 1980s, associated with emergence of a clonal group of strains of the M1T1 serotype. Among other virulence attributes, the M1T1 clone secretes NAD+-glycohydrolase (NADase). When GAS binds to epithelial cells in vitro, NADase is translocated into the cytosol in a process mediated by streptolysin O (SLO), and expression of these two toxins is associated with enhanced GAS intracellular survival. Because SLO is required for NADase translocation, it has been difficult to distinguish pathogenic effects of NADase from those of SLO. To resolve the effects of the two proteins, we made use of anthrax toxin as an alternative means to deliver NADase to host cells, independently of SLO. We developed a novel method for purification of enzymatically active NADase fused to an amino-terminal fragment of anthrax toxin lethal factor (LFn-NADase) that exploits the avid, reversible binding of NADase to its endogenous inhibitor. LFn-NADase was translocated across a synthetic lipid bilayer in vitro in the presence of anthrax toxin protective antigen in a pH-dependent manner. Exposure of human oropharyngeal keratinocytes to LFn-NADase in the presence of protective antigen resulted in cytosolic delivery of NADase activity, inhibition of protein synthesis, and cell death, whereas a similar construct of an enzymatically inactive point mutant had no effect. Anthrax toxin-mediated delivery of NADase in an amount comparable to that observed during in vitro infection with live GAS rescued the defective intracellular survival of NADase-deficient GAS and increased the survival of SLO-deficient GAS. Confocal microscopy demonstrated that delivery of LFn-NADase prevented intracellular trafficking of NADase-deficient GAS to lysosomes. We conclude that NADase mediates cytotoxicity and promotes intracellular survival of GAS in host cells. PMID:26938870

  20. NAD+-Glycohydrolase Promotes Intracellular Survival of Group A Streptococcus.

    PubMed

    Sharma, Onkar; O'Seaghdha, Maghnus; Velarde, Jorge J; Wessels, Michael R

    2016-03-01

    A global increase in invasive infections due to group A Streptococcus (S. pyogenes or GAS) has been observed since the 1980s, associated with emergence of a clonal group of strains of the M1T1 serotype. Among other virulence attributes, the M1T1 clone secretes NAD+-glycohydrolase (NADase). When GAS binds to epithelial cells in vitro, NADase is translocated into the cytosol in a process mediated by streptolysin O (SLO), and expression of these two toxins is associated with enhanced GAS intracellular survival. Because SLO is required for NADase translocation, it has been difficult to distinguish pathogenic effects of NADase from those of SLO. To resolve the effects of the two proteins, we made use of anthrax toxin as an alternative means to deliver NADase to host cells, independently of SLO. We developed a novel method for purification of enzymatically active NADase fused to an amino-terminal fragment of anthrax toxin lethal factor (LFn-NADase) that exploits the avid, reversible binding of NADase to its endogenous inhibitor. LFn-NADase was translocated across a synthetic lipid bilayer in vitro in the presence of anthrax toxin protective antigen in a pH-dependent manner. Exposure of human oropharyngeal keratinocytes to LFn-NADase in the presence of protective antigen resulted in cytosolic delivery of NADase activity, inhibition of protein synthesis, and cell death, whereas a similar construct of an enzymatically inactive point mutant had no effect. Anthrax toxin-mediated delivery of NADase in an amount comparable to that observed during in vitro infection with live GAS rescued the defective intracellular survival of NADase-deficient GAS and increased the survival of SLO-deficient GAS. Confocal microscopy demonstrated that delivery of LFn-NADase prevented intracellular trafficking of NADase-deficient GAS to lysosomes. We conclude that NADase mediates cytotoxicity and promotes intracellular survival of GAS in host cells. PMID:26938870

  1. Synthesis of a Poly-hydroxypyrolidine-Based inhibitor of Mycobacterium tuberculosis GlgE

    PubMed Central

    2015-01-01

    Long treatment times, poor drug compliance, and natural selection during treatment of Mycobacterium tuberculosis (Mtb) have given rise to extensively drug-resistant tuberculosis (XDR-TB). As a result, there is a need to identify new antituberculosis drug targets. Mtb GlgE is a maltosyl transferase involved in α-glucan biosynthesis. Mutation of GlgE in Mtb increases the concentration of maltose-1-phosphate (M1P), one substrate for GlgE, causing rapid cell death. We have designed 2,5-dideoxy-3-O-α-d-glucopyranosyl-2,5-imino-d-mannitol (9) to act as an inhibitor of GlgE. Compound 9 was synthesized using a convergent synthesis by coupling thioglycosyl donor 14 and 5-azido-3-O-benzyl-5-deoxy-1,2-O-isopropylidene-β-d-fructopyranose (23) to form disaccharide 24. A reduction and intramolecular reductive amination transformed the intermediate disaccharide 24 to the desired pyrolidine 9. Compound 9 inhibited both Mtb GlgE and a variant of Streptomyces coelicolor (Sco) GlgEI with Ki = 237 ± 27 μM and Ki = 102 ± 7.52 μM, respectively. The results confirm that a Sco GlgE-V279S variant can be used as a model for Mtb GlgE. In conclusion, we designed a lead transition state inhibitor of GlgE, which will be instrumental in further elucidation of the enzymatic mechanism of Mtb GlgE. PMID:25137149

  2. The DNA methylation inhibitor 5-azacytidine decreases melanin synthesis by inhibiting CREB phosphorylation.

    PubMed

    Shin, Jun Seob; Jeong, Hyo-Soon; Kim, Myo-Kyoung; Yun, Hye-Young; Baek, Kwang Jin; Kwon, Nyoun Soo; Kim, Dong-Seok

    2015-10-01

    Here we examined the effects of a DNA methylation inhibitor, 5-azacytidine, on melanogenesis in Mel-Ab cells. We found that 5-azacytidine decreased the melanin content and tyrosinase activity in these cells in a dose-dependent manner; importantly, 5-azacytidine was not cytotoxic at the concentrations used in these experiments. On the other hand, 5-azacytidine did not affect tyrosinase activity in a cell-free system, indicating that 5-azacytidine is not a direct tyrosinase inhibitor. Instead, 5-azacytidine decreased the protein levels of microphthalmia-associated transcription factor (MITF) and tyrosinase. Thus, we investigated the effects of 5-azacytidine on signal transduction pathways related to melanogenesis. However, 5-azacytidine did not have any effect on either Akt or glycogen synthase kinase 3β (GSK3β) phosphorylation. The phosphorylation of cAMP response element-binding protein (CREB) is well known to regulate MITF expression, thereby also regulating tyrosinase expression. We found that 5-azacytidine decreased the phosphorylation of CREB. Therefore, we propose that 5-azacytidine may decrease melanin synthesis by downregulating MITF and tyrosinase via CREB inactivation. PMID:26601420

  3. Synthesis and characterization of phosphocitric acid, a potent inhibitor of hydroxylapatite crystal growth.

    PubMed

    Tew, W P; Mahle, C; Benavides, J; Howard, J E; Lehninger, A L

    1980-04-29

    Human urine and extracts of rat liver mitochondria contain apparently identical agents capable of inhibiting the precipitation or crystallization of calcium phosphate. Its general properties, as well as 1H NMR and mass spectra, have suggested that the agent is phosphocitric acid. This paper reports the synthesis of phosphocitric acid via the phosphorylation of triethyl citrate with o-phenylene phosphochloridate, hydrogenolysis of the product to yield triethyl phosphocitrate, hydrolytic removal of the blocking ethyl groups and also chromatographic purification. An enzymatic assay of phosphocitrate is described. Synthetic phosphocitrate was found to be an exceedingly potent inhibitor of the growth of hydroxylapatite seed crystals in a medium supersaturated with respect to Ca2+ and phosphate. Comparative assays showed phosphocitrate to be much more potent than the most active precipitation-crystallization inhibitors previously reported, which include pyrophosphate and ATP. 14C-Labeled phosphocitrate was bound very tightly to hydroxylapatite crystals. Such binding appeared to be essential for its inhibitory activity on crystal growth. Citrate added before but not after, phosphocitrate greatly enhanced the inhibitory potency of the latter. This enhancement effect was not given by other tricarboxylic acids. The monoethyl ester of phosphocitrate had no inhibitory effect on hydroxylapatite crystal growth. PMID:7378389

  4. Design and synthesis of potent and multifunctional aldose reductase inhibitors based on quinoxalinones.

    PubMed

    Qin, Xiangyu; Hao, Xin; Han, Hui; Zhu, Shaojuan; Yang, Yanchun; Wu, Bobin; Hussain, Saghir; Parveen, Shagufta; Jing, Chaojun; Ma, Bing; Zhu, Changjin

    2015-02-12

    Quinoxalin-2(1H)-one based design and synthesis produced several series of aldose reductase (ALR2) inhibitor candidates. In particular, phenolic structure was installed in the compounds for the combination of antioxidant activity and strengthening the ability to fight against diabetic complications. Most of the series 6 showed potent and selective effects on ALR2 inhibition with IC50 values in the range of 0.032-0.468 μM, and 2-(3-(2,4-dihydroxyphenyl)-7-fluoro-2-oxoquinoxalin-1(2H)-yl)acetic acid (6e) was the most active. More significantly, most of the series 8 revealed not only good activity in the ALR2 inhibition but also potent antioxidant activity, and 2-(3-(3-methoxy-4-hydroxystyryl)-2-oxoquinoxalin-1(2H)-yl)acetic acid (8d) was even as strong as the well-known antioxidant Trolox at a concentration of 100 μM, verifying the C3 p-hydroxystyryl side chain as the key structure for alleviating oxidative stress. These results therefore suggest an achievement of multifunctional ALR2 inhibitors having both potency for ALR2 inhibition and as antioxidants. PMID:25602762

  5. Stimulators and inhibitors of lymphocyte DNA synthesis in supernatants from human lymphoid cell lines.

    PubMed

    Vesole, D H; Goust, J M; Fett, J W; Fudenberg, H H

    1979-09-01

    Some T and B lymphoid cell lines (LCL) were found to secrete into their supernatants a substance able to stimulate lymphocyte proliferation. This substance produced an increase in [3H]thymidine uptake by mononuclear cells when added to unstimulated cultures (mitogenic effect) or when added to cultures stimulated with phytohemagglutinin (PHA) or pokeweed mitogen (PWM) (potentiating effect). When complete supernatants were used, the potentiating effect was sometimes masked by an inhibitor of DNA synthesis. Fractionation on Sephadex G-100 separated these two activities. The stimulatory substance eluted at a m.w. range of 15,000 to 30,000, and the inhibitor eluted with the albumin peak. B cells with or without monocytes were the most sensitive to the mitogenic effect, whereas T cells were unaffected. Responses to PHA and PWM were potentiated when T cells were present, but the maximum effect was observed when the proportion of T cells was less than 50%. The stimulatory material may be similar to lymphocyte mitogenic factor and may function as a T cell-replacing factor in B cell stimulation. PMID:313950

  6. Synthesis and biological activities of vitamin D-like inhibitors of CYP24 hydroxylase

    PubMed Central

    Chiellini, Grazia; Rapposelli, Simona; Zhu, Jinge; Massarelli, Ilaria; Saraceno, Marilena; Bianucci, Anna Maria; Plum, Lori A.; Clagett-Dame, Margaret; DeLuca, Hector F.

    2012-01-01

    Selective inhibitors of CYP24A1 represent an important synthetic target in a search for novel vitamin D compounds of therapeutic value. In the present work, we show the synthesis and biological properties of two novel side chain modified 2-methylene-19-nor-1,25(OH)2D3 analogs, the 22-imidazole-1-yl derivative 2 (VIMI) and the 25-N-cyclopropylamine compound 3 (CPA1), which were efficiently prepared in convergent syntheses utilizing the Lythgoe type Horner–Wittig olefination reaction. When tested in a cell-free assay, both compounds were found to be potent competitive inhibitors of CYP24A1, with the cyclopropylamine analog 3 exhibiting an 80–1 selective inhibition of CYP24A1 over CYP27B1. Addition of 3 to a mouse osteoblast culture sustained the level of 1,25(OH)2D3, further demonstrating its effectiveness in CYP24A1 inhibition. Importantly, the in vitro effects on human promyeloid leukemia (HL-60) cell differentiation by 3 were nearly identical to those of 1,25(OH)2D3 and in vivo the compound showed low calcemic activity. Finally, the results of preliminary theoretical studies provide useful insights to rationalize the ability of analog 3 to selectively inhibit the cytochrome P450 isoform CYP24A1. PMID:22133546

  7. The Use of Ascorbate as an Oxidation Inhibitor in Prebiotic Amino Acid Synthesis: A Cautionary Note

    NASA Astrophysics Data System (ADS)

    Kuwahara, Hideharu; Eto, Midori; Kawamoto, Yukinori; Kurihara, Hironari; Kaneko, Takeo; Obayashi, Yumiko; Kobayashi, Kensei

    2012-12-01

    It is generally thought that the terrestrial atmosphere at the time of the origin of life was CO2-rich and that organic compounds such as amino acids would not have been efficiently formed abiotically under such conditions. It has been pointed out, however, that the previously reported low yields of amino acids may have been partially due to oxidation by nitrite/nitrate during acid hydrolysis. Specifically, the yield of amino acids was found to have increased significantly (by a factor of several hundred) after acid hydrolysis with ascorbic acid as an oxidation inhibitor. However, it has not been shown that CO2 was the carbon source for the formation of the amino acids detected after acid hydrolysis with ascorbic acid. We therefore reinvestigated the prebiotic synthesis of amino acids in a CO2-rich atmosphere using an isotope labeling experiment. Herein, we report that ascorbic acid does not behave as an appropriate oxidation inhibitor, because it contributes amino acid contaminants as a consequence of its reactions with the nitrogen containing species and formic acid produced during the spark discharge experiment. Thus, amino acids are not efficiently formed from a CO2-rich atmosphere under the conditions studied.

  8. Design, Synthesis, and Biological Evaluation of 14-Substituted Aromathecins as Topoisomerase I Inhibitors

    PubMed Central

    Cinelli, Maris A.; Morrell, Andrew; Dexheimer, Thomas S.; Scher, Evan S.; Pommier, Yves; Cushman, Mark

    2008-01-01

    The aromathecin or “rosettacin” class of topoisomerase I (top1) inhibitors is effectively a “composite” of the natural products camptothecin and luotonin A and the synthetic indenoisoquinolines. The aromathecins have aroused considerable interest following the isolation and total synthesis of 22-hydroxyacuminatine, a rare cytotoxic natural product containing the 12H-5,11a-diazadibenzo[b,h]fluoren-11-one system. We have developed two novel syntheses of this system and prepared a series of 14-substituted aromathecins as novel antiproliferative topoisomerase I poisons. These inhibitors are proposed to act via an intercalation and “poisoning” mechanism identical to camptothecin and the indenoisoquinolines. Many of these compounds possess greater antiproliferative activity and anti-top1 activity than the parent unsubstituted compound (rosettacin) and previously synthesized aromathecins, as well as greater top1 inhibitory activity than 22-hydroxyacuminatine. In addition to potentially aiding solubility and localization to the DNA–enzyme complex, nitrogenous substituents located at the 14-position of the aromathecin system have been proposed to project into the major groove of the top1–DNA complex and hydrogen bond to major-groove amino acids, thereby stabilizing the ternary complex. PMID:18630891

  9. New MKLP-2 inhibitors in the paprotrain series: Design, synthesis and biological evaluations.

    PubMed

    Labrière, Christophe; Talapatra, Sandeep K; Thoret, Sylviane; Bougeret, Cécile; Kozielski, Frank; Guillou, Catherine

    2016-02-15

    Members of the kinesin superfamily are involved in key functions during intracellular transport and cell division. Their involvement in cell division makes certain kinesins potential targets for drug development in cancer chemotherapy. The two most advanced kinesin targets are Eg5 and CENP-E with inhibitors in clinical trials. Other mitotic kinesins are also being investigated for their potential as prospective drug targets. One recently identified novel potential cancer therapeutic target is the Mitotic kinesin-like protein 2 (MKLP-2), a member of the kinesin-6 family, which plays an essential role during cytokinesis. Previous studies have shown that inhibition of MKLP-2 leads to binucleated cells due to failure of cytokinesis. We have previously identified compound 1 (paprotrain) as the first selective inhibitor of MKLP-2. Herein we describe the synthesis and biological evaluation of new analogs of 1. Our structure-activity relationship (SAR) study reveals the key chemical elements in the paprotrain family necessary for MKLP-2 inhibition. We have successfully identified one MKLP-2 inhibitor 9a that is more potent than paprotrain. In addition, in vitro analysis of a panel of kinesins revealed that this compound is selective for MKLP-2 compared to other kinesins tested and also does not have an effect on microtubule dynamics. Upon testing in different cancer cell lines, we find that the more potent paprotrain analog is also more active than paprotrain in 10 different cancer cell lines. Increased selectivity and higher potency is therefore a step forward toward establishing MKLP-2 as a potential cancer drug target. PMID:26778612

  10. Secondary NAD+ deficiency in the inherited defect of glutamine synthetase.

    PubMed

    Hu, Liyan; Ibrahim, Khalid; Stucki, Martin; Frapolli, Michele; Shahbeck, Noora; Chaudhry, Farrukh A; Görg, Boris; Häussinger, Dieter; Penberthy, W Todd; Ben-Omran, Tawfeg; Häberle, Johannes

    2015-11-01

    Glutamine synthetase (GS) deficiency is an ultra-rare inborn error of amino acid metabolism that has been described in only three patients so far. The disease is characterized by neonatal onset of severe encephalopathy, low levels of glutamine in blood and cerebrospinal fluid, chronic moderate hyperammonemia, and an overall poor prognosis in the absence of an effective treatment. Recently, enteral glutamine supplementation was shown to be a safe and effective therapy for this disease but there are no data available on the long-term effects of this intervention. The amino acid glutamine, severely lacking in this disorder, is central to many metabolic pathways in the human organism and is involved in the synthesis of nicotinamide adenine dinucleotide (NAD(+)) starting from tryptophan or niacin as nicotinate, but not nicotinamide. Using fibroblasts, leukocytes, and immortalized peripheral blood stem cells (PBSC) from a patient carrying a GLUL gene point mutation associated with impaired GS activity, we tested whether glutamine deficiency in this patient results in NAD(+) depletion and whether it can be rescued by supplementation with glutamine, nicotinamide or nicotinate. The present study shows that congenital GS deficiency is associated with NAD(+) depletion in fibroblasts, leukocytes and PBSC, which may contribute to the severe clinical phenotype of the disease. Furthermore, it shows that NAD(+) depletion can be rescued by nicotinamide supplementation in fibroblasts and leukocytes, which may open up potential therapeutic options for the treatment of this disorder. PMID:25896882

  11. The NADPH oxidase inhibitor apocynin induces nitric oxide synthesis via oxidative stress

    SciTech Connect

    Riganti, Chiara

    2008-05-01

    We have recently shown that apocynin elicits an oxidative stress in N11 mouse glial cells and other cell types. Here we report that apocynin increased the accumulation of nitrite, the stable derivative of nitric oxide (NO), in the extracellular medium of N11 cell cultures, and the NO synthase (NOS) activity in cell lysates. The increased synthesis of NO was associated with increased expression of inducible NOS (iNOS) mRNA, increased nuclear translocation of the redox-sensitive transcription factor NF-{kappa}B and decreased intracellular level of its inhibitor IkB{alpha}. These effects, accompanied by increased production of H{sub 2}O{sub 2}, were very similar to those observed after incubation with bacterial lipopolysaccharide (LPS) and were inhibited by catalase. These results suggest that apocynin, similarly to LPS, induces increased NO synthesis by eliciting a generation of reactive oxygen species (ROS), which in turn causes NF-{kappa}B activation and increased expression of iNOS. Therefore, the increased bioavailability of NO reported in the literature after in vivo or in vitro treatments with apocynin might depend, at least partly, on the drug-elicited induction of iNOS, and not only on the inhibition of NADPH oxidase and the subsequent decreased scavenging of NO by oxidase-derived ROS, as it is often supposed.

  12. The synthesis of lysylfluoromethanes and their properties as inhibitors of trypsin, plasmin and cathepsin B.

    PubMed Central

    Angliker, H; Wikstrom, P; Rauber, P; Shaw, E

    1987-01-01

    The synthesis of two lysylfluoromethanes is described by an extension of the synthesis method of Rauber, Angliker, Walker & Shaw [(1986) Biochem. J. 239, 633-640]. Ala-Phe-Lys-CH2F was found to be an active-centre-directed inhibitor of plasmin and trypsin, as is the corresponding chloromethane. However, the rate of covalent-bond formation is about an order of magnitude lower at 25 degrees C for the fluoro derivative. It was, in addition, an extremely effective inactivator of cathepsin B at pH 5.4 and 6.4. The chemical reactivity of fluoromethanes was compared with that of chloromethanes as alkylators of GSH. At pH 7.4 and 37 degrees C, a fluoromethane has 1/500th the reactivity of a chloromethane. A comparison of the rates of reaction of the fluoromethane with cathepsin B and with GSH at pH 6.4 revealed an enhancement of 10(8)-fold for the alkylation of the enzyme, ascribable largely to a proximity effect. PMID:2954536

  13. Tributyltin induces mitochondrial fission through NAD-IDH dependent mitofusin degradation in human embryonic carcinoma cells.

    PubMed

    Yamada, Shigeru; Kotake, Yaichiro; Nakano, Mizuho; Sekino, Yuko; Kanda, Yasunari

    2015-08-01

    Organotin compounds, such as tributyltin (TBT), are well-known endocrine disruptors. TBT acts at the nanomolar level through genomic pathways via the peroxisome proliferator activated receptor (PPAR)/retinoid X receptor (RXR). We recently reported that TBT inhibits cell growth and the ATP content in the human embryonic carcinoma cell line NT2/D1 via a non-genomic pathway involving NAD(+)-dependent isocitrate dehydrogenase (NAD-IDH), which metabolizes isocitrate to α-ketoglutarate. However, the molecular mechanisms by which NAD-IDH mediates TBT toxicity remain unclear. In the present study, we evaluated the effects of TBT on mitochondrial NAD-IDH and energy production. Staining with MitoTracker revealed that nanomolar TBT levels induced mitochondrial fragmentation. TBT also degraded the mitochondrial fusion proteins, mitofusins 1 and 2. Interestingly, apigenin, an inhibitor of NAD-IDH, mimicked the effects of TBT. Incubation with an α-ketoglutarate analogue partially recovered TBT-induced mitochondrial dysfunction, supporting the involvement of NAD-IDH. Our data suggest that nanomolar TBT levels impair mitochondrial quality control via NAD-IDH in NT2/D1 cells. Thus, mitochondrial function in embryonic cells could be used to assess cytotoxicity associated with metal exposure. PMID:25909344

  14. Regulation of the Nampt-mediated NAD salvage pathway and its therapeutic implications in pancreatic cancer.

    PubMed

    Ju, Huai-Qiang; Zhuang, Zhuo-Nan; Li, Hao; Tian, Tian; Lu, Yun-Xin; Fan, Xiao-Qiang; Zhou, Hai-Jun; Mo, Hai-Yu; Sheng, Hui; Chiao, Paul J; Xu, Rui-Hua

    2016-08-28

    Nicotinamide adenine dinucleotide (NAD) is a crucial cofactor for the redox reactions in the metabolic pathways of cancer cells that have elevated aerobic glycolysis (Warburg effect). Cancer cells are reported to rely on NAD recycling and inhibition of the NAD salvage pathway causes metabolic collapse and cell death. However, the underlying regulatory mechanisms and clinical implications for the NAD salvage pathway in pancreatic ductal adenocarcinoma (PDAC) remain unclear. This study showed that the expression of Nampt, the rate-limiting enzyme of the NAD salvage pathway, was significantly increased in PDAC cells and PDAC tissues. Additionally, inhibition of Nampt impaired tumor growth in vitro and tumorigenesis in vivo, which was accompanied by a decreased cellular NAD level and glycolytic activity. Mechanistically, the Nampt expression was independent of Kras and p16 status, but it was directly regulated by miR-206, which was inversely correlated with the expression of Nampt in PDAC tissues. Importantly, pharmacological inhibition of Nampt by its inhibitor, FK866, significantly enhanced the antitumor activity of gemcitabine in PDAC cells and in orthotopic xenograft mouse models. In conclusion, the present study revealed a novel regulatory mechanism for Nampt in PDAC and suggested that Nampt inhibition may override gemcitabine resistance by decreasing the NAD level and suppressing glycolytic activity, warranting further clinical investigation for pancreatic cancer treatment. PMID:27233476

  15. Modulation of the equilibrative nucleoside transporter by inhibitors of DNA synthesis.

    PubMed Central

    Pressacco, J.; Wiley, J. S.; Jamieson, G. P.; Erlichman, C.; Hedley, D. W.

    1995-01-01

    Expression of the equilibrative, S-(p-nitrobenzyl)-6-thioinosine (NBMPR)-sensitive nucleoside transporter (es), a component of the nucleoside salvage pathway, was measured during unperturbed growth and following exposure to various antimetabolites at growth-inhibitory concentrations. The probe 5-(SAENTA-x8)-fluorescein is a highly modified form of adenosine incorporating a fluorescein molecule. It binds. with high affinity and specificity to the (es) nucleoside transporter at a 1:1 stoichiometry, allowing reliable estimates of es expression by flow cytometry. Using a dual labelling technique which combined the vital DNA dye Hoechst-33342 and 5-(SAENTA-x8)-fluorescein, we found that surface expression of es approximately doubled between G1 and G2 + M phases of the cell cycle. To address the question of whether es expression could be modulated in cells exposed to drugs which inhibit de novo synthesis of nucleotides, cells were exposed to antimetabolite drugs having different modes of action. Hydroxyurea and 5-fluorouracil (5-FU), which inhibit the de novo synthesis of DNA precursors, produced increases in the expression of es. In contrast, cytosine arabinoside (ara-C) and aphidicolin, which directly inhibit DNA synthesis, produced no significant increase in es expression. Thymidine (TdR), which is an allosteric inhibitor of ribonucleotide reductase that depletes dATP, dCTP and dGTP pools while repleting the dTTP pool, had no significant effect on es expression. These data suggest that surface expression of the es nucleoside transporter is regulated by a mechanism which is sensitive to the supply of deoxynucleotides. Because 5-FU (which specifically depletes dTTP pools) causes a large increase in expression whereas TdR (which depletes all precursors except dTTP) does not, this mechanism might be particularly sensitive to dTTP pools. PMID:7547244

  16. Rational Design Synthesis and Evaluation of New Selective Inhibitors of Microbial Class II (Zinc Dependent) Fructose Bis-phosphate Aldolases

    SciTech Connect

    R Daher; M Coincon; M Fonvielle; P Gest; M Guerin; M Jackson; J Sygusch; M Therisod

    2011-12-31

    We report the synthesis and biochemical evaluation of several selective inhibitors of class II (zinc dependent) fructose bis-phosphate aldolases (Fba). The products were designed as transition-state analogues of the catalyzed reaction, structurally related to the substrate fructose bis-phosphate (or sedoheptulose bis-phosphate) and based on an N-substituted hydroxamic acid, as a chelator of the zinc ion present in active site. The compounds synthesized were tested on class II Fbas from various pathogenic microorganisms and, by comparison, on a mammalian class I Fba. The best inhibitor shows Ki against class II Fbas from various pathogens in the nM range, with very high selectivity (up to 105). Structural analyses of inhibitors in complex with aldolases rationalize and corroborate the enzymatic kinetics results. These inhibitors represent lead compounds for the preparation of new synthetic antibiotics, notably for tuberculosis prophylaxis.

  17. Acquisition of a Potent and Selective TC-PTP Inhibitor via a Stepwise Fluorophoretagged Combinatorial Synthesis and Screening Strategy

    PubMed Central

    Zhang, Sheng; Chen, Lan; Luo, Yong; Gunawan, Andrea; Lawrence, David S.; Zhang, Zhong-Yin

    2009-01-01

    Protein tyrosine phosphatases (PTPs) regulate a broad range of cellular processes including proliferation, differentiation, migration, apoptosis, and the immune responses. Dysfunction of PTP activity is associated with cancers, metabolic syndromes, and autoimmune disorders. Consequently, small molecule PTP inhibitors should not only serve as powerful tools to delineate the physiological roles of these enzymes in vivo, but also as lead compounds for therapeutic development. We describe a novel stepwise fluorophore-tagged combinatorial library synthesis and competitive fluorescence polarization screening approach that transforms a weak and general PTP inhibitor into an extremely potent and selective TC-PTP inhibitor with highly efficacious cellular activity. The result serves as a proof-of-concept in PTP inhibitor development, as it demonstrates the feasibility of acquiring potent, yet highly selective, cell permeable PTP inhibitory agents. Given the general nature of the approach, this strategy should be applicable to other PTP targets. PMID:19737019

  18. Design, synthesis, molecular docking studies and in vitro screening of ethyl 4-(3-benzoylthioureido) benzoates as urease inhibitors.

    PubMed

    Saeed, Aamer; Khan, Muhammad Siraj; Rafique, Hummera; Shahid, Mohammad; Iqbal, Jamshed

    2014-02-01

    Thioureas are exceptionally versatile building blocks towards the synthesis of wide variety of heterocyclic systems, which also possess extensive range of pharmacological activities. The substituted benzoic acids were converted into corresponding acid chlorides, these acid chlorides were then treated with potassium thiocyanate in acetone and then the reaction mixture was refluxed for 1-2h afford ethyl 4-(3-benzoylthioureido)benzoates thioureas in good yields. All the newly synthesized compounds were evaluated for their urease inhibitory activities and were found to be potent inhibitors of urease enzyme. Compounds 1f and 1g were identified as the most potent urease inhibitors (IC50 0.21 and 0.13 μM, respectively), and was 100-fold more potent than the standard inhibitors. Further molecular docking studies were carried out using the crystal structure of urease to find out the binding mode of the inhibitors with the enzyme. PMID:24269986

  19. Regulatory Mechanisms in Anthocyanin Biosynthesis in First Internodes of Sorghum vulgare: Effect of Presumed Inhibitors of Protein Synthesis 1

    PubMed Central

    Stafford, Helen A.

    1966-01-01

    There was a 6 to 24-hour lag in the production of anthocyanins in the light after excision of 4-day-old etiolated internodes of Sorghum vulgare variety Wheatland milo. In internodes infiltrated with water, apigeninidin was formed first at 12 to 24 hours and continued to be produced slowly. Luteolinidin was formed slightly later, but its formation rapidly exceeded that of apigeninidin. Cyanidin was the last type to be produced, but equaled the amounts of luteolinidin by 4 days. In noninfiltrated internodes, the production of cyanidin was greatly accelerated, beginning at about 6 hours. Data from experiments with inhibitors that presumably affect protein synthesis at different loci indicated that protein synthesis was necessary for maximum production of all 3 anthocyanins, but that different steps were rate limiting. Light independent synthesis of apigeninidin and luteolinidin was inhibited by chloramphenicol and l-ethionine but not by actinomycin D and 8-azaguanine. However, the synthesis of these 2 anthocyanins was not inhibited by puromycin, but was sometimes stimulated. The light-induced synthesis of cyanidin was inhibited by actinomycin, azaguanine, chloramphenicol and ethionine. Actinomycin no longer was inhibitory if added after incubation for 6 hours in air. All inhibitors were capable of inhibiting to various degrees either the incorporation of 14C-uracil into RNA or 14C-leucine into protein. The inhibitor data suggest that the light insensitive synthesis of apigeninidin and luteolinidin may be controlled by enzyme synthesis at the level of ribosomes via stable mRNA, while the light-induced production of cyanidin is dependent initially on the production of mRNA. The latter hypothesis is similar to that recently proposed by Lange and Mohr for a cyanidin produced in Sinapis seedlings. PMID:16656361

  20. Intracellular NAD+ levels are associated with LPS-induced TNF-α release in pro-inflammatory macrophages

    PubMed Central

    Al-Shabany, Abbas Jawad; Moody, Alan John; Foey, Andrew David; Billington, Richard Andrew

    2016-01-01

    Metabolism and immune responses have been shown to be closely linked and as our understanding increases, so do the intricacies of the level of linkage. NAD+ has previously been shown to regulate tumour necrosis factor-α (TNF-α) synthesis and TNF-α has been shown to regulate NAD+ homoeostasis providing a link between a pro-inflammatory response and redox status. In the present study, we have used THP-1 differentiation into pro- (M1-like) and anti- (M2-like) inflammatory macrophage subset models to investigate this link further. Pro- and anti-inflammatory macrophages showed different resting NAD+ levels and expression levels of NAD+ homoeostasis enzymes. Challenge with bacterial lipopolysaccharide, a pro-inflammatory stimulus for macrophages, caused a large, biphasic and transient increase in NAD+ levels in pro- but not anti-inflammatory macrophages that were correlated with TNF-α release and inhibition of certain NAD+ synthesis pathways blocked TNF-α release. Lipopolysaccharide stimulation also caused changes in mRNA levels of some NAD+ homoeostasis enzymes in M1-like cells. Surprisingly, despite M2-like cells not releasing TNF-α or changing NAD+ levels in response to lipopolysaccharide, they showed similar mRNA changes compared with M1-like cells. These data further strengthen the link between pro-inflammatory responses in macrophages and NAD+. The agonist-induced rise in NAD+ shows striking parallels to well-known second messengers and raises the possibility that NAD+ is acting in a similar manner in this model. PMID:26764408

  1. NAD+ metabolism in health and disease.

    PubMed

    Belenky, Peter; Bogan, Katrina L; Brenner, Charles

    2007-01-01

    Nicotinamide adenine dinucleotide (NAD(+)) is both a coenzyme for hydride-transfer enzymes and a substrate for NAD(+)-consuming enzymes, which include ADP-ribose transferases, poly(ADP-ribose) polymerases, cADP-ribose synthases and sirtuins. Recent results establish protective roles for NAD(+) that might be applicable therapeutically to prevent neurodegenerative conditions and to fight Candida glabrata infection. In addition, the contribution that NAD(+) metabolism makes to lifespan extension in model systems indicates that therapies to boost NAD(+) might promote some of the beneficial effects of calorie restriction. Nicotinamide riboside, the recently discovered nucleoside precursor of NAD(+) in eukaryotic systems, might have advantages as a therapy to elevate NAD(+) without inhibiting sirtuins, which is associated with high-dose nicotinamide, or incurring the unpleasant side-effects of high-dose nicotinic acid. PMID:17161604

  2. Induction of α-Amylase Inhibitor Synthesis in Barley Embryos and Young Seedlings by Abscisic Acid and Dehydration Stress 1

    PubMed Central

    Robertson, Masumi; Walker-Simmons, M.; Munro, Doug; Hill, Robert D.

    1989-01-01

    An endogenous α-amylase inhibitor was found to be synthesized in embryos of developing barley grain (Hordeum vulgare cv Bonanza). Accumulation of this protein occurred late in development (stage IV), at the same time that endogenous abscisic acid (ABA) showed a large increase. The inhibitor could be induced up to 23-fold in isolated immature embryos (stage III) by culture in ABA. Precocious germination was also blocked in stage III embryos by ABA. Dehydration stress on the isolated immature embryos also induced higher levels of the inhibitor and ABA. An even greater response to dehydration stress was observed in young seedlings, where inhibitor content increased 20-fold and ABA increased 80-fold during water stress. The high degree of correlation between ABA and inhibitor contents in in situ embryos, dehydrated embryos and young seedlings, as well as the increase in inhibitor caused by exogenously applied ABA to isolated embryos, suggests that increased α-amylase inhibitor synthesis in response to dehydration stress is mediated by ABA. PMID:16667035

  3. Identification of a pentatricopeptide repeat protein implicated in splicing of intron 1 of mitochondrial nad7 transcripts.

    PubMed

    Koprivova, Anna; des Francs-Small, Catherine Colas; Calder, Grant; Mugford, Sam T; Tanz, Sandra; Lee, Bok-Rye; Zechmann, Bernd; Small, Ian; Kopriva, Stanislav

    2010-10-15

    Splicing of plant organellar transcripts is facilitated by members of a large protein family, the pentatricopeptide repeat proteins. We have identified a pentatricopeptide repeat protein in a genetic screen for mutants resistant to inhibition of root growth by buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis and consequently named BIR6 (BSO-insensitive roots 6). BIR6 is involved in splicing of intron 1 of the mitochondrial nad7 transcript. Loss-of-function mutations in BIR6 result in a strongly reduced accumulation of fully processed nad7 transcript. This affects assembly of Complex I and results in moderate growth retardation. In agreement with disruption of Complex I function, the genes encoding alternative NADH oxidizing enzymes are induced in the mutant, and the mutant plants are less sensitive to mannitol and salt stress. Mutation in the BIR6 gene allowed normal root growth in presence of BSO and strongly attenuated depletion of glutathione content at these conditions. The same phenotype was observed with other mutants affected in function of Complex I, thus reinforcing the importance of Complex I function for cellular redox homeostasis. PMID:20682769

  4. Identification of a Pentatricopeptide Repeat Protein Implicated in Splicing of Intron 1 of Mitochondrial nad7 Transcripts

    PubMed Central

    Koprivova, Anna; des Francs-Small, Catherine Colas; Calder, Grant; Mugford, Sam T.; Tanz, Sandra; Lee, Bok-Rye; Zechmann, Bernd; Small, Ian; Kopriva, Stanislav

    2010-01-01

    Splicing of plant organellar transcripts is facilitated by members of a large protein family, the pentatricopeptide repeat proteins. We have identified a pentatricopeptide repeat protein in a genetic screen for mutants resistant to inhibition of root growth by buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis and consequently named BIR6 (BSO-insensitive roots 6). BIR6 is involved in splicing of intron 1 of the mitochondrial nad7 transcript. Loss-of-function mutations in BIR6 result in a strongly reduced accumulation of fully processed nad7 transcript. This affects assembly of Complex I and results in moderate growth retardation. In agreement with disruption of Complex I function, the genes encoding alternative NADH oxidizing enzymes are induced in the mutant, and the mutant plants are less sensitive to mannitol and salt stress. Mutation in the BIR6 gene allowed normal root growth in presence of BSO and strongly attenuated depletion of glutathione content at these conditions. The same phenotype was observed with other mutants affected in function of Complex I, thus reinforcing the importance of Complex I function for cellular redox homeostasis. PMID:20682769

  5. Nicotinamide mononucleotide synthetase is the key enzyme for an alternative route of NAD biosynthesis in Francisella tularensis

    PubMed Central

    Sorci, Leonardo; Martynowski, Dariusz; Rodionov, Dmitry A.; Eyobo, Yvonne; Zogaj, Xhavit; Klose, Karl E.; Nikolaev, Evgeni V.; Magni, Giulio; Zhang, Hong; Osterman, Andrei L.

    2009-01-01

    Enzymes involved in the last 2 steps of nicotinamide adenine dinucleotide (NAD) cofactor biosynthesis, which catalyze the adenylylation of the nicotinic acid mononucleotide (NaMN) precursor to nicotinic acid dinucleotide (NaAD) followed by its amidation to NAD, constitute promising drug targets for the development of new antibiotics. These enzymes, NaMN adenylyltransferase (gene nadD) and NAD synthetase (gene nadE), respectively, are indispensable and conserved in nearly all bacterial pathogens. However, a comparative genome analysis of Francisella tularensis allowed us to predict the existence of an alternative route of NAD synthesis in this category A priority pathogen, the causative agent of tularaemia. In this route, the amidation of NaMN to nicotinamide mononucleotide (NMN) occurs before the adenylylation reaction, which converts this alternative intermediate to the NAD cofactor. The first step is catalyzed by NMN synthetase, which was identified and characterized in this study. A crystal structure of this enzyme, a divergent member of the NadE family, was solved at 1.9-Å resolution in complex with reaction products, providing a rationale for its unusual substrate preference for NaMN over NaAD. The second step is performed by NMN adenylyltransferase of the NadM family. Here, we report validation of the predicted route (NaMN → NMN → NAD) in F. tularensis including mathematical modeling, in vitro reconstitution, and in vivo metabolite analysis in comparison with a canonical route (NaMN → NaAD → NAD) of NAD biosynthesis as represented by another deadly bacterial pathogen, Bacillus anthracis. PMID:19204287

  6. Nicotinamide mononucleotide synthetase is the key enzyme for an alternative route of NAD biosynthesis in Francisella tularensis.

    PubMed

    Sorci, Leonardo; Martynowski, Dariusz; Rodionov, Dmitry A; Eyobo, Yvonne; Zogaj, Xhavit; Klose, Karl E; Nikolaev, Evgeni V; Magni, Giulio; Zhang, Hong; Osterman, Andrei L

    2009-03-01

    Enzymes involved in the last 2 steps of nicotinamide adenine dinucleotide (NAD) cofactor biosynthesis, which catalyze the adenylylation of the nicotinic acid mononucleotide (NaMN) precursor to nicotinic acid dinucleotide (NaAD) followed by its amidation to NAD, constitute promising drug targets for the development of new antibiotics. These enzymes, NaMN adenylyltransferase (gene nadD) and NAD synthetase (gene nadE), respectively, are indispensable and conserved in nearly all bacterial pathogens. However, a comparative genome analysis of Francisella tularensis allowed us to predict the existence of an alternative route of NAD synthesis in this category A priority pathogen, the causative agent of tularaemia. In this route, the amidation of NaMN to nicotinamide mononucleotide (NMN) occurs before the adenylylation reaction, which converts this alternative intermediate to the NAD cofactor. The first step is catalyzed by NMN synthetase, which was identified and characterized in this study. A crystal structure of this enzyme, a divergent member of the NadE family, was solved at 1.9-A resolution in complex with reaction products, providing a rationale for its unusual substrate preference for NaMN over NaAD. The second step is performed by NMN adenylyltransferase of the NadM family. Here, we report validation of the predicted route (NaMN --> NMN --> NAD) in F. tularensis including mathematical modeling, in vitro reconstitution, and in vivo metabolite analysis in comparison with a canonical route (NaMN --> NaAD --> NAD) of NAD biosynthesis as represented by another deadly bacterial pathogen, Bacillus anthracis. PMID:19204287

  7. Novel 2-oxoimidazolidine-4-carboxylic acid derivatives as Hepatitis C virus NS3-4A serine protease inhibitors: synthesis, activity, and X-ray crystal structure of an enzyme inhibitor complex

    SciTech Connect

    Arasappan, Ashok; Njoroge, F. George; Parekh, Tejal N.; Yang, Xiaozheng; Pichardo, John; Butkiewicz, Nancy; Prongay, Andrew; Yao, Nanhua; Girijavallabhan, Viyyoor

    2008-06-30

    Synthesis and HCV NS3 serine protease inhibitory activity of some novel 2-oxoimidazolidine-4-carboxylic acid derivatives are reported. Inhibitors derived from this new P2 core exhibited activity in the low {micro}M range. X-ray structure of an inhibitor, 15c bound to the protease is presented.

  8. In vitro characterization of the NAD(+) synthetase NadE1 from Herbaspirillum seropedicae.

    PubMed

    Laskoski, Kerly; Santos, Adrian R S; Bonatto, Ana C; Pedrosa, Fábio O; Souza, Emanuel M; Huergo, Luciano F

    2016-05-01

    Nicotinamide adenine dinucleotide synthetase enzyme (NadE) catalyzes the amination of nicotinic acid adenine dinucleotide (NaAD) to form NAD(+). This reaction represents the last step in the majority of the NAD(+) biosynthetic routes described to date. NadE enzymes typically use either glutamine or ammonium as amine nitrogen donor, and the reaction is energetically driven by ATP hydrolysis. Given the key role of NAD(+) in bacterial metabolism, NadE has attracted considerable interest as a potential target for the development of novel antibiotics. The plant-associative nitrogen-fixing bacteria Herbaspirillum seropedicae encodes two putative NadE, namely nadE1 and nadE2. The nadE1 gene is linked to glnB encoding the signal transduction protein GlnB. Here we report the purification and in vitro characterization of H. seropedicae NadE1. Gel filtration chromatography analysis suggests that NadE1 is an octamer. The NadE1 activity was assayed in vitro, and the Michaelis-Menten constants for substrates NaAD, ATP, glutamine and ammonium were determined. Enzyme kinetic and in vitro substrate competition assays indicate that H. seropedicae NadE1 uses glutamine as a preferential nitrogen donor. PMID:26802007

  9. Structure-based Design and In-Parallel Synthesis of Inhibitors of AmpC b-lactamase

    SciTech Connect

    Tondi, D.; Powers, R.A.; Negri, M.C.; Caselli, M.C.; Blazquez, J.; Costi, M.P.; Shoichet, B.K.

    2010-03-08

    Group I {beta}-lactamases are a major cause of antibiotic resistance to {beta}-lactams such as penicillins and cephalosporins. These enzymes are only modestly affected by classic {beta}-lactam-based inhibitors, such as clavulanic acid. Conversely, small arylboronic acids inhibit these enzymes at sub-micromolar concentrations. Structural studies suggest these inhibitors bind to a well-defined cleft in the group I {beta}-lactamase AmpC; this cleft binds the ubiquitous R1 side chain of {beta}-lactams. Intriguingly, much of this cleft is left unoccupied by the small arylboronic acids. To investigate if larger boronic acids might take advantage of this cleft, structure-guided in-parallel synthesis was used to explore new inhibitors of AmpC. Twenty-eight derivatives of the lead compound, 3-aminophenylboronic acid, led to an inhibitor with 80-fold better binding (2; K{sub i} 83 nM). Molecular docking suggested orientations for this compound in the R1 cleft. Based on the docking results, 12 derivatives of 2 were synthesized, leading to inhibitors with K{sub i} values of 60 nM and with improved solubility. Several of these inhibitors reversed the resistance of nosocomial Gram-positive bacteria, though they showed little activity against Gram-negative bacteria. The X-ray crystal structure of compound 2 in complex with AmpC was subsequently determined to 2.1 {angstrom} resolution. The placement of the proximal two-thirds of the inhibitor in the experimental structure corresponds with the docked structure, but a bond rotation leads to a distinctly different placement of the distal part of the inhibitor. In the experimental structure, the inhibitor interacts with conserved residues in the R1 cleft whose role in recognition has not been previously explored. Combining structure-based design with in-parallel synthesis allowed for the rapid exploration of inhibitor functionality in the R1 cleft of AmpC. The resulting inhibitors differ considerably from {beta}-lactams but

  10. 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. PMID:26397189

  11. Design, Synthesis, and Evaluation of Donepezil-Like Compounds as AChE and BACE-1 Inhibitors.

    PubMed

    Costanzo, Paola; Cariati, Luca; Desiderio, Doriana; Sgammato, Roberta; Lamberti, Anna; Arcone, Rosaria; Salerno, Raffaele; Nardi, Monica; Masullo, Mariorosario; Oliverio, Manuela

    2016-05-12

    An ecofriendly synthetic pathway for the synthesis of donepezil precursors is described. Alternative energy sources were used for the total synthesis in order to improve yields, regioselectively, and rate of each synthetic step and to reduce the coproduction of waste at the same time. For all products, characterized by an improved structural rigidity respect to donepezil, the inhibitor activity on AChE, the selectivity vs BuChE, the side-activity on BACE-1, and the effect on SHSY-5Y neuroblastoma cells viability were tested. Two potential new lead compounds for a dual therapeutic strategy against Alzheimer's disease were envisaged. PMID:27190595

  12. Discovery of a novel nicotinamide phosphoribosyl transferase (NAMPT) inhibitor via in silico screening.

    PubMed

    Takeuchi, Mikio; Niimi, Tatsuya; Masumoto, Mari; Orita, Masaya; Yokota, Hiroyuki; Yamamoto, Tomoko

    2014-01-01

    Nicotinamide phosphoribosyl transferase (NAMPT) is a key enzyme in the salvage pathway of mammalian nicotinamide adenine dinucleotide (NAD) biosynthesis, catalyzing the synthesis of nicotinamide mononucleotide from nicotinamide (Nam). The diverse functions of NAD suggest that NAMPT inhibitors are potential drug candidates as anticancer agents, immunomodulators, or other agents. However, difficulty in conducting high-throughput NAMPT assay with good sensitivity has hampered the discovery of novel anti-NAMPT drugs with improved profiles. We combined an in silico screening strategy with a radioisotope (RI)-based enzyme assay and rationally identified promising NAMPT inhibitors with novel structures. AS1604498 was the most potent inhibitor, with an IC50 of 44 nM, and inhibited THP-1 and K562 cell line growth with the IC50 of 198 nM and 673 nM, respectively. The mode of action was found to reduce intracellular NAD following apoptosis, suggesting that these compounds inhibit NAMPT in cell-based assay. This strategy can be used to discover new drug candidates with targets which are difficult to assess through high-throughput screening. Our hit compounds may be used as seed compounds for developing new therapeutics with NAMPT. PMID:24389478

  13. Microwave-Assisted Synthesis of a MK2 Inhibitor by Suzuki-Miyaura Coupling for Study in Werner Syndrome Cells

    PubMed Central

    Bagley, Mark C.; Baashen, Mohammed; Chuckowree, Irina; Dwyer, Jessica E.; Kipling, David; Davis, Terence

    2015-01-01

    Microwave-assisted Suzuki-Miyaura cross-coupling reactions have been employed towards the synthesis of three different MAPKAPK2 (MK2) inhibitors to study accelerated aging in Werner syndrome (WS) cells, including the cross-coupling of a 2-chloroquinoline with a 3-pyridinylboronic acid, the coupling of an aryl bromide with an indolylboronic acid and the reaction of a 3-amino-4-bromopyrazole with 4-carbamoylphenylboronic acid. In all of these processes, the Suzuki-Miyaura reaction was fast and relatively efficient using a palladium catalyst under microwave irradiation. The process was incorporated into a rapid 3-step microwave-assisted method for the synthesis of a MK2 inhibitor involving 3-aminopyrazole formation, pyrazole C-4 bromination using N-bromosuccinimide (NBS), and Suzuki-Miyaura cross-coupling of the pyrazolyl bromide with 4-carbamoylphenylboronic acid to give the target 4-arylpyrazole in 35% overall yield, suitable for study in WS cells. PMID:26046488

  14. Synthesis, docking, and biological studies of phenanthrene β-diketo acids as novel HIV-1 integrase inhibitors

    PubMed Central

    Sharma, Horrick; Sanchez, Tino W.; Neamati, Nouri; Detorio, Mervi; Schinazi, Raymond F.; Cheng, Xiaolin; Buolamwini, John K.

    2013-01-01

    In the present study we report the synthesis of halogen-substituted phenanthrene β-diketo acids as new HIV-1 integrase inhibitors. The target phenanthrenes were obtained using both standard thermal- and microwave-assisted synthesis. 4-(6-Chlorophenanthren-2-yl)-2,4-dioxobutanoic acid (18) was the most active compound of the series that inhibited both 3′-end processing (3′-P) and strand transfer (ST) with IC50 values of 5 and 1.3 μM, respectively. Docking studies revealed two predominant binding modes that were distinct from the binding modes of raltegravir and elvitegravir, and suggest a novel binding region in the IN active site. Moreover, these compounds do not interact significantly with some of the key amino acids (Q148 and N155) implicated in viral resistance. Therefore, this series of compounds can further be investigated for a possible chemotype to circumvent resistance to clinical HIV-1 IN inhibitors. PMID:24091080

  15. Effect of protein synthesis inhibitors on viral mRNA's synthesized early in adenovirus type 2 infection.

    PubMed Central

    Eggerding, F; Raskas, H J

    1978-01-01

    Viral mRNA species synthesized early in adenovirus type 2 infection in the presence of cycloheximide were compared with those synthesized in the absence of drug or in the presence of the DNA synthesis inhibitor 1-beta-D-arabinofuranosylcytosine. Cycloheximide caused approximately a 10-fold stimulation in the accumulation of [3H]uridine into early viral mRNA species. The only exception was a 24s mRNA transcribed from the transforming end of the genome; in the presence of cycloheximide, accumulation of this mRNA species was stimulated no more than 2-fold. Treatment with cycloheximide also resulted in the accumulation of polyadenylated RNAs transcribed from EcoRI-C that are heterogeneous and smaller than the 20S mRNA. Other translation inhibitors were shown to have similar effects, suggesting that inhibition of protein synthesis early after infection induces alterations in the metabolism of specific RNA sequences. PMID:621786

  16. Design, synthesis and characterization of novel inhibitors against mycobacterial β-ketoacyl CoA reductase FabG4.

    PubMed

    Banerjee, Deb Ranjan; Dutta, Debajyoti; Saha, Baisakhee; Bhattacharyya, Sudipta; Senapati, Kalyan; Das, Amit K; Basak, Amit

    2014-01-01

    We report the design and synthesis of triazole-polyphenol hybrid compounds 1 and 2 as inhibitors of the FabG4 (Rv0242c) enzyme of Mycobacterium tuberculosis for the first time. A major advance in this field occurred only a couple of years ago with the X-ray crystal structure of FabG4, which has helped us to design these inhibitors by the computational fragment-based drug design (FBDD) approach. Compound 1 has shown competitive inhibition with an inhibition constant (Ki) value of 3.97 ± 0.02 μM. On the other hand, compound 2 has been found to be a mixed type inhibitor with a Ki value of 0.88 ± 0.01 μM. Thermodynamic analysis using isothermal titration calorimetry (ITC) reveals that both inhibitors bind at the NADH co-factor binding domain. Their MIC values, as determined by resazurin assay against M. smegmatis, indicated their good anti-mycobacterial properties. A preliminary structure-activity relationship (SAR) study supports the design of these inhibitors. These compounds may be possible candidates as lead compounds for alternate anti-tubercular drugs. All of the reductase enzymes of the Mycobacterium family have a similar ketoacyl reductase (KAR) domain. Hence, this work may be extrapolated to find structure-based inhibitors of other reductase enzymes. PMID:24129589

  17. Chiral Proton Catalysis of Secondary Nitroalkane Additions to Azomethine: Synthesis of a Potent GlyT1 Inhibitor

    PubMed Central

    Davis, Tyler A.; Danneman, Michael W.; Johnston, Jeffrey N.

    2014-01-01

    The first enantioselective synthesis of a potent GlyT1 inhibitor is described. A 3-nitroazetidine donor is used in an enantioselective aza-Henry reaction catalyzed by a bis(amidine)-triflic acid salt organocatalyst, delivering the key intermediate with 92% ee. This adduct is reductively denitrated and converted to the target through a short sequence, thereby allowing assignment of the absolute configuration of the more potent enantiomer. PMID:22543734

  18. Synthesis and SAR of novel isoxazoles as potent c-jun N-terminal kinase (JNK) Inhibitors

    PubMed Central

    He, Yuanjun; Duckett, Derek; Chen, Weimin; Ling, Yuan Yuan; Cameron, Michael D.; Lin, Li; Ruiz, Claudia H.; LoGrasso, Philip V.; Kamenecka, Theodore M.; Koenig, Marcel

    2014-01-01

    The design and synthesis of isoxazole 3 is described, a potent JNK inhibitor with two fold selectivity over p38. Optimization of this scaffold led to compounds 27 and 28 which showed greatly improved selectivity over p38 by maintaining the JNK3 potency of compound 3. Extensive SAR studies will be described as well as preliminary in vivo data of the two lead compounds. PMID:24332487

  19. Synthesis and SAR of 4-substituted-2-aminopyrimidines as novel c-Jun N-terminal kinase (JNK) inhibitors.

    PubMed

    Humphries, Paul S; Lafontaine, Jennifer A; Agree, Charles S; Alexander, David; Chen, Ping; Do, Quyen-Quyen T; Li, Lilian Y; Lunney, Elizabeth A; Rajapakse, Ranjan J; Siegel, Karen; Timofeevski, Sergei L; Wang, Tianlun; Wilhite, David M

    2009-04-15

    The development of a series of novel 4-substituted-2-aminopyrimidines as inhibitors of c-Jun N-terminal kinases is described. The synthesis, in vitro inhibitory values for JNK1, and the in vitro inhibitory value for a c-Jun cellular assay are discussed. Optimization of microsomal clearance led to the identification of 9c, whose kinase selectivity is reported. PMID:19327989

  20. Synthesis and Biological Evaluation of Benzochromenopyrimidinones as Cholinesterase Inhibitors and Potent Antioxidant, Non-Hepatotoxic Agents for Alzheimer's Disease.

    PubMed

    Dgachi, Youssef; Bautista-Aguilera, Oscar M; Benchekroun, Mohamed; Martin, Hélène; Bonet, Alexandre; Knez, Damijan; Godyń, Justyna; Malawska, Barbara; Gobec, Stanislav; Chioua, Mourad; Janockova, Jana; Soukup, Ondrej; Chabchoub, Fakher; Marco-Contelles, José; Ismaili, Lhassane

    2016-01-01

    We report herein the straightforward two-step synthesis and biological assessment of novel racemic benzochromenopyrimidinones as non-hepatotoxic, acetylcholinesterase inhibitors with antioxidative properties. Among them, compound 3Bb displayed a mixed-type inhibition of human acetylcholinesterase (IC50 = 1.28 ± 0.03 μM), good antioxidant activity, and also proved to be non-hepatotoxic on human HepG2 cell line. PMID:27187348

  1. Solid-Phase synthesis of a library constructed of aromatic phosphate, long alkyl chains and tryptophane components, and identification of potent dipeptide telomerase inhibitors.

    PubMed

    Sasaki, S; Ehara, T; Alam, M R; Fujino, Y; Harada, N; Kimura, J; Nakamura, H; Maeda, M

    2001-10-01

    Telomerase inhibitors are expected as a new candidate of therapeutic agents for cancer. Recently, we have found novel inhibitors based on the bisindole skeleton. In this study, solid-phase synthesis was applied to construct a library of inhibitors having aromatic phosphate, long alkyl chain and tryptophane components, from which a D,D-ditryptophane derivative has been identified as a new potent telomerase inhibitor with IC(50) values of 0.3 microM. A hypothetical binding model for the new inhibitors has been proposed based on the structure-activity relationship. PMID:11551754

  2. The glutathione synthesis inhibitor buthionine sulfoximine synergistically enhanced melphalan activity against preclinical models of multiple myeloma.

    PubMed

    Tagde, A; Singh, H; Kang, M H; Reynolds, C P

    2014-01-01

    Melphalan (L-PAM) has been an integral part of multiple myeloma (MM) treatment as a conditioning regimen before stem cell transplant (SCT). After initial response, most treated patients experience relapse with an aggressive phenotype. Increased glutathione (GSH) in MM may mediate resistance to L-PAM. We demonstrated that the GSH synthesis inhibitor buthionine sulfoximine (BSO) synergistically enhanced L-PAM activity (inducing 2-4 logs of cell kill) against nine MM cell lines (also in the presence of marrow stroma or cytokines) and in seven primary MM samples (combination indices <1.0). In MM cell lines, BSO significantly (P<0.05) depleted GSH, increased L-PAM-induced single-strand DNA breaks, mitochondrial depolarization, caspase cleavage and apoptosis. L-PAM depleted GSH, but GSH rapidly recovered in a L-PAM-resistant MM cell line unless also treated with BSO. Treatment with N-acetylcysteine antagonized BSO+L-PAM cytotoxicity without increasing GSH. In human MM xenografted into beige-nude-xid mice, BSO significantly depleted MM intracellular GSH and significantly increased apoptosis compared with L-PAM alone. BSO+L-PAM achieved complete responses (CRs) in three MM xenograft models including maintained CRs >100 days, and significantly increased the median event-free survival relative to L-PAM alone. Combining BSO with L-PAM warrants clinical testing in advanced MM. PMID:25036800

  3. Synthesis and activity of benzimidazole-1,3-dioxide inhibitors of separase.

    PubMed

    Do, Ha T; Zhang, Nenggang; Pati, Debananda; Gilbertson, Scott R

    2016-09-15

    Due to the oncogenic activity of cohesin protease, separase in human cancer cells, modulation of separase enzymatic activity could constitute a new therapeutic strategy for targeting resistant, separase-overexpressing aneuploid tumors. Herein, we report the synthesis, structural information, and structure-activity relationship (SAR) of separase inhibitors based on modification of the lead molecule 2,2-dimethyl-5-nitro-2H-benzimidazole-1,3-dioxide, named Sepin-1, (1) identified from a high-throughput-screen. Replacement of -NO2 at C5 with other functional groups reduce the inhibitory activity in separase enzymatic assay. Substitution of the two methyl groups with other alkyl chains at the C2 moderately improves the effects on the inhibitory activity of those compounds. Modifications on 2H-benzimidazole-1,3-dioxide or the skeleton have variable effect on inhibition of separase enzymatic activity. Density-functional theory (DFT) calculations suggest there may be a correlation between the charges on the oxide moieties on these compounds and their activity in inhibiting separase enzyme. PMID:27530289

  4. Synthesis and Pharmacokinetic Evaluation of Siderophore Biosynthesis Inhibitors for Mycobacterium tuberculosis

    PubMed Central

    Nelson, Kathryn M.; Viswanathan, Kishore; Dawadi, Surendra; Duckworth, Benjamin P.; Boshoff, Helena I.; Barry, Clifton E.; Aldrich, Courtney C.

    2015-01-01

    MbtA catalyzes the first committed biosynthetic step of the mycobactins, which are important virulence factors associated with iron acquisition in Mycobacterium tuberculosis. MbtA is a validated therapeutic target for antitubercular drug development. 5′-O-[N-(salicyl)sulfamoyl]adenosine (1) is a bisubstrate inhibitor of MbtA and exhibits exceptionally potent biochemical and antitubercular activity. However, 1 suffers from sub-optimal drug disposition properties resulting in a short half-life (t1/2), low exposure (AUC), and low bioavailability (F). Four strategies were pursued to address these liabilities including the synthesis of prodrugs, increasing the pKa of the acyl-sulfonyl moiety, modulation of the lipophilicity, and strategic introduction of fluorine into 1. Complete pharmacokinetic (PK) analysis of all compounds was performed. The most successful modifications involved fluorination of the nucleoside that provided substantial improvements in t1/2 and AUC. Increasing the pKa of the acyl-sulfonyl linker yielded incremental enhancements while modulation of the lipophilicity and prodrug approaches led to substantially poorer PK parameters. PMID:26110337

  5. Efficacy of chitin synthesis inhibitors on nymphal German cockroaches (Dictyoptera: Blattellidae).

    PubMed

    DeMark, J J; Bennett, G W

    1989-12-01

    Second- and fifth-instar Blattella germanica (L.), fed the chitin synthesis inhibitors triflumuron, chlorfluazuron, hexafluron, and UC 84572 (structure not disclosed) were examined for mortality and developmental abnormalities. All compounds were active against B. germanica (L.), with lower diet concentrations being required to kill second instars compared with fifth instars. Chlorfluazuron was significantly more active against second and fifth instars (LC50 = 0.000191 and 0.000363% AI, respectively for the second and fifth instars). UC 84572 also killed nymphs at extremely low concentrations (LC50 = 0.000508 and 0.000754% AI, respectively, for second and fifth instars). LC50's for hexafluron and triflumuron against fifth instars were more than 1,000 times higher than that for chlorfluazuron. Sensitive periods of exposure were determined by comparing effects when four different age classes of fifth instars (1-, 4-, 7-, and 10-d old) fed on the compounds for 3 d. Triflumuron was most effective when ingested during the first three age classes and hexafluron was most effective during the last three age classes. Chlorfluazuron and UC 84572 were most effective when ingested during the second age class (days 4-6). Adults surviving exposure during the fifth instar were often deformed and weak; they died at a greater rate than the controls. However, most surviving adults were able to reproduce normally. PMID:2607029

  6. Effect of the chitin synthesis inhibitor triflumuron on the development, viability and reproduction of Aedes aegypti.

    PubMed

    Belinato, Thiago Affonso; Martins, Ademir Jesus; Lima, José Bento Pereira; Lima-Camara, Tamara Nunes de; Peixoto, Alexandre Afrânio; Valle, Denise

    2009-02-01

    The control of Aedes aegypti is impaired due to the development of resistance to chemical insecticides. Insect Growth Regulators (IGR) exhibit distinct mechanisms of action and are considered potential vector control alternatives. Studies regarding the effects of sublethal IGR doses on the viability of resulting adults will contribute to eval-uating their impact in the field. We analyzed several aspects of Ae. aegypti adults surviving exposure to a partially lethal dose of triflumuron, a chitin synthesis inhibitor. A highly significant difference in the proportion of males and females was noted in the triflumuron-exposed group (65.0% males) compared to the controls (50.2% males). Triflumuron affected adult longevity, particularly for females; after 16 days, only 29.2% of males and 13.8% of females were alive, in contrast with 94% survival of the control mosquitoes. The locomotor activity was reduced and the blood-feeding ability of the treated females was also affected (90.4% and 48.4% of the control and triflumuron-exposed females, respectively, successfully ingested blood). Triflumuron-surviving females ingested roughly 30% less blood and laid 25% fewer eggs than the control females. The treated males and females exhibited a diminished ability to copulate, resulting in less viable eggs. PMID:19274375

  7. Synthesis and biological evaluation of enantiomerically pure glyceric acid derivatives as LpxC inhibitors.

    PubMed

    Tangherlini, Giovanni; Torregrossa, Tullio; Agoglitta, Oriana; Köhler, Jens; Melesina, Jelena; Sippl, Wolfgang; Holl, Ralph

    2016-03-01

    Inhibitors of the UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine deacetylase (LpxC) represent a promising class of novel antibiotics, selectively combating Gram-negative bacteria. In order to elucidate the impact of the hydroxymethyl groups of diol (S,S)-4 on the inhibitory activity against LpxC, glyceric acid ethers (R)-7a, (S)-7a, (R)-7b, and (S)-7b, lacking the hydroxymethyl group in benzylic position, were synthesized. The compounds were obtained in enantiomerically pure form by a chiral pool synthesis and a lipase-catalyzed enantioselective desymmetrization, respectively. The enantiomeric hydroxamic acids (R)-7b (Ki=230nM) and (S)-7b (Ki=390nM) show promising enzyme inhibition. However, their inhibitory activities do not substantially differ from each other leading to a low eudismic ratio. Generally, the synthesized glyceric acid derivatives 7 show antibacterial activities against two Escherichia coli strains exceeding the ones of their respective regioisomes 6. PMID:26827141

  8. Pyridopyrimidine derivatives as inhibitors of cyclic nucleotide synthesis: Application for treatment of diarrhea

    PubMed Central

    Kots, Alexander Y.; Choi, Byung-Kwon; Estrella-Jimenez, Maria E.; Warren, Cirle A.; Gilbertson, Scott R.; Guerrant, Richard L.; Murad, Ferid

    2008-01-01

    Acute secretory diarrhea induced by infection with enterotoxigenic strains of Escherichia coli involves binding of stable toxin (STa) to its receptor on the intestinal brush border, guanylyl cyclase type C (GC-C). Intracellular cGMP is elevated, inducing increase in chloride efflux and subsequent accumulation of fluid in the intestinal lumen. We have screened a library of compounds and identified a pyridopyrimidine derivatives {5-(3-bromophenyl)-1,3-dimethyl-5,11-dihydro-1H-indeno[2′,1′:5,6]pyrido[2,3-d]pyrimidine-2,4,6-trione; BPIPP} as an inhibitor of GC-C that can suppress STa-stimulated cGMP accumulation by decreasing GC-C activation in intact T84 human colorectal carcinoma cells. BPIPP inhibited stimulation of guanylyl cyclases, including types A and B and soluble isoform in various cells. BPIPP suppressed stimulation of adenylyl cyclase and significantly decreased the activities of adenylyl cyclase toxin of Bordetella pertussis and edema toxin of Bacillus anthracis. The effects of BPIPP on cyclic nucleotide synthesis were observed only in intact cells. The mechanism of BPIPP-dependent inhibition appears to be complex and indirect, possibly associated with phospholipase C and tyrosine-specific phosphorylation. BPIPP inhibited chloride-ion transport stimulated by activation of guanylyl or adenylyl cyclases and suppressed STa-induced fluid accumulation in an in vivo rabbit intestinal loop model. Thus, BPIPP may be a promising lead compound for treatment of diarrhea and other diseases. PMID:18559851

  9. Synthesis of three bromophenols from red algae as PTP1B inhibitors

    NASA Astrophysics Data System (ADS)

    Guo, Shuju; Li, Jing; Li, Ting; Shi, Dayong; Han, Lijun

    2011-01-01

    Bromophenols are a set of natural products widely distributed in seaweed, most of which exhibit interesting and useful biological activities. To develop a reliable and efficient synthetic route to these natural bromophenols, three of them, 3,4-dibromo-5-(2'-bromo-3',4'-dihydroxy-6'-methoxymethyl-benzyl)-benzene-1,2-diol (compound 9), 3,4-dibromo-5-(2'-bromo-6'-ethoxy methyl-3',4'-dihydroxybenzyl)-benzene-1,2-diol (compound 10), and 3-bromo-4-(3'-bromo-4',5'-dihydroxy benzyl)-5-(ethoxymethyl)-benzene-1,2-diol (compound 14), isolated from red marine algae, have been synthesized in eight steps with an overall yield of 14.4%, 14.4%, and 18.2% respectively, via a practical approach employing bromination, Wolff-Kishner-Huang reduction and a Friedel-Crafts reaction as key steps. The protein tyrosine phosphatase 1B (PTP1B) inhibitory activities of the synthetic compounds were evaluated by the colorimetric assay. The results show that these compounds are moderate PTP1B inhibitors. The synthesis of these bromophenol derivatives makes in vivo studies of their structure-activity relationships and inhibition activity against PTP1B possible.

  10. Functionalized imidazolium and benzimidazolium salts as paraoxonase 1 inhibitors: Synthesis, characterization and molecular docking studies.

    PubMed

    Karataş, Mert Olgun; Uslu, Harun; Alıcı, Bülent; Gökçe, Başak; Gencer, Nahit; Arslan, Oktay; Arslan, N Burcu; Özdemir, Namık

    2016-03-15

    Paraoxonase (PON) is a key enzyme in metabolism of living organisms and decreased activity of PON1 was acknowledged as a risk for atherosclerosis and organophosphate toxicity. The present study describes the synthesis, characterization, PON1 inhibitory properties and molecular docking studies of functionalized imidazolium and benzimidazolium salts (1a-5g). The structures of all compounds were elucidated by IR, NMR, elemental analysis and structures of compounds 2b and 2c were characterized by single-crystal X-ray diffraction. Compound 1c, a coumarin substituted imidazolium salt showed the best inhibitory effect on the activity of PON1 with good IC50 value (6.37 μM). Kinetic investigation was evaluated for this compound and results showed that this compound is competitive inhibitor of PON1 with Ki value of 2.39 μM. Molecular docking studies were also performed for most active compound 1c and one of least active compound 2c in order to determine the probable binding model into active site of PON1 and validation of the experimental results. PMID:26879855

  11. Evaluation of Two Formulated Chitin Synthesis Inhibitors, Hexaflumuron and Lufenuron Against the Raisin Moth, Ephestia figulilella

    PubMed Central

    Khajepour, Simin; Izadi, Hamzeh; Asari, Mohammad Javad

    2012-01-01

    The raisin moth, Ephestia figulilella Gregson (Lepidoptera: Pyralidae), has a nearly cosmopolitan distribution, and causes severe quantitative and qualitative losses throughout the world. The larvae attack various drying and dried fruits, fallen figs, and damaged or moldy clusters of grapes on vines. Control of this pest in storage depends mostly on synthetic pesticides with several adverse side effects. To mitigate the adverse effects of these pesticides, investigations have focused on the development of compounds with more selectivity, and short residual life. In this research, insecticidal effects of two chitin synthesis inhibitors, hexaflumuron and lufenuron, were investigated against E. figulilella. Graded concentrations of each pesticide were prepared with distilled water. One-day-old fifth instar were sprayed by Potter's precision spray tower. Application of hexaflumuron and lufenuron on last instar larvae of E. figulilella caused not only mortality in larval stage, but also caused defects in pupal and adult stages. Larval mortality increased as concentration increased. The longevity of the fifth instars in both hexaflumuron and lufenuron treatments, in comparison with the controls, increased by more than 12 days. The longevity of adults decreased by about 10 days. Probit analysis data revealed that the sensitivity of the test insect to hexaflumuron (EC50 = 95.38 ppm) was greater than lufenuron (EC50= 379.21 ppm). PMID:23425138

  12. Disruption of reproductive activity of Coptotermes formosanus (Isoptera: Rhinotermitidae) primary reproductives by three chitin synthesis inhibitors.

    PubMed

    Rojas, M Guadalupe; Morales-Ramos, Juan A

    2004-12-01

    Effects of the chitin synthesis inhibitors (CSIs) diflubenzuron, hexaflumuron, and lufenuron on the Formosan subterranean termite, Coptotermes formosanus Shiraki, primary reproductives were studied in the laboratory. Incipient colonies were established by collecting and pairing C. formosanus alates and placing them in dishes containing an artificial diet. Three groups of 56 colonies each were fed with a diet containing 10 ppm of one of each of the CSIs and were compared with a control group fed with an untreated diet. All eggs oviposited by treated young queens failed to hatch at the end of 6 mo. Estimated queen fecundity was significantly lower in the lufenuron treatment compared with the control group. Fecundity of hexaflumuron-treated queens did not differ significantly from that of queens from the control group and the other treatments. Adult mortality was significantly higher in the diflubenzuron and lufenuron treatments than in the control group at the end of 6 mo., but not in the hexaflumuron treatment. All the pairs died within 8 mo. in the diflubenzuron and lufenuron treatments, even after treatment was suspended at the end of 6 mo. Mortality in the hexaflumuron treatment was significantly higher than in the control group by the end of 9 mo. The three CSIs tested eliminated reproduction in C. formosanus by preventing egg hatching and induced adult mortality. Possible mechanisms by which CSIs induce termite adult death are discussed. PMID:15666759

  13. Evaluation of two formulated chitin synthesis inhibitors, hexaflumuron and lufenuron against the raisin moth, Ephestia figulilella.

    PubMed

    Khajepour, Simin; Izadi, Hamzeh; Asari, Mohammad Javad

    2012-01-01

    The raisin moth, Ephestia figulilella Gregson (Lepidoptera: Pyralidae), has a nearly cosmopolitan distribution, and causes severe quantitative and qualitative losses throughout the world. The larvae attack various drying and dried fruits, fallen figs, and damaged or moldy clusters of grapes on vines. Control of this pest in storage depends mostly on synthetic pesticides with several adverse side effects. To mitigate the adverse effects of these pesticides, investigations have focused on the development of compounds with more selectivity, and short residual life. In this research, insecticidal effects of two chitin synthesis inhibitors, hexaflumuron and lufenuron, were investigated against E. figulilella. Graded concentrations of each pesticide were prepared with distilled water. One-day-old fifth instar were sprayed by Potter's precision spray tower. Application of hexaflumuron and lufenuron on last instar larvae of E. figulilella caused not only mortality in larval stage, but also caused defects in pupal and adult stages. Larval mortality increased as concentration increased. The longevity of the fifth instars in both hexaflumuron and lufenuron treatments, in comparison with the controls, increased by more than 12 days. The longevity of adults decreased by about 10 days. Probit analysis data revealed that the sensitivity of the test insect to hexaflumuron (EC(50) = 95.38 ppm) was greater than lufenuron (EC(50)= 379.21 ppm). PMID:23425138

  14. Design and synthesis of HIV-1 protease inhibitors for a long-acting injectable drug application.

    PubMed

    Kesteleyn, Bart; Amssoms, Katie; Schepens, Wim; Hache, Geerwin; Verschueren, Wim; Van De Vreken, Wim; Rombauts, Klara; Meurs, Greet; Sterkens, Patrick; Stoops, Bart; Baert, Lieven; Austin, Nigel; Wegner, Jörg; Masungi, Chantal; Dierynck, Inge; Lundgren, Stina; Jönsson, Daniel; Parkes, Kevin; Kalayanov, Genadiy; Wallberg, Hans; Rosenquist, Asa; Samuelsson, Bertil; Van Emelen, Kristof; Thuring, Jan Willem

    2013-01-01

    The design and synthesis of novel HIV-1 protease inhibitors (PIs) (1-22), which display high potency against HIV-1 wild-type and multi-PI-resistant HIV-mutant clinical isolates, is described. Lead optimization was initiated from compound 1, a Phe-Phe hydroxyethylene peptidomimetic PI, and was directed towards the discovery of new PIs suitable for a long-acting (LA) injectable drug application. Introducing a heterocyclic 6-methoxy-3-pyridinyl or a 6-(dimethylamino)-3-pyridinyl moiety (R(3)) at the para-position of the P1' benzyl fragment generated compounds with antiviral potency in the low single digit nanomolar range. Halogenation or alkylation of the metabolic hot spots on the various aromatic rings resulted in PIs with high stability against degradation in human liver microsomes and low plasma clearance in rats. Replacing the chromanolamine moiety (R(1)) in the P2 protease binding site by a cyclopentanolamine or a cyclohexanolamine derivative provided a series of high clearance PIs (16-22) with EC(50)s on wild-type HIV-1 in the range of 0.8-1.8 nM. PIs 18 and 22, formulated as nanosuspensions, showed gradual but sustained and complete release from the injection site over two months in rats, and were therefore identified as interesting candidates for a LA injectable drug application for treating HIV/AIDS. PMID:23177258

  15. 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

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

    PubMed

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

    1996-09-01

    The essential role of HIV-1 protease (HIV-1 PR) in the viral life cycle makes it an attractive target for the development of substrate-based inhibitors that may find efficacy as anti-AIDS drugs. However, resistance has arisen to potent peptidomimetic drugs necessitating the further development of novel chemical backbones for diversity based chemistry focused on probing the active site for inhibitor interactions and binding modes that evade protease resistance. AQ148 is a potent inhibitor of HIV-1 PR and represents a new class of transition state analogues incorporating an aminimide peptide isostere. A 3-D crystallographic structure of AQ148, a tetrapeptide isostere, has been determined in complex with its target HIV-1 PR to a resolution of 2.5 A and used to evaluate the specific structural determinants of AQ148 potency and to correlate structure-activity relationships within the class of related compounds. AQ148 is a competitive inhibitor of HIV-1 PR with a Ki value of 137 nM. Twenty-nine derivatives have been synthesized and chemical modifications have been made at the P1, P2, P1', and P2' sites. The atomic resolution structure of AQ148 bound to HIV-1 PR reveals both an inhibitor binding mode that closely resembles that of other peptidomimetic inhibitors and specific protein/inhibitor interactions that correlate with structure-activity relationships. The structure provides the basis for the design, synthesis and evaluation of the next generation of hydroxyethyl aminimide inhibitors. The aminimide peptide isostere is a scaffold with favorable biological properties well suited to both the combinatorial methods of peptidomimesis and the rational design of potent and specific substrate-based analogues. PMID:8894111

  17. Pharmacological NAD-Boosting Strategies Improve Mitochondrial Homeostasis in Human Complex I-Mutant Fibroblasts.

    PubMed

    Felici, Roberta; Lapucci, Andrea; Cavone, Leonardo; Pratesi, Sara; Berlinguer-Palmini, Rolando; Chiarugi, Alberto

    2015-06-01

    Mitochondrial disorders are devastating genetic diseases for which efficacious therapies are still an unmet need. Recent studies report that increased availability of intracellular NAD obtained by inhibition of the NAD-consuming enzyme poly(ADP-ribose) polymerase (PARP)-1 or supplementation with the NAD-precursor nicotinamide riboside (NR) ameliorates energetic derangement and symptoms in mouse models of mitochondrial disorders. Whether these pharmacological approaches also improve bioenergetics of human cells harboring mitochondrial defects is unknown. It is also unclear whether the same signaling cascade is prompted by PARP-1 inhibitors and NR supplementation to improve mitochondrial homeostasis. Here, we show that human fibroblasts mutant for the NADH dehydrogenase (ubiquinone) Fe-S protein 1 (NDUFS1) subunit of respiratory complex I have similar ATP, NAD, and mitochondrial content compared with control cells, but show reduced mitochondrial membrane potential. Interestingly, mutant cells also show increased transcript levels of mitochondrial DNA but not nuclear DNA respiratory complex subunits, suggesting activation of a compensatory response. At variance with prior work in mice, however, NR supplementation, but not PARP-1 inhibition, increased intracellular NAD content in NDUFS1 mutant human fibroblasts. Conversely, PARP-1 inhibitors, but not NR supplementation, increased transcription of mitochondrial transcription factor A and mitochondrial DNA-encoded respiratory complexes constitutively induced in mutant cells. Still, both NR and PARP-1 inhibitors restored mitochondrial membrane potential and increased organelle content as well as oxidative activity of NDUFS1-deficient fibroblasts. Overall, data provide the first evidence that in human cells harboring a mitochondrial respiratory defect exposure to NR or PARP-1, inhibitors activate different signaling pathways that are not invariantly prompted by NAD increases, but equally able to improve energetic

  18. Synthesis of a 10,000-membered library of molecules resembling carpanone and discovery of vesicular traffic inhibitors.

    PubMed

    Goess, Brian C; Hannoush, Rami N; Chan, Lawrence K; Kirchhausen, Tomas; Shair, Matthew D

    2006-04-26

    Split-and-pool synthesis of a 10,000-membered library of molecules resembling the natural product carpanone has been achieved. The synthesis features development of solid-phase multicomponent reactions between nitrogen nucleophiles, enones, and hydroxylamines, and a solid-phase application of the Huisgen cycloaddition affording substituted triazoles. The synthesis was performed in high-capacity (500 microm) polystyrene beads using a one bead-one stock solution strategy that enabled phenotypic screens of the resulting library. Using whole-cell fluorescence imaging, we discovered a series of molecules from the carpanone-based library that inhibit exocytosis from the Golgi apparatus. The most potent member of this series has an IC(50) of 14 microM. We also report structure-activity relationships for the molecules exhibiting this interesting phenotype. These inhibitors of exocytosis may be useful reagents for the study of vesicular traffic. PMID:16620111

  19. Synthesis and anti-HIV activity of some [Nucleoside Reverse Transcriptase Inhibitor]-C5'-linker-[Integrase Inhibitor] heterodimers as inhibitors of HIV replication.

    PubMed

    Sugeac, Elena; Fossey, Christine; Ladurée, Daniel; Schmidt, Sylvie; Laumond, Geraldine; Aubertin, Anne-Marie

    2004-12-01

    Selected for their expected ability to inhibit HIV replication, a series of eight heterodimers containing a Nucleoside Reverse Transcriptase Inhibitor (NRTI) and an Integrase Inhibitor (INI), bound by a linker, were designed and synthesized. For the NRTIs, d4U, d2U and d4T were chosen. For the INIs, 4-[1-(4-fluorobenzyl)-1H-pyrrol-2-yl]-2,4-dioxobutyric acid (6) and 4-(3,5-dibenzyloxyphenyl)-2,4-dioxobutyric acid (9) (belonging to the beta-diketo acids class) were chosen. The conjugation of the two different inhibitors (NRTI and INI) was performed using an amino acid (glycine or beta-alanine) as a cleavable linker. PMID:15662954

  20. Kinetics and regulation of hepatoma mitochondrial NAD(P) malic enzyme.

    PubMed

    Teller, J K; Fahien, L A; Davis, J W

    1992-05-25

    Kinetic studies of Morris 7777 hepatoma mitochondrial NAD(P) malic enzyme were consistent with an ordered mechanism where NAD adds to the enzyme before malate and dissociation of NADH from the enzyme is rate-limiting. In addition to its active site, malate apparently also associates with a lower affinity with an activator site. The activator fumarate competes with malate at the activator site and facilitates dissociation of NADH from the enzyme. The ratio of NAD(P) malic enzyme to malate dehydrogenase activity in the hepatoma mitochondrial extract was found to be too low, even in the presence of known inhibitors of malate dehydrogenase, to account for the known ability of NAD(P) malic enzyme to intercept exogenous malate from malate dehydrogenase in intact tumor mitochondria (Moreadith, R.W., and Lehninger, A.L. (1984) J. Biol. Chem. 259, 6215-6221). However, NAD(P) malic enzyme may be able to intercept exogenous malate because according to the present results, it can associate with the pyruvate dehydrogenase complex, which could localize NAD(P) malic enzyme in the vicinity of the inner mitochondrial membrane. The activity levels of some key metabolic enzymes were found to be different in Morris 7777 mitochondria than in liver or mitochondria of other rapidly dividing tumors. These results are discussed in terms of differences among tumors in their ability to utilize malate, glutamate, and citrate as respiratory fuels. PMID:1587826

  1. Nicotinamide riboside promotes Sir2 silencing and extends lifespan via Nrk and Urh1/Pnp1/Meu1 pathways to NAD+.

    PubMed

    Belenky, Peter; Racette, Frances G; Bogan, Katrina L; McClure, Julie M; Smith, Jeffrey S; Brenner, Charles

    2007-05-01

    Although NAD(+) biosynthesis is required for Sir2 functions and replicative lifespan in yeast, alterations in NAD(+) precursors have been reported to accelerate aging but not to extend lifespan. In eukaryotes, nicotinamide riboside is a newly discovered NAD(+) precursor that is converted to nicotinamide mononucleotide by specific nicotinamide riboside kinases, Nrk1 and Nrk2. In this study, we discovered that exogenous nicotinamide riboside promotes Sir2-dependent repression of recombination, improves gene silencing, and extends lifespan without calorie restriction. The mechanism of action of nicotinamide riboside is totally dependent on increased net NAD(+) synthesis through two pathways, the Nrk1 pathway and the Urh1/Pnp1/Meu1 pathway, which is Nrk1 independent. Additionally, the two nicotinamide riboside salvage pathways contribute to NAD(+) metabolism in the absence of nicotinamide-riboside supplementation. Thus, like calorie restriction in the mouse, nicotinamide riboside elevates NAD(+) and increases Sir2 function. PMID:17482543

  2. A rise in NAD precursor nicotinamide mononucleotide (NMN) after injury promotes axon degeneration

    PubMed Central

    Di Stefano, M; Nascimento-Ferreira, I; Orsomando, G; Mori, V; Gilley, J; Brown, R; Janeckova, L; Vargas, M E; Worrell, L A; Loreto, A; Tickle, J; Patrick, J; Webster, J R M; Marangoni, M; Carpi, F M; Pucciarelli, S; Rossi, F; Meng, W; Sagasti, A; Ribchester, R R; Magni, G; Coleman, M P; Conforti, L

    2015-01-01

    NAD metabolism regulates diverse biological processes, including ageing, circadian rhythm and axon survival. Axons depend on the activity of the central enzyme in NAD biosynthesis, nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2), for their maintenance and degenerate rapidly when this activity is lost. However, whether axon survival is regulated by the supply of NAD or by another action of this enzyme remains unclear. Here we show that the nucleotide precursor of NAD, nicotinamide mononucleotide (NMN), accumulates after nerve injury and promotes axon degeneration. Inhibitors of NMN-synthesising enzyme NAMPT confer robust morphological and functional protection of injured axons and synapses despite lowering NAD. Exogenous NMN abolishes this protection, suggesting that NMN accumulation within axons after NMNAT2 degradation could promote degeneration. Ectopic expression of NMN deamidase, a bacterial NMN-scavenging enzyme, prolongs survival of injured axons, providing genetic evidence to support such a mechanism. NMN rises prior to degeneration and both the NAMPT inhibitor FK866 and the axon protective protein WldS prevent this rise. These data indicate that the mechanism by which NMNAT and the related WldS protein promote axon survival is by limiting NMN accumulation. They indicate a novel physiological function for NMN in mammals and reveal an unexpected link between new strategies for cancer chemotherapy and the treatment of axonopathies. PMID:25323584

  3. The Importance of NAD in Multiple Sclerosis

    PubMed Central

    Penberthy, W. Todd; Tsunoda, Ikuo

    2009-01-01

    The etiology of multiple sclerosis (MS) is unknown but it manifests as a chronic inflammatory demyelinating disease in the central nervous system (CNS). During chronic CNS inflammation, nicotinamide adenine dinucleotide (NAD) concentrations are altered by (T helper) Th1-derived cytokines through the coordinated induction of both indoleamine 2,3-dioxygenase (IDO) and the ADP cyclase CD38 in pathogenic microglia and lymphocytes. While IDO activation may keep auto-reactive T cells in check, hyper-activation of IDO can leave neuronal CNS cells starving for extracellular sources of NAD. Existing data indicate that glia may serve critical functions as an essential supplier of NAD to neurons during times of stress. Administration of pharmacological doses of non-tryptophan NAD precursors ameliorates pathogenesis in animal models of MS. Animal models of MS involve artificially stimulated autoimmune attack of myelin by experimental autoimmune encephalomyelitis (EAE) or by viral-mediated demyelination using Thieler's murine encephalomyelitis virus (TMEV). The WldS mouse dramatically resists razor axotomy mediated axonal degeneration. This resistance is due to increased efficiency of NAD biosynthesis that delays stress-induced depletion of axonal NAD and ATP. Although the WldS genotype protects against EAE pathogenesis, TMEV-mediated pathogenesis is exacerbated. In this review, we contrast the role of NAD in EAE versus TMEV demyelinating pathogenesis to increase our understanding of the pharmacotherapeutic potential of NAD signal transduction pathways. We speculate on the importance of increased SIRT1 activity in both PARP-1 inhibition and the potentially integral role of neuronal CD200 interactions through glial CD200R with induction of IDO in MS pathogenesis. A comprehensive review of immunomodulatory control of NAD biosynthesis and degradation in MS pathogenesis is presented. Distinctive pharmacological approaches designed for NAD-complementation or targeting NAD

  4. Tissue Inhibitor of Metalloproteinase-2 Suppresses Collagen Synthesis in Cultured Keloid Fibroblasts

    PubMed Central

    Dohi, Teruyuki; Aoki, Masayo; Ogawa, Rei; Akaishi, Satoshi; Shimada, Takashi; Okada, Takashi; Hyakusoku, Hiko

    2015-01-01

    Background: Keloids are defined as a kind of dermal fibroproliferative disorder resulting from the accumulation of collagen. In the remodeling of extracellular matrix, the balance between matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) is as critical as the proper production of extracellular matrix. We investigate the role of TIMPs and MMPs in the pathogenesis of keloids and examine the therapeutic potential of TIMP-2. Methods: The expression of TIMPs and MMPs in most inflamed parts of cultured keloid fibroblasts (KFs) and peripheral normal skin fibroblasts (PNFs) in the same individuals and the reactivity of KFs to cyclic mechanical stretch were analyzed by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay (n = 7). To evaluate the effect of treating KFs with TIMP-2, collagen synthesis was investigated by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay, and microscopic analysis was used to examine the treatment effects of TIMP-2 on ex vivo cultures of keloid tissue (n = 6). Results: TIMP-2 was downregulated in cultured KFs compared with PNFs in the same individuals, and the reduction in TIMP-2 was exacerbated by cyclic mechanical stretch. Administration of TIMP-2 (200 or 300 ng/mL) significantly suppressed expression of Col1A2 and Col3A1 mRNA and collagen type I protein in KFs. TIMP-2 also significantly reduced the skin dermal and collagen bundle thickness in ex vivo cultures of keloid tissue. Conclusion: These results indicated that downregulation of TIMP-2 in KFs is a crucial event in the pathogenesis of keloids, and the TIMP-2 would be a promising candidate for the treatment of keloids. PMID:26495233

  5. Hazards and uptake of chitin synthesis inhibitors in bumblebees Bombus terrestris.

    PubMed

    Mommaerts, Veerle; Sterk, Guido; Smagghe, Guy

    2006-08-01

    This research project examined the potential hazards of a major class of insect growth regulators (IGRs) to survival, reproduction and larval growth in bumblebees Bombus terrestris L. Eight chitin synthesis inhibitors (CSIs) were tested: buprofezin, cyromazine, diflubenzuron, flucycloxuron, flufenoxuron, lufenuron, novaluron and teflubenzuron. These different IGRs, which are important in the control of pest insects in greenhouses, were applied via three different routes of exposure under laboratory conditions: dermal contact, and orally via the drinking of sugar/water and via pollen. The compounds were tested at their respective maximum field recommended concentrations (MFRC) and also in dose-response assays to calculate LC(50) values. In general, none of the CSIs showed acute worker toxicity. However, there was a dramatic reduction in brood production, especially after oral treatment with pollen and sugar/water. Conspicuously, egg fertility was reduced in all treatments with diflubenzuron and teflubenzuron. In addition to egg mortality, the worker bumblebees removed larvae from the treated nest, and in most cases these individuals were dead first-second instars. Under a binocular microscope, such larvae showed an abnormally formed cuticle leading to mechanical weakness and death. In another series of experiments using (14)C-diflubenzuron and (14)C-flufenoxuron, cuticular penetration in workers was studied for a better understanding of the differences in toxicity. With (14)C-diflubenzuron, transovarial transport and accumulation in the deposited eggs supported the strong reproductive effects. Overall, the present results suggest that CSIs should be applied with caution in combination with bumblebees. The compatibility of each compound to be used in combination with B. terrestris is discussed in relation to calculated LC(50) values, routes of uptake and effects. PMID:16786494

  6. Physiological and Morphological Aspects of Aedes aegypti Developing Larvae: Effects of the Chitin Synthesis Inhibitor Novaluron

    PubMed Central

    Farnesi, Luana C.; Brito, José M.; Linss, Jutta G.; Pelajo-Machado, Marcelo; Valle, Denise; Rezende, Gustavo L.

    2012-01-01

    Population control of the dengue vector mosquito, Aedes aegypti, is difficult due to many reasons, one being the development of resistance to neurotoxic insecticides employed. The biosynthesis of chitin, a major constituent of insect cuticle, is a novel target for population control. Novaluron is a benzoylphenylurea (BPU) that acts as a chitin synthesis inhibitor, already used against mosquitoes. However, information regarding BPU effects on immature mosquito stages and physiological parameters related with mosquito larval development are scarce. A set of physiological parameters were recorded in control developing larvae and novaluron was administered continuously to Ae. aegypti larvae, since early third instar. Larval instar period duration was recorded from third instar until pupation. Chitin content was measured during third and fourth instars. Fourth instars were processed histochemically at the mesothorax region, stained with hematoxylin and eosin (HE) for assessment of internal tissues, and labeled with WGA-FITC to reveal chitinized structures. In control larvae: i) there is a chitin content increase during both third and fourth instars where late third instars contain more chitin than early fourth instars; ii) thoracic organs and a continuous cuticle, closely associated with the underlying epidermis were observed; iii) chitin was continuously present throughout integument cuticle. Novaluron treatment inhibited adult emergence, induced immature mortality, altered adult sex ratio and caused delay in larval development. Moreover, novaluron: i) significantly affected chitin content during larval development; ii) induced a discontinuous and altered cuticle in some regions while epidermis was often thinner or missing; iii) rendered chitin cuticle presence discontinuous and less evident. In both control and novaluron larvae, chitin was present in the peritrophic matrix. This study showed quantitatively and qualitatively evidences of novaluron effects on Ae

  7. Synthesis and Evaluation of Heterocyclic Catechol Mimics as Inhibitors of Catechol-O-methyltransferase (COMT)

    PubMed Central

    2015-01-01

    3-Hydroxy-4-pyridinones and 5-hydroxy-4-pyrimidinones were identified as inhibitors of catechol-O-methyltransferase (COMT) in a high-throughput screen. These heterocyclic catechol mimics exhibit potent inhibition of the enzyme and an improved toxicity profile versus the marketed nitrocatechol inhibitors tolcapone and entacapone. Optimization of the series was aided by X-ray cocrystal structures of the novel inhibitors in complex with COMT and cofactors SAM and Mg2+. The crystal structures suggest a mechanism of inhibition for these heterocyclic inhibitors distinct from previously disclosed COMT inhibitors. PMID:25815153

  8. Effect of protein-synthesis inhibitors on testosterone production in rat testis interstitial tissue and Leydig-cell preparations.

    PubMed Central

    Cooke, B A; Janszen, F H; Clotscher, W F; van der Molen, H J

    1975-01-01

    Luteinizing-hormone-stimulated testosterone biosynthesis was inhibited by cycloheximide during incubation of rat testis intersitial tissue in vitro and also by puromycin and cycloheximide during incubation of Leydig-cell preparations, but not by chloramphenicol. These results suggest that a protein regualtor(s) formed by cytoplasmic protein synthesis is involved in steroidogenesis in the rat testis. The specific effect of cycloheximide and puromycin on protein synthesis rather than on other non-specific processes is suggested by the inhibition of protein synthesis and steroidogenesis with different doses of the inhibitors and the lack of effect of cycloheximide on luteinizing-hormone-induced adenosine 3':5'-cyclic monophosphate production. Stimulation of testosterone production by luteinizing hormone during superfusion of interstitial tissue was detectable within 10-20 min and reached a maximum of 120 min, and thereafter slowly decreased. Cycloheximide added at maximum steroid production caused a rapid decrease in testosterone synthesis which followed first-order kinetics (half-life 13 min), thus indicating that the protein regulator(s) has a short half-life. No effect of cycloheximide, puromycin or chloramphenicol on testosterone production in the absence of added luteinizing hormone was found, suggesting that the basal production of testosterone is independent of protein synthesis. PMID:174545

  9. Synthesis of Chromone, Quinolone, and Benzoxazinone Sulfonamide Nucleosides as Conformationally Constrained Inhibitors of Adenylating Enzymes Required for Siderophore Biosynthesis

    PubMed Central

    Engelhart, Curtis A.; Aldrich, Courtney C.

    2013-01-01

    MbtA catalyzes the first committed step of mycobactin biosynthesis in Mycobacterium tuberculosis (Mtb) and is responsible for the incorporation of salicylic acid into the mycobactin siderophores. 5′-O-[N-(Salicyl)sulfamoyl]adenosine (Sal-AMS) is an extremely potent nucleoside inhibitor of MbtA that possesses excellent activity against whole-cell Mtb, but suffers from poor bioavailability. In an effort to improve the bioavailability, we have designed four conformationally constrained analogues of Sal-AMS that remove two rotatable bonds and the ionized sulfamate group based on computational and structural studies. Herein we describe the synthesis, biochemical, and microbiological evaluation of chromone-, quinolone-, and benzoxazinone-3-sulfonamide derivatives of Sal-AMS. We developed new chemistry to assemble these three heterocycles from common β-ketosulfonamide intermediates. The synthesis of the chromone- and quinolone-3-sulfonamide intermediates features formylation of a β-ketosulfonamide employing dimethylformamide dimethyl acetal to afford an enaminone that can react intramolecularly with a phenol or intermolecularly with a primary amine via addition-elimination reaction(s). The benzoxazinone-3-sulfonamide was prepared by nitrosation of a β-ketosulfonamide followed by intramolecular nucleophilic aromatic substitution. Mitsunobu coupling of these bicyclic sulfonamides with a protected adenosine derivative followed by global deprotection provides a concise synthesis of the respective inhibitors. PMID:23805993

  10. Synthesis of C1 inhibitor in fibroblasts from patients with type I and type II hereditary angioneurotic edema.

    PubMed Central

    Kramer, J; Katz, Y; Rosen, F S; Davis, A E; Strunk, R C

    1991-01-01

    Patients with hereditary angioneurotic edema (HANE) have serum levels of functionally active inhibitor of the first component of complement (C1 INH) between 5 and 30% of normal, instead of the 50% expected from the single normal allele. Increases in rates of catabolism have been documented in patients with HANE and certainly account for some of decrease in C1 INH level. A possible role for a decrease in synthesis of C1 INH in producing serum levels of C1 INH below the expected 50% of normal has not been well studied. We studied the synthesis of C1 INH in skin fibroblast lines, which produce easily detectable amounts of C1 INH. In type I HANE cells, C1 INH synthesis was 19.6 +/- 4.0% (mean +/- SD) of normal, much less than the 50% predicted. In type II HANE cells, the total amount of C1 INH synthesis (functional and dysfunctional) was 98.9 +/- 17% of normal; the functional protein comprised 43% of the total. Thus, type II HANE cells synthesized functional C1 INH at a much greater rate than for the type I cells. In both type I and II HANE cells, amounts of steady-state C1 INH mRNA levels paralleled rates of C1 INH synthesis, indicating that control of C1 INH synthesis occurred at pretranslational levels. Both type I and type II fibroblasts synthesized normal amounts of C1r and C1s. These data suggest that the lower than expected amounts of functionally active C1 INH in type I HANE may be due, in part, to a decrease in rate of synthesis of the protein, and that the expressions of the normal C1 INH allele in HANE is influenced by the type of abnormal allele present. Images PMID:1902490