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Sample records for nad synthesis inhibitor

  1. nadA and nadB of Shigella flexneri 5a are antivirulence loci responsible for the synthesis of quinolinate, a small molecule inhibitor of Shigella pathogenicity.

    PubMed

    Prunier, Anne-Laure; Schuch, Raymond; Fernández, Reinaldo E; Mumy, Karen L; Kohler, Henrik; McCormick, Beth A; Maurelli, Anthony T

    2007-07-01

    The evolution of bacterial pathogens from commensal organisms involves virulence gene acquisition followed by pathoadaptation to the new host, including inactivation of antivirulence loci (AVL). AVL are core ancestral genes whose expression is incompatible with the pathogenic lifestyle. Previous studies identified cadA (encoding lysine decarboxylase) as an AVL of Shigella spp. In this study, AVL of Shigella were identified by examining a phenotypic difference from its non-pathogenic ancestor, Escherichia coli. Unlike most E. coli strains, Shigella spp. are nicotinic acid auxotrophs, the pathway for the de novo synthesis of NAD being uniformly defective. In Shigella flexneri, this defect is due to alterations in the nadA and/or nadB genes encoding the enzyme complex that converts L-aspartate to quinolinate, a precursor to NAD synthesis. Quinolinate was found to inhibit invasion and cell-to-cell spread of Sh. flexneri 5a and its ability to induce polymorphonuclear neutrophil transepithelial migration. Virulence of other Shigella species was also inhibited by quinolinate. Introduction of functional nadA and nadB genes from E. coli K-12 into Sh. flexneri 5a restored its ability to synthesize quinolinate but also resulted in strong attenuation of virulence in this strain. The results define nadA and nadB as AVL in Shigella and validate the concept of pathoadaptive evolution of bacteria from commensal ancestors by inactivation of AVL. They also suggest that studies focusing on this form of bacterial evolution can identify novel inhibitors of virulence in other bacterial pathogens.

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

    PubMed

    Denu, John M

    2007-05-04

    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.

  3. 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

  4. 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.

  5. Flavonoid Apigenin Is an Inhibitor of the NAD+ase CD38

    PubMed Central

    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.

    2013-01-01

    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

  6. 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

  7. Novel concept of enzyme selective nicotinamide adenine dinucleotide (NAD)-modified inhibitors based on enzyme taxonomy from the diphosphate conformation of NAD.

    PubMed

    Fujii, Mikio; Kitagawa, Yasuyuki; Iida, Shui; Kato, Keisuke; Ono, Machiko

    2015-11-15

    The dihedral angle θ of the diphosphate part of NAD(P) were investigated to distinguish the differences in the binding-conformation of NAD(P) to enzymes and to create an enzyme taxonomy. Furthermore, new inhibitors with fixed dihedral angles showed that enzymes could recognize the differences in the dihedral angle θ. We suggest the taxonomy and the dihedral angle θ are important values for chemists to consider when designing inhibitors and drugs that target enzymes.

  8. 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

  9. 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. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Molecular biology of pyridine nucleotide biosynthesis in Escherichia coli. Cloning and characterization of quinolinate synthesis genes nadA and nadB.

    PubMed

    Flachmann, R; Kunz, N; Seifert, J; Gütlich, M; Wientjes, F J; Läufer, A; Gassen, H G

    1988-08-01

    The two genes, nadA and nadB, responsible for quinolinate biosynthesis from aspartate and dihydroxyacetone phosphate in Escherichia coli were cloned and characterized. Quinolinate (pyridine-2,3-dicarboxylate) is the biosynthetic precursor of the pyridine ring of NAD. Gene nadA was identified by complementation in three different nadA mutant strains. Sequence analysis provided an 840-bp open reading frame coding for a 31,555-Da protein. Gene nadB was identified by complementation in a nadB mutant strain and by the L-aspartate oxidase activity of its gene product. Sequence analysis showed a 1620-bp open reading frame coding for a 60,306-Da protein. For both genes, promoter regions and ribosomal binding sites were assigned by comparison to consensus sequences. The nadB gene product, L-aspartate oxidase, was purified to homogeneity and the N-terminal sequence of 19 amino acids was determined. The enzyme was shown to be specific for L-aspartate. High-copy-number vectors, carrying either gene nadA, nadB or nadA + nadB, increased quinolinate production 1.5-fold, 2.0-fold and 15-fold respectively. Both gene products seem to be equally rate-limiting in quinolinate synthesis.

  11. Discovery of bacterial NAD+-dependent DNA ligase inhibitors: optimization of antibacterial activity.

    PubMed

    Stokes, Suzanne S; Huynh, Hoan; Gowravaram, Madhusudhan; Albert, Robert; Cavero-Tomas, Marta; Chen, Brendan; Harang, Jenna; Loch, James T; Lu, Min; Mullen, George B; Zhao, Shannon; Liu, Ce-Feng; Mills, Scott D

    2011-08-01

    Optimization of adenosine analog inhibitors of bacterial NAD(+)-dependent DNA ligase is discussed. Antibacterial activity against Streptococcus pneumoniae and Staphylococcus aureus was improved by modification of the 2-position substituent on the adenine ring and 3'- and 5'-substituents on the ribose. Compounds with logD values 1.5-2.5 maximized potency and maintained drug-like physical properties.

  12. Lung contains an inhibitor for nicotinatemononucleotide pyrophosphorylase (carboxylating) of NAD biosynthesis.

    PubMed

    Seither, R L; Brown, O R; Babu, B V

    1991-01-01

    Rat, cow and foal lung extracts contained an inhibitor for the liver NAD biosynthetic-pathway enzyme, nicotinatemononucleotide pyrophosphorylase (carboxylating) [EC 2.4.2.19]. The inhibitor was not dialyzable, was labile at 100 degrees C, was retained by a 30,000 dalton pore size Amicon membrane and, when partially purified by precipitation at 40-100% ammonium sulfate, inhibited the enzyme stoichiometrically. Lung reportedly does not contain nicotinate-mononucleotide pyrophosphorylase or make NAD de novo. However, the inhibitor would mask detection of the enzyme in lung extracts. We detected a low nicotinatemononucleotide pyrophosphorylase-like activity (0.003 +/- 0.001 nanomoles CO2 produced from quinolinic acid per mg of extract protein) in rat lung but none in foal or cow lung.

  13. Inhibitor and NAD+ binding to poly(ADP-ribose) polymerase as derived from crystal structures and homology modeling.

    PubMed

    Ruf, A; de Murcia, G; Schulz, G E

    1998-03-17

    Inhibitors of poly(ADP-ribose) polymerase (PARP, EC 2.4.2.30) are of clinical interest because they have potential for improving radiation therapy and chemotherapy of cancer. The refined binding structures of four such inhibitors are reported together with the refined structure of the unligated catalytic fragment of the enzyme. Following their design, all inhibitors bind at the position of the nicotinamide moiety of the substrate NAD+. The observed binding mode suggests inhibitor improvements that avoid other NAD(+)-binding enzymes. Because the binding pocket of NAD+ has been strongly conserved during evolution, the homology with ADP-ribosylating bacterial toxins could be used to extend the bound nicotinamide, which is marked by the inhibitors, to the full NAD+ molecule.

  14. 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

  15. Discovery of 4-Amino-8-quinoline Carboxamides as Novel, Submicromolar Inhibitors of NAD-Hydrolyzing Enzyme CD38.

    PubMed

    Becherer, J David; Boros, Eric E; Carpenter, Tiffany Y; Cowan, David J; Deaton, David N; Haffner, Curt D; Jeune, Michael R; Kaldor, Istvan W; Poole, J Chuck; Preugschat, Frank; Rheault, Tara R; Schulte, Christie A; Shearer, Barry G; Shearer, Todd W; Shewchuk, Lisa M; Smalley, Terrence L; Stewart, Eugene L; Stuart, J Darren; Ulrich, John C

    2015-09-10

    Starting from the micromolar 8-quinoline carboxamide high-throughput screening hit 1a, a systematic exploration of the structure-activity relationships (SAR) of the 4-, 6-, and 8-substituents of the quinoline ring resulted in the identification of approximately 10-100-fold more potent human CD38 inhibitors. Several of these molecules also exhibited pharmacokinetic parameters suitable for in vivo animal studies, including low clearances and decent oral bioavailability. Two of these CD38 inhibitors, 1ah and 1ai, were shown to elevate NAD tissue levels in liver and muscle in a diet-induced obese (DIO) C57BL/6 mouse model. These inhibitor tool compounds will enable further biological studies of the CD38 enzyme as well as the investigation of the therapeutic implications of NAD enhancement in disease models of abnormally low NAD.

  16. 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.

  17. Synthesis of influenza neuraminidase inhibitors.

    PubMed

    Abdel-Magid, A F; Maryanoff, C A; Mehrman, S J

    2001-11-01

    Influenza neuraminidase inhibitors provide a means to combat flu, a potentially very serious disease. For the first time, there is a way to treat influenza by blocking the influenza enzyme neuraminidase to stop or slow the progression of infection. The diverse structures and synthesis of several influenza neuraminidase inhibitors are discussed. Contributions from chemical process development groups are highlighted for those compounds that have reached the market, such as zanamivir and oseltamivir phosphate.

  18. 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

  19. 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

  20. Regulation of SIRT2-dependent α-tubulin deacetylation by cellular NAD levels.

    PubMed

    Skoge, Renate Hvidsten; Dölle, Christian; Ziegler, Mathias

    2014-11-01

    Acetylation of α-tubulin on lysine 40 is one of the major posttranslational modifications of microtubules. The acetylation reaction is catalyzed by alpha-tubulin N-acetyltransferase and the modification can be reversed by either the NAD-independent class II histone deacetylase HDAC6 or the NAD-dependent deacetylase SIRT2. In this study, we assessed to what extent cellular NAD levels are involved in the regulation of the α-tubulin acetylation state. Cells were subjected to different treatments known to influence cellular NAD content. In response to NAD depletion caused by inhibition of NAD synthesis from nicotinamide, α-tubulin was hyperacetylated. Under these conditions, the normal tubulin acetylation state could be restored by providing the cells with alternative NAD precursors. Likewise, decreasing the rate of endogenous NAD consumption using an inhibitor of poly-ADP-ribosylation also stabilized the acetylation of α-tubulin. Conversely, the level of acetylated α-tubulin decreased when NAD synthesis was enhanced by overexpression of an NAD biosynthetic enzyme. Combined, these results show that the tubulin acetylation status is reciprocally regulated by cellular NAD levels. Furthermore, we provide evidence confirming that the NAD-dependent regulation of tubulin acetylation is mediated by SIRT2.

  1. Conserved water-mediated recognition and dynamics of NAD+ (carboxamide group) to hIMPDH enzyme: water mimic approach toward the design of isoform-selective inhibitor.

    PubMed

    Bairagya, Hridoy R; Mishra, Deepak K; Mukhopadhyay, Bishnu P; Sekar, K

    2014-01-01

    Inosine monophosphate dehydrogenase (IMPDH) enzyme involves in GMP biosynthesis pathway. Type I hIMPDH is expressed at lower levels in all cells, whereas type II is especially observed in acute myelogenous leukemia, chronic myelogenous leukemia cancer cells, and 10 ns simulation of the IMP-NAD(+) complex structures (PDB ID. 1B3O and 1JCN) have revealed the presence of a few conserved hydrophilic centers near carboxamide group of NAD(+). Three conserved water molecules (W1, W, and W1') in di-nucleotide binding pocket of enzyme have played a significant role in the recognition of carboxamide group (of NAD(+)) to D274 and H93 residues. Based on H-bonding interaction of conserved hydrophilic (water molecular) centers within IMP-NAD(+)-enzyme complexes and their recognition to NAD(+), some covalent modification at carboxamide group of di-nucleotide (NAD(+)) has been made by substituting the -CONH2group by -CONHNH2 (carboxyl hydrazide group) using water mimic inhibitor design protocol. The modeled structure of modified ligand may, though, be useful for the development of antileukemic agent or it could be act as better inhibitor for hIMPDH-II.

  2. Novel bacterial NAD+-dependent DNA ligase inhibitors with broad-spectrum activity and antibacterial efficacy in vivo.

    PubMed

    Mills, Scott D; Eakin, Ann E; Buurman, Ed T; Newman, Joseph V; Gao, Ning; Huynh, Hoan; Johnson, Kenneth D; Lahiri, Sushmita; Shapiro, Adam B; Walkup, Grant K; Yang, Wei; Stokes, Suzanne S

    2011-03-01

    DNA ligases are indispensable enzymes playing a critical role in DNA replication, recombination, and repair in all living organisms. Bacterial NAD+-dependent DNA ligase (LigA) was evaluated for its potential as a broad-spectrum antibacterial target. A novel class of substituted adenosine analogs was discovered by target-based high-throughput screening (HTS), and these compounds were optimized to render them more effective and selective inhibitors of LigA. The adenosine analogs inhibited the LigA activities of Escherichia coli, Haemophilus influenzae, Mycoplasma pneumoniae, Streptococcus pneumoniae, and Staphylococcus aureus, with inhibitory activities in the nanomolar range. They were selective for bacterial NAD+-dependent DNA ligases, showing no inhibitory activity against ATP-dependent human DNA ligase 1 or bacteriophage T4 ligase. Enzyme kinetic measurements demonstrated that the compounds bind competitively with NAD+. X-ray crystallography demonstrated that the adenosine analogs bind in the AMP-binding pocket of the LigA adenylation domain. Antibacterial activity was observed against pathogenic Gram-positive and atypical bacteria, such as S. aureus, S. pneumoniae, Streptococcus pyogenes, and M. pneumoniae, as well as against Gram-negative pathogens, such as H. influenzae and Moraxella catarrhalis. The mode of action was verified using recombinant strains with altered LigA expression, an Okazaki fragment accumulation assay, and the isolation of resistant strains with ligA mutations. In vivo efficacy was demonstrated in a murine S. aureus thigh infection model and a murine S. pneumoniae lung infection model. Treatment with the adenosine analogs reduced the bacterial burden (expressed in CFU) in the corresponding infected organ tissue as much as 1,000-fold, thus validating LigA as a target for antibacterial therapy.

  3. Influence of GSH synthesis inhibition on temporal distribution of NAD+/NADH during vascular endothelial cells proliferation.

    PubMed

    Busu, C; Atanasiu, V; Caldito, G; Aw, T Y

    2014-01-01

    Pathological conditions states such as stroke, diabetes mellitus, hypertension, dyslipidemia are associated with increased levels of free radicals that alter normal function of the vascular endothelium and perturb vascular homeostasis. The redox couples reduced glutathione (GSH)/oxidized glutathione (GSSG), NADH/NAD+, and NADPH/NADP+ play major functions in the intracellular redox balance. Any decrease in tissue or systemic GSH levels under the aforementioned pathologies would enhance oxidative damage to the vascular endothelium. Beside their role as coenzyme that participate in cellular metabolism, pyridine nucleotides serve also as substrate for enzymes involved in DNA repair and longevity. There is scant data on NAD+/NADH kinetics and distribution during human cells proliferation. Here, we determined the influence of cellular GSH status on the early dynamics of nuclear-to-cytosol (N-to-C) NAD+ and nuclear NADH kinetics (6 h interval) over 72 h of endothelial cell proliferation. The IHEC cell line was used as a surrogate for human brain micro vascular endothelial cells. Inhibition of GSH synthesis by buthionine sulfoximine (BSO) and sustained low cellular GSH significantly increased nuclear NADH levels (p<0.01), which correlated with lower nuclear GSH and prolonged cell cycle S-phase. When BSO was removed the pattern of nuclear NAD+ resembled that of control group, but nuclear NADH concentrations remained elevated, as in GSH deficient cells (p<0.01). The coincidence of high nuclear NADH and lower nuclear NAD+ with S-phase prolongation are suggestive of CtBP and NAD+-dependent DNA repair enzyme activation under conditions of decreased cellular GSH. These results provide important insights into GSH control of vascular endothelial growth and restitution, key processes in the restoration of the endothelium adjacent to the post-injury lesion site.

  4. 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.

  5. NAD+-dependent Deacetylase SIRT3 Regulates Mitochondrial Protein Synthesis by Deacetylation of the Ribosomal Protein MRPL10*

    PubMed Central

    Yang, Yongjie; Cimen, Huseyin; Han, Min-Joon; Shi, Tong; Deng, Jian-Hong; Koc, Hasan; Palacios, Orsolya M.; Montier, Laura; Bai, Yidong; Tong, Qiang; Koc, Emine C.

    2010-01-01

    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 protein in the mitochondrial ribosome. Ribosome-associated SIRT3 was found to be responsible for deacetylation of MRPL10 in an NAD+-dependent manner. We mapped the acetylated Lys residues by tandem mass spectrometry and determined the role of these residues in acetylation of MRPL10 by site-directed mutagenesis. Furthermore, we observed that the increased acetylation of MRPL10 led to an increase in translational activity of mitochondrial ribosomes in Sirt3−/− mice. In a similar manner, ectopic expression and knockdown of SIRT3 in C2C12 cells resulted in the suppression and enhancement of mitochondrial protein synthesis, respectively. Our findings constitute the first evidence for the regulation of mitochondrial protein synthesis by the reversible acetylation of the mitochondrial ribosome and characterize MRPL10 as a novel substrate of the NAD+-dependent deacetylase, SIRT3. PMID:20042612

  6. NAD+-dependent DNA Ligase (Rv3014c) from Mycobacterium tuberculosis. Crystal structure of the adenylation domain and identification of novel inhibitors.

    PubMed

    Srivastava, Sandeep Kumar; Tripathi, Rama Pati; Ramachandran, Ravishankar

    2005-08-26

    DNA ligases utilize either ATP or NAD+ as cofactors to catalyze the formation of phosphodiester bonds in nicked DNA. Those utilizing NAD+ are attractive drug targets because of the unique cofactor requirement for ligase activity. We report here the crystal structure of the adenylation domain of the Mycobacterium tuberculosis NAD+-dependent ligase with bound AMP. The adenosine nucleoside moiety of AMP adopts a syn-conformation. The structure also captures a new spatial disposition between the two subdomains of the adenylation domain. Based on the crystal structure and an in-house compound library, we have identified a novel class of inhibitors for the enzyme using in silico docking calculations. The glycosyl ureide-based inhibitors were able to distinguish between NAD+- and ATP-dependent ligases as evidenced by in vitro assays using T4 ligase and human DNA ligase I. Moreover, assays involving an Escherichia coli strain harboring a temperature-sensitive ligase mutant and a ligase-deficient Salmonella typhimurium strain suggested that the bactericidal activity of the inhibitors is due to inhibition of the essential ligase enzyme. The results can be used as the basis for rational design of novel antibacterial agents.

  7. A critical role of autophagy in antileukemia/lymphoma effects of APO866, an inhibitor of NAD biosynthesis.

    PubMed

    Ginet, Vanessa; Puyal, Julien; Rummel, Coralie; Aubry, Dominique; Breton, Caroline; Cloux, Anne-Julie; Majjigapu, Somi R; Sordat, Bernard; Vogel, Pierre; Bruzzone, Santina; Nencioni, Alessio; Duchosal, Michel A; Nahimana, Aimable

    2014-04-01

    APO866, an inhibitor of NAD biosynthesis, exhibits potent antitumor properties in various malignancies. Recently, it has been shown that APO866 induces apoptosis and autophagy in human hematological cancer cells, but the role of autophagy in APO866-induced cell death remains unclear. Here, we report studies on the molecular mechanisms underlying APO866-induced cell death with emphasis on autophagy. Treatment of leukemia and lymphoma cells with APO866 induced both autophagy, as evidenced by an increase in autophagosome formation and in SQSTM1/p62 degradation, but also increased caspase activation as revealed by CASP3/caspase 3 cleavage. As an underlying mechanism, APO866-mediated autophagy was found to deplete CAT/catalase, a reactive oxygen species (ROS) scavenger, thus promoting ROS production and cell death. Inhibition of autophagy by ATG5 or ATG7 silencing prevented CAT degradation, ROS production, caspase activation, and APO866-induced cell death. Finally, supplementation with exogenous CAT also abolished APO866 cytotoxic activity. Altogether, our results indicated that autophagy is essential for APO866 cytotoxic activity on cells from hematological malignancies and also indicate an autophagy-dependent CAT degradation, a novel mechanism for APO866-mediated cell killing. Autophagy-modulating approaches could be a new way to enhance the antitumor activity of APO866 and related agents.

  8. The crystal structure of human alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase in complex with 1,3-dihydroxyacetonephosphate suggests a regulatory link between NAD synthesis and glycolysis.

    PubMed

    Garavaglia, Silvia; Perozzi, Silvia; Galeazzi, Luca; Raffaelli, Nadia; Rizzi, Menico

    2009-11-01

    The enzyme alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD) is a zinc-dependent amidohydrolase that participates in picolinic acid (PA), quinolinic acid (QA) and NAD homeostasis. Indeed, the enzyme stands at a branch point of the tryptophan to NAD pathway, and determines the final fate of the amino acid, i.e. transformation into PA, complete oxidation through the citric acid cycle, or conversion into NAD through QA synthesis. Both PA and QA are key players in a number of physiological and pathological conditions, mainly affecting the central nervous system. As their relative concentrations must be tightly controlled, modulation of ACMSD activity appears to be a promising prospect for the treatment of neurological disorders, including cerebral malaria. Here we report the 2.0 A resolution crystal structure of human ACMSD in complex with the glycolytic intermediate 1,3-dihydroxyacetonephosphate (DHAP), refined to an R-factor of 0.19. DHAP, which we discovered to be a potent enzyme inhibitor, resides in the ligand binding pocket with its phosphate moiety contacting the catalytically essential zinc ion through mediation of a solvent molecule. Arg47, Asp291 and Trp191 appear to be the key residues for DHAP recognition in human ACMSD. Ligand binding induces a significant conformational change affecting a strictly conserved Trp-Met couple, and we propose that these residues are involved in controlling ligand admission into ACMSD. Our data may be used for the design of inhibitors with potential medical interest, and suggest a regulatory link between de novo NAD biosynthesis and glycolysis.

  9. 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.

  10. Asymmetric Synthesis of Akt Kinase Inhibitor Ipatasertib.

    PubMed

    Han, Chong; Savage, Scott; Al-Sayah, Mohammad; Yajima, Herbert; Remarchuk, Travis; Reents, Reinhard; Wirz, Beat; Iding, Hans; Bachmann, Stephan; Fantasia, Serena M; Scalone, Michelangelo; Hell, André; Hidber, Pirmin; Gosselin, Francis

    2017-09-15

    A highly efficient asymmetric synthesis of the Akt kinase inhibitor ipatasertib (1) is reported. The bicyclic pyrimidine 2 starting material was prepared via a nitrilase biocatalytic resolution, halogen-metal exchange/anionic cyclization, and a highly diastereoselective biocatalytic ketone reduction as key steps. The route also features a halide activated, Ru-catalyzed asymmetric hydrogenation of a vinylogous carbamic acid to produce α-aryl-β-amino acid 3 in high yield and enantioselectivity. The API was assembled in a convergent manner through a late-stage amidation/deprotection/monohydrochloride salt formation sequence.

  11. The isoniazid-NAD adduct is a slow, tight-binding inhibitor of InhA, the Mycobacterium tuberculosis enoyl reductase: adduct affinity and drug resistance.

    PubMed

    Rawat, Richa; Whitty, Adrian; Tonge, Peter J

    2003-11-25

    Isoniazid (INH), a frontline antitubercular drug, inhibits InhA, the enoyl reductase from Mycobacterium tuberculosis, by forming a covalent adduct with the NAD cofactor. Here, we report that the INH-NAD adduct is a slow, tight-binding competitive inhibitor of InhA. Demonstration that the adduct binds to WT InhA by a two-step enzyme inhibition mechanism, with initial, weak binding (K(-1) = 16 +/- 11 nM) followed by slow conversion to a final inhibited complex (EI*) with overall Ki = 0.75 +/- 0.08 nM, reconciles existing contradictory values for the inhibitory potency of INH-NAD for InhA. The first order rate constant for conversion of the initial EI complex to EI* (k2 = 0.13 +/- 0.01 min(-1)) is similar to the maximum rate constant observed for InhA inhibition in reaction mixtures containing InhA, INH, NADH, and the INH-activating enzyme KatG (catalase/peroxidase from M. tuberculosis), consistent with an inhibition mechanism in which the adduct forms in solution rather than on the enzyme. Importantly, three mutations that correlate with INH resistance, I21V, I47T, and S94A, have little impact on the inhibition constants. Thus, drug resistance does not result simply from a reduction in affinity of INH-NAD for pure InhA. Instead, we hypothesize that protein-protein interactions within the FASII complex are critical to the mechanism of INH action. Finally, for M161V, an InhA mutation that correlates with resistance to the common biocide triclosan in Mycobacterium smegmatis, binding to form the initial EI complex is significantly weakened, explaining why this mutant inactivates more slowly than WT InhA when incubated with INH, NADH, and KatG.

  12. De-novo NAD+ synthesis regulates SIRT1-FOXO1 apoptotic pathway in response to NQO1 substrates in lung cancer cells

    PubMed Central

    Cheng, Xuefang; Li, Qingran; Liu, Fang; Ye, Hui; Zhao, Min; Wang, Hong; Wang, Guangji; Hao, Haiping

    2016-01-01

    Tryptophan metabolism is essential in diverse kinds of tumors via regulating tumor immunology. However, the direct role of tryptophan metabolism and its signaling pathway in cancer cells remain largely elusive. Here, we establish a mechanistic link from L-type amino acid transporter 1 (LAT1) mediated transport of tryptophan and the subsequent de-novo NAD+ synthesis to SIRT1-FOXO1 regulated apoptotic signaling in A549 cells in response to NQO1 activation. In response to NQO1 activation, SIRT1 is repressed leading to the increased cellular accumulation of acetylated FOXO1 that transcriptionally activates apoptotic signaling. Decreased uptake of tryptophan due to the downregulation of LAT1 coordinates with PARP-1 hyperactivation to induce rapid depletion of NAD+ pool. Particularly, the LAT1-NAD+-SIRT1 signaling is activated in tumor tissues of patients with non-small cell lung cancer. Because NQO1 activation is characterized with oxidative challenge induced DNA damage, these results suggest that LAT1 and de-novo NAD+ synthesis in NSCLC cells may play essential roles in sensing excessive oxidative stress. PMID:27566573

  13. 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

  14. Practical synthesis of a p38 MAP kinase inhibitor.

    PubMed

    Achmatowicz, Michał; Thiel, Oliver R; Wheeler, Philip; Bernard, Charles; Huang, Jinkun; Larsen, Robert D; Faul, Margaret M

    2009-01-16

    p38 MAP kinase inhibitors have attracted considerable interest as potential agents for the treatment of inflammatory diseases. Herein, we describe a concise and efficient synthesis of inhibitor 1 that is based on a phthalazine scaffold. Highlights of our approach include a practical synthesis of a 1,6-disubstituted phthalazine building block 24 as well as the one-pot formation of boronic acid 27. Significant synthetic work to understand the reactivity principles of the intermediates helped in selection of the final synthetic route. Subsequent optimization of the individual steps of the final sequence led to a practical synthesis of 1.

  15. Complexes of bacterial nicotinate mononucleotide adenylyltransferase with inhibitors: implication for structure-based drug design and improvement§

    PubMed Central

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

    2010-01-01

    Bacterial nicotinate mononucleotide adenylyltransferase encoded by the essential gene nadD plays a central role in the synthesis of the redox cofactor NAD+. 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. PMID:20578699

  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. 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

  18. Base-modified NAD and AMP derivatives and their activity against bacterial DNA ligases.

    PubMed

    Pergolizzi, Giulia; Cominetti, Marco M D; Butt, Julea N; Field, Robert A; Bowater, Richard P; Wagner, Gerd K

    2015-06-14

    We report the chemical synthesis and conformational analysis of a collection of 2-, 6- and 8-substituted derivatives of β-NAD(+) and AMP, and their biochemical evaluation against NAD(+)-dependent DNA ligases from Escherichia coli and Mycobacterium tuberculosis. Bacterial DNA ligases are validated anti-microbial targets, and new strategies for their inhibition are therefore of considerable scientific and practical interest. Our study includes several pairs of β-NAD(+) and AMP derivatives with the same substitution pattern at the adenine base. This has enabled the first direct comparison of co-substrate and inhibitor behaviour against bacterial DNA ligases. Our results suggest that an additional substituent in position 6 or 8 of the adenine base in β-NAD(+) is detrimental for activity as either co-substrate or inhibitor. In contrast, substituents in position 2 are not only tolerated, but appear to give rise to a new mode of inhibition, which targets the conformational changes these DNA ligases undergo during catalysis. Using a molecular modelling approach, we highlight that these findings have important implications for our understanding of ligase mechanism and inhibition, and may provide a promising starting point for the rational design of a new class of inhibitors against NAD(+)-dependent DNA ligases.

  19. 8-Thioalkyl-adenosine derivatives inhibit Listeria monocytogenes NAD kinase through a novel binding mode.

    PubMed

    Paoletti, Julie; Assairi, Liliane; Gelin, Muriel; Huteau, Valérie; Nahori, Marie-Anne; Dussurget, Olivier; Labesse, Gilles; Pochet, Sylvie

    2016-11-29

    Increased resistance of pathogens to existing antibiotics necessitates the search for novel targets to develop potent antimicrobials. Biosynthetic pathways of several cofactors important for bacterial growth, such as nicotinamide adenine dinucleotide phosphate (NADP), have been proposed as a promising source of antibiotic targets. Nicotinamide adenine dinucleotide kinases (NADK; EC 2.7.1.23) are attractive for inhibitor development, since they catalyze the phosphorylation of NAD to NADP, which is an essential step of NADP metabolism. We previously synthesized diadenosine derivatives that inhibited NADK from two human pathogens, Listeria monocytogenes and Staphylococcus aureus, in the micromolar range. They behave as NAD mimics with the 5',5'-diphosphate group substituted by a 8,5' thioglycolic bridge. In an attempt to improve inhibitory potency, we designed new NAD mimics based on a single adenosine moiety harboring a larger derivatization attached to the C8 position and a small group at the 5' position. Here we report the synthesis of a series of 8-thioalkyl-adenosine derivatives containing various aryl and heteroaryl moieties and their evaluation as inhibitors of L. monocytogenes NADK1, S. aureus NADK and their human counterpart. Novel, sub-micromolar inhibitors of LmNADK1 were identified. Surprisingly, most LmNADK1 inhibitors demonstrated a high selectivity index against the close staphylococcal ortholog and the human NADK. Structural characterization of enzyme-inhibitor complexes revealed the original binding mode of these novel NAD mimics.

  20. 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

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

    USDA-ARS?s Scientific Manuscript database

    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 ...

  2. Synthesis of Berberine–Efflux Pump Inhibitor Hybrid Antibacterials

    PubMed Central

    Bremner, John B.; Kelso, Michael J.

    2010-01-01

    This paper reports the compact synthesis of two isomeric dual-action hybrid antimicrobials where the 13-position of the antibacterial berberine has been linked via 3'- and 4'-methylene bridges to INF55 (5-nitro-2-phenylindole), an inhibitor of the bacterial NorA multidrug-resistance pump. PMID:21311737

  3. Catastrophic NAD+ Depletion in Activated T Lymphocytes through Nampt Inhibition Reduces Demyelination and Disability in EAE

    PubMed Central

    Ferrando, Tiziana; Poggi, Alessandro; Garuti, Anna; D'Urso, Agustina; Selmo, Martina; Benvenuto, Federica; Cea, Michele; Zoppoli, Gabriele; Moran, Eva; Soncini, Debora; Ballestrero, Alberto; Sordat, Bernard; Patrone, Franco; Mostoslavsky, Raul; Uccelli, Antonio; Nencioni, Alessio

    2009-01-01

    Nicotinamide phosphoribosyltransferase (Nampt) inhibitors such as FK866 are potent inhibitors of NAD+ synthesis that show promise for the treatment of different forms of cancer. Based on Nampt upregulation in activated T lymphocytes and on preliminary reports of lymphopenia in FK866 treated patients, we have investigated FK866 for its capacity to interfere with T lymphocyte function and survival. Intracellular pyridine nucleotides, ATP, mitochondrial function, viability, proliferation, activation markers and cytokine secretion were assessed in resting and in activated human T lymphocytes. In addition, we used experimental autoimmune encephalomyelitis (EAE) as a model of T-cell mediated autoimmune disease to assess FK866 efficacy in vivo. We show that activated, but not resting, T lymphocytes undergo massive NAD+ depletion upon FK866-mediated Nampt inhibition. As a consequence, impaired proliferation, reduced IFN-γ and TNF-α production, and finally autophagic cell demise result. We demonstrate that upregulation of the NAD+-degrading enzyme poly-(ADP-ribose)-polymerase (PARP) by activated T cells enhances their susceptibility to NAD+ depletion. In addition, we relate defective IFN-γ and TNF-α production in response to FK866 to impaired Sirt6 activity. Finally, we show that FK866 strikingly reduces the neurological damage and the clinical manifestations of EAE. In conclusion, Nampt inhibitors (and possibly Sirt6 inhibitors) could be used to modulate T cell-mediated immune responses and thereby be beneficial in immune-mediated disorders. PMID:19936064

  4. An NAD+-dependent transcriptional program governs self-renewal and radiation resistance in glioblastoma

    PubMed Central

    Gujar, Amit D.; Le, Son; Mao, Diane D.; Dadey, David Y. A.; Turski, Alice; Sasaki, Yo; Aum, Diane; Luo, Jingqin; Dahiya, Sonika; Yuan, Liya; Rich, Keith M.; Milbrandt, Jeffrey; Hallahan, Dennis E.; Tran, David D.; Kim, Albert H.

    2016-01-01

    Accumulating evidence suggests cancer cells exhibit a dependency on metabolic pathways regulated by nicotinamide adenine dinucleotide (NAD+). Nevertheless, how the regulation of this metabolic cofactor interfaces with signal transduction networks remains poorly understood in glioblastoma. Here, we report nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting step in NAD+ synthesis, is highly expressed in glioblastoma tumors and patient-derived glioblastoma stem-like cells (GSCs). High NAMPT expression in tumors correlates with decreased patient survival. Pharmacological and genetic inhibition of NAMPT decreased NAD+ levels and GSC self-renewal capacity, and NAMPT knockdown inhibited the in vivo tumorigenicity of GSCs. Regulatory network analysis of RNA sequencing data using GSCs treated with NAMPT inhibitor identified transcription factor E2F2 as the center of a transcriptional hub in the NAD+-dependent network. Accordingly, we demonstrate E2F2 is required for GSC self-renewal. Downstream, E2F2 drives the transcription of members of the inhibitor of differentiation (ID) helix–loop–helix gene family. Finally, we find NAMPT mediates GSC radiation resistance. The identification of a NAMPT-E2F2-ID axis establishes a link between NAD+ metabolism and a self-renewal transcriptional program in glioblastoma, with therapeutic implications for this formidable cancer. PMID:27930300

  5. Design, synthesis, and evaluation of inhibitors of pyruvate phosphate dikinase.

    PubMed

    Wu, Chun; Dunaway-Mariano, Debra; Mariano, Patrick S

    2013-03-01

    Pyruvate phosphate dikinase (PPDK) catalyzes the phosphorylation reaction of pyruvate that forms phosphoenolpyruvate (PEP) via two partial reactions: PPDK + ATP + P(i) → PPDK-P + AMP + PP(i) and PPDK-P + pyruvate → PEP + PPDK. Based on its role in the metabolism of microbial human pathogens, PPDK is a potential drug target. A screen of substances that bind to the PPDK ATP-grasp domain active site revealed that flavone analogues are potent inhibitors of the Clostridium symbiosum PPDK. In silico modeling studies suggested that placement of a 3–6 carbon-tethered ammonium substituent at the 3′- or 4′-positions of 5,7-dihydroxyflavones would result in favorable electrostatic interactions with the PPDK Mg-ATP binding site. As a result, polymethylene-tethered amine derivatives of 5,7-dihydroxyflavones were prepared. Steady-state kinetic analysis of these substances demonstrates that the 4′-aminohexyl-5,7-dyhydroxyflavone 10 is a potent competitive PPDK inhibitor (K(i) = 1.6 ± 0.1 μM). Single turnover experiments were conducted using 4′-aminopropyl-5,7-dihydroxyflavone 7 to show that this flavone specifically targets the ATP binding site and inhibits catalysis of only the PPDK + ATP + P(i) → PPDK-P + AMP PP(i) partial reaction. Finally, the 4′-aminopbutyl-5,7-dihydroxyflavone 8 displays selectivity for inhibition of PPDK versus other enzymes that utilize ATP and NAD.

  6. Synthesis of macrocyclic trypanosomal cysteine protease inhibitors.

    PubMed

    Chen, Yen Ting; Lira, Ricardo; Hansell, Elizabeth; McKerrow, James H; Roush, William R

    2008-11-15

    The importance of cysteine proteases in parasites, compounded with the lack of redundancy compared to their mammalian hosts makes proteases attractive targets for the development of new therapeutic agents. The binding mode of K11002 to cruzain, the major cysteine protease of Trypanosoma cruzi was used in the design of conformationally constrained inhibitors. Vinyl sulfone-containing macrocycles were synthesized via olefin ring-closing metathesis and evaluated against cruzain and the closely related cysteine protease, rhodesain.

  7. Screening and in situ synthesis using crystals of a NAD kinase lead to a potent antistaphylococcal compound.

    PubMed

    Gelin, Muriel; Poncet-Montange, Guillaume; Assairi, Liliane; Morellato, Laurence; Huteau, Valérie; Dugué, Laurence; Dussurget, Olivier; Pochet, Sylvie; Labesse, Gilles

    2012-06-06

    Making new ligands for a given protein by in situ ligation of building blocks (or fragments) is an attractive method. However, it suffers from inherent limitations, such as the limited number of available chemical reactions and the low information content of usual chemical library deconvolution. Here, we describe a focused screening of adenosine derivatives using X-ray crystallography. We discovered an unexpected and biocompatible chemical reactivity and have simultaneously identified the mode of binding of the resulting products. We observed that the NAD kinase from Listeria monocytogenes (LmNADK1) can promote amide formation between 5'-amino-5'-deoxyadenosine and carboxylic acid groups. This unexpected reactivity allowed us to bridge in situ two adenosine derivatives to fully occupy the active NAD site. This guided the design of a close analog showing micromolar inhibition of two human pathogenic NAD kinases and potent bactericidal activity against Staphylococcus aureus in vitro. Copyright © 2012 Elsevier Ltd. All rights reserved.

  8. Glutamine versus Ammonia Utilization in the NAD Synthetase Family

    PubMed Central

    Shatalin, Konstantin; Gelfand, Mikhail S.; Osterman, Andrei L.; Sorci, Leonardo

    2012-01-01

    NAD is a ubiquitous and essential metabolic redox cofactor which also functions as a substrate in certain regulatory pathways. The last step of NAD synthesis is the ATP-dependent amidation of deamido-NAD by NAD synthetase (NADS). Members of the NADS family are present in nearly all species across the three kingdoms of Life. In eukaryotic NADS, the core synthetase domain is fused with a nitrilase-like glutaminase domain supplying ammonia for the reaction. This two-domain NADS arrangement enabling the utilization of glutamine as nitrogen donor is also present in various bacterial lineages. However, many other bacterial members of NADS family do not contain a glutaminase domain, and they can utilize only ammonia (but not glutamine) in vitro. A single-domain NADS is also characteristic for nearly all Archaea, and its dependence on ammonia was demonstrated here for the representative enzyme from Methanocaldococcus jannaschi. However, a question about the actual in vivo nitrogen donor for single-domain members of the NADS family remained open: Is it glutamine hydrolyzed by a committed (but yet unknown) glutaminase subunit, as in most ATP-dependent amidotransferases, or free ammonia as in glutamine synthetase? Here we addressed this dilemma by combining evolutionary analysis of the NADS family with experimental characterization of two representative bacterial systems: a two-subunit NADS from Thermus thermophilus and a single-domain NADS from Salmonella typhimurium providing evidence that ammonia (and not glutamine) is the physiological substrate of a typical single-domain NADS. The latter represents the most likely ancestral form of NADS. The ability to utilize glutamine appears to have evolved via recruitment of a glutaminase subunit followed by domain fusion in an early branch of Bacteria. Further evolution of the NADS family included lineage-specific loss of one of the two alternative forms and horizontal gene transfer events. Lastly, we identified NADS structural

  9. Screening of Zulu medicinal plants for prostaglandin-synthesis inhibitors.

    PubMed

    Jäger, A K; Hutchings, A; van Staden, J

    1996-06-01

    Aqueous and ethanolic extracts of 39 plants used in traditional Zulu medicine to treat headache or inflammatory diseases were screened for prostaglandin-synthesis inhibitors. Extracts were tested in an in vitro assay for cyclooxygenase inhibitors. In general, ethanolic extracts caused higher inhibition than aqueous extracts. Two-thirds of the plants screened had high inhibitory activity. The highest inhibition was obtained with ethanolic extracts of Bidens pilosa, Eucomis autumnalis, Harpephyllum caffrum, Helichrysum nudifolium, Leonotis intermedia, L. leonorus, Ocotea bullata, Rumex saggitatus, Solanum mauritianum, Synadenium cupulare and Trichilia dregeana.

  10. 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.

  11. 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.

  12. 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.

  13. Regulation of NAD metabolism in Salmonella typhimurium: molecular sequence analysis of the bifunctional nadR regulator and the nadA-pnuC operon.

    PubMed Central

    Foster, J W; Park, Y K; Penfound, T; Fenger, T; Spector, M P

    1990-01-01

    In Salmonella typhimurium, de novo synthesis of NAD is regulated through the transcriptional control of the nadA and nadB loci. Likewise, the pyridine nucleotide salvage pathway is controlled at pncB. The transcriptional expression of these three loci is coordinately regulated by the product of nadR. However, there is genetic evidence suggesting that NadR is bifunctional, serving in both regulatory and transport capacities. One class of mutations in the nadR locus imparts a transport-defective PnuA- phenotype. These mutants retain regulation properties but are unable to transport nicotinamide mononucleotide (NMN) intact across the cell membrane. Other nadR mutants lose both regulatory and transport capabilities, while a third class loses only regulatory ability. The unusual NMN transport activity requires both the PnuC and NadR proteins, with the pnuC locus residing in an operon with nadA. To prove that nadR encoded a single protein and to gain insight into a regulatory target locus, the nadR and nadA pnuC loci were cloned and sequenced. A DNA fragment which complemented both regulatory and transport mutations was found to contain a single open reading frame capable of encoding a 409-amino-acid protein (47,022 daltons), indicating that NadR is indeed bifunctional. Confirmation of the operon arrangement for nadA and pnuC was obtained through the sequence analysis of a 2.4-kilobase DNA fragment which complemented both NadA and PnuC mutant phenotypes. The nadA product, confirmed in maxicells, was a 365-amino-acid protein (40,759 daltons), while pnuC encoded a 322-amino-acid protein (36,930 daltons). The extremely hydrophobic (71%) nature of the PnuC protein indicated that it was an integral membrane protein, consistent with its central role in the transport of NMN across the cytoplasmic membrane. The results presented here and in previous studies suggest a hypothetical model in which NadR interacts with PnuC at low internal NAD levels, permitting transport of NMN

  14. Regulation of NAD metabolism in Salmonella typhimurium: molecular sequence analysis of the bifunctional nadR regulator and the nadA-pnuC operon.

    PubMed

    Foster, J W; Park, Y K; Penfound, T; Fenger, T; Spector, M P

    1990-08-01

    In Salmonella typhimurium, de novo synthesis of NAD is regulated through the transcriptional control of the nadA and nadB loci. Likewise, the pyridine nucleotide salvage pathway is controlled at pncB. The transcriptional expression of these three loci is coordinately regulated by the product of nadR. However, there is genetic evidence suggesting that NadR is bifunctional, serving in both regulatory and transport capacities. One class of mutations in the nadR locus imparts a transport-defective PnuA- phenotype. These mutants retain regulation properties but are unable to transport nicotinamide mononucleotide (NMN) intact across the cell membrane. Other nadR mutants lose both regulatory and transport capabilities, while a third class loses only regulatory ability. The unusual NMN transport activity requires both the PnuC and NadR proteins, with the pnuC locus residing in an operon with nadA. To prove that nadR encoded a single protein and to gain insight into a regulatory target locus, the nadR and nadA pnuC loci were cloned and sequenced. A DNA fragment which complemented both regulatory and transport mutations was found to contain a single open reading frame capable of encoding a 409-amino-acid protein (47,022 daltons), indicating that NadR is indeed bifunctional. Confirmation of the operon arrangement for nadA and pnuC was obtained through the sequence analysis of a 2.4-kilobase DNA fragment which complemented both NadA and PnuC mutant phenotypes. The nadA product, confirmed in maxicells, was a 365-amino-acid protein (40,759 daltons), while pnuC encoded a 322-amino-acid protein (36,930 daltons). The extremely hydrophobic (71%) nature of the PnuC protein indicated that it was an integral membrane protein, consistent with its central role in the transport of NMN across the cytoplasmic membrane. The results presented here and in previous studies suggest a hypothetical model in which NadR interacts with PnuC at low internal NAD levels, permitting transport of NMN

  15. Preventing NAD+ Depletion Protects Neurons against Excitotoxicity

    PubMed Central

    Liu, Dong; Pitta, Michael; Mattson, Mark P.

    2008-01-01

    Neurons are excitable cells that require large amounts of energy to support their survival and functions and are therefore prone to excitotoxicity, which involves energy depletion. By examining bioenergetic changes induced by glutamate, we found that the cellular nicotinamide adenine dinucleotide (NAD+) level is a critical determinant of neuronal survival. The bioenergetic effects of mitochondrial uncoupling and caloric restriction were also examined in cultured neurons and rodent brain. 2, 4-dinitrophenol (DNP) is a chemical mitochondrial uncoupler that stimulates glucose uptake and oxygen consumption on cultured neurons, which accelerates oxidation of NAD(P)H to NAD+ in mitochondria. The NAD+-dependent histone deacetylase sirtulin 1 (SIRT1) and glucose transporter 1 (GLUT1) mRNA are upregulated mouse brain under caloric restriction. To examine whether NAD+ mediates neuroprotective effects, nicotinamide, a precursor of NAD+ and inhibitor of SIRT1 and poly (ADP-ribose) polymerase 1 (PARP1) (two NAD+-dependent enzymes), was employed. Nicotinamide attenuated excitotoxic death and preserved cellular NAD+ levels to support SIRT1 and PARP 1 activities. Our findings suggest that mild mitochondrial uncoupling and caloric restriction exert hormetic effects by stimulating bioenergetics in neurons thereby increasing tolerance of neurons to metabolic stress. PMID:19076449

  16. 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.

  17. Enantioselective synthesis of new oxazolidinylthiazolidines as enzyme inhibitors.

    PubMed

    Saiz, Cecilia; Villamil, Valentina; González, Mariano M; Rossi, Ma Agustina; Martínez, Lorena; Suescun, Leopoldo; Vila, Alejandro J; Mahler, Graciela

    2017-01-15

    The synthesis of new oxazolidinylthiazolidines bicycles, oxygen analogues of bisthiazolidines, also known as metallo-β-lactamase inhibitors is described. The reaction of β-aminoalcohols and 2,5-dihydroxy-1,4-dithiane led to oxazolidinylthiazolidines and/or dithia-azabicycles as the main products. The distribution pattern depends mainly on the aminoalcohol substituents. In a one-pot reaction, four new bonds are formed in good yields and with high atom efficiency. When the oxazolidinylthiazolidines are formed, two stereogenic centres are generated with high enantiospecificity. The reaction mechanism is discussed based on crystallographic data and interconversion studies. Two oxazolidinylthiazolidines were evaluated as inhibitors of the potent lactamase NDM-1 and compound 4f displayed competitive inhibition with Ki = 1.6 ± 0.6 µM.

  18. Inhibitors of Angiogenesis in Cancer Therapy - Synthesis and Biological Activity.

    PubMed

    Gensicka, Monika; Głowacka, Agnieszka; Dzierzbicka, Krystyna; Cholewinski, Grzegorz

    2015-01-01

    Angiogenesis is the process of formation of new capillaries from preexisting blood vessels. Angiogenesis is involved in normal physiological processes, and plays an important role in tumor invasion and development of metastases. Vascular endothelial growth factor (VEGF) plays a key role in angiogenesis. VEGF is a mitogen for vascular endothelial cells and stimulates their proliferation. By inhibiting the biological activity of VEGF, and then signal cascades with neutralizing VEGF antibodies and signal inhibitors, may negatively regulate the growth and metastasis. Anti-angiogenesis therapy is less toxic than chemotherapy. Angiogenesis is a multistep and multifactorial process, and therefore, can be blocked at different levels. In this review article, the authors present the synthesis of novel inhibitors of angiogenesis, together with the results of biological tests in vitro, and in some cases, state trials.

  19. Synthesis of antifungal glucan synthase inhibitors from enfumafungin.

    PubMed

    Zhong, Yong-Li; Gauthier, Donald R; Shi, Yao-Jun; McLaughlin, Mark; Chung, John Y L; Dagneau, Philippe; Marcune, Benjamin; Krska, Shane W; Ball, Richard G; Reamer, Robert A; Yasuda, Nobuyoshi

    2012-04-06

    An efficient, new, and scalable semisynthesis of glucan synthase inhibitors 1 and 2 from the fermentation product enfumafungin 3 is described. The highlights of the synthesis include a high-yielding ether bond-forming reaction between a bulky sulfamidate 17 and alcohol 4 and a remarkably chemoselective, improved palladium(II)-mediated Corey-Yu allylic oxidation at the highly congested C-12 position of the enfumafungin core. Multi-hundred gram quantities of the target drug candidates 1 and 2 were prepared, in 12 linear steps with 25% isolated yield and 13 linear steps with 22% isolated yield, respectively.

  20. Design, synthesis and in vitro evaluation of potent, novel, small molecule inhibitors of plasminogen activator inhibitor-1.

    PubMed

    Folkes, Adrian; Brown, S David; Canne, Lynne E; Chan, Jocelyn; Engelhardt, Erin; Epshteyn, Sergey; Faint, Richard; Golec, Julian; Hanel, Art; Kearney, Patrick; Leahy, James W; Mac, Morrison; Matthews, David; Prisbylla, Michael P; Sanderson, Jason; Simon, Reyna J; Tesfai, Zerom; Vicker, Nigel; Wang, Shouming; Webb, Robert R; Charlton, Peter

    2002-04-08

    We have synthesized and evaluated a series of tetramic acid-based and hydroxyquinolinone-based inhibitors of plasminogen activator inhibitor-1 (PAI-1). These studies resulted in the identification of several compounds which showed excellent potency against PAI-1. The design, synthesis and SAR of these compounds are described.

  1. Transport of NAD+ in Percoll-Purified Potato Tuber Mitochondria

    PubMed Central

    Neuburger, Michel; Day, David A.; Douce, Roland

    1985-01-01

    A mechanism by which intact potato (Solanum tuberosum) mitochondria may regulate the matrix NAD content was studied in vitro. If mitochondria were incubated with NAD+ at 25°C in 0.3 molar mannitol, 10 millimolar phosphate buffer (pH 7.4), 5 millimolar MgCl2, and 5 millimolar α-ketoglutarate, the NAD pool size increased with time. In the presence of uncouplers, net uptake was not only inhibited, but NAD+ efflux was observed instead. Furthermore, the rate of NAD+ accumulation in the matrix space was strongly inhibited by the analog N-4-azido-2-nitrophenyl-4-aminobutyryl-3′-NAD+. When suspended in a medium that avoided rupture of the outer membrane, intact purified mitochondria progressively lost their NAD+ content. This led to a slow decrease of NAD+-linked substrates oxidation by isolated mitochondria The rate of NAD+ efflux from the matrix space was strongly temperature dependent and was inhibited by the analog inhibitor of NAD+ transport indicating that a carrier was required for net flux in either direction. It is proposed that uptake and efflux operate to regulate the total matrix NAD pool size. PMID:16664254

  2. Identification of a repressor gene involved in the regulation of NAD de novo biosynthesis in Salmonella typhimurium.

    PubMed Central

    Zhu, N; Olivera, B M; Roth, J R

    1988-01-01

    Mutations at the nadI locus affect expression of the first two genes of NAD synthesis, nadA and nadB, which are unlinked. Genetic data imply that the regulatory effects of nadI mutations are not due to indirect consequences of physiological alterations. Two types of mutations map in the nadI region. Common null mutations (nadI) show constitutive high-level expression of the nadB and nadA genes. Rare nadIs mutations cause constitutive low-level expression of nadB and nadA. Some nadIs mutations shut off the expression of the biosynthetic genes sufficiently to cause a nicotinic acid auxotrophy. Spontaneous revertants of auxotrophic nadIs mutants have a NadI- phenotype, including some with deletions of the nadI locus. The nadI locus encodes a repressor protein acting on the unlinked nadA and nadB genes. PMID:3275606

  3. Thromboxane synthesis inhibitors and postprandial jejunal capillary exchange capacity.

    PubMed

    Mangino, M J; Chou, C C

    1988-05-01

    The effects of thromboxane synthesis inhibitors (imidazole and U 63557A; Upjohn) and the cyclooxygenase inhibitor, mefenamic acid, on jejunal capillary filtration coefficients (Kfc) were determined in dogs before and during the presence of predigested food in the jejunal lumen. The jejunal Kfc increased significantly soon after the placement of a predigested test food containing all major constituents of diet. The Kfc remained elevated as long as the food was present in the lumen (15 min). Mefenamic acid (10 mg/kg iv) did not significantly alter resting jejunal Kfc or alter the food-induced increase in Kfc. Imidazole (5.0 mg/min ia) or U 63557A (5.0 mg/kg iv) per se significantly increased jejunal Kfc. Placement of digested food further increased the Kfc to levels significantly higher than those observed before administration of the two thromboxane synthase inhibitors. Production of thromboxane B2 by jejunal tissue was significantly reduced and 6-ketoprostaglandin F1 alpha (the stable hydrolysis product of prostacyclin) production was significantly increased after administration of U 63557A. Our study indicates that the relative production of endogenous thromboxanes and other prostanoids modulates jejunal capillary exchange capacity in the absence or presence of digested food in the jejunal lumen.

  4. The chemistry of nicotinamide adenine dinucleotide (NAD) analogues containing C-nucleosides related to nicotinamide riboside.

    PubMed

    Pankiewicz, Krzysztof W; Watanabe, Kyoichi A; Lesiak-Watanabe, Krystyna; Goldstein, Barry M; Jayaram, Hiremagalur N

    2002-04-01

    Oncolytic C-nucleosides, tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) and benzamide riboside (3-beta-D-ribofuranosylbenzamide) are converted in cell into active metabolites thiazole-4-carboxamide- and benzamide adenine dinucleotide, TAD and BAD, respectively. TAD and BAD as NAD analogues were found to bind at the nicotinamide adenine dinucleotide (cofactor NAD) site of inosine monophosphate dehydrogenase (IMPDH), an important target in cancer treatment. The synthesis and evaluation of anticancer activity of a number of C-nucleosides related to tiazofurin and nicotinamide riboside then followed and are reviewed herein. Interestingly, pyridine C-nucleosides (such as C-nicotinamide riboside) are not metabolized into the corresponding NAD analogues in cell. Their conversion by chemical methods is described. As dinucleotides these compounds show inhibition of IMPDH in low micromolar level. Also, the synthesis of BAD in metabolically stable bis(phosphonate) form is discussed indicating the usefulness of such preformed inhibitors in drug development. Among tiazofurin analogues, Franchetti and Grifantini found, that the replacement of the sulfur by oxygen (as in oxazafurin) but not the removal of nitrogen (tiophenfurin) of the thiazole ring resulted in inactive compounds. The anti cancer activity of their synthetic dinucleotide analogues indicate that inactive compounds are not only poorly metabolized in cell but also are weak inhibitors of IMPDH as dinucleotides.

  5. A mutant of Saccharomyces cerevisiae lacking catabolic NAD-specific glutamate dehydrogenase. Growth characteristics of the mutant and regulation of enzyme synthesis in the wild-type strain.

    PubMed

    Middelhoven, W J; van Eijk, J; van Renesse, R; Blijham, J M

    1978-01-01

    NAD-specific glutamate dehydrogenase (GDH-B) was induced in a wild-type strain derived of alpha-sigma 1278b by alpha-amino acids, the nitrogen of which according to known degradative pathways is transferred to 2-oxoglutarate. A recessive mutant (gdhB) devoid of GDH-B activity grew more slowly than the wild type if one of these amino acids was the sole source of nitrogen. Addition of ammonium chloride, glutamine, asparagine or serine to growth media with inducing alpha-amino acids as the main nitrogen source increased the growth rate of the gdhB mutant to the wild-type level and repressed GDH-B synthesis in the wild type. Arginine, urea and allantoin similarly increased the growth rate of the gdhB mutant and repressed GDH-B synthesis in the presence of glutamate, but not in the presence of aspartate, alanine or proline as the main nitrogen source. These observations are consistent with the view that GDH-B in vivo deaminates glutamate. Ammonium ions are required for the biosynthesis of glutamine, asparagine, arginine, histidine and purine and pyrimidine bases. Aspartate and alanine apparently are more potent inducers of GDH-B than glutamate. Anabolic NADP-specific glutamate dehydrogenase (GDH-A) can not fulfil the function of GDH-B in the gdhB mutant. This is concluded from the equal growth rates in glutamate, aspartate and proline media as observed with a gdhB mutant and with a gdhA, gdhB double mutant in which both glutamate dehydrogenases area lacking. The double mutant showed an anomalous growth behaviour, growth rates on several nitrogen sources being unexpectedly low.

  6. 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.

  7. Studies on the de novo biosynthesis of NAD in Escherichia coli. The separation of the nadB gene product from the nadA gene product and its purification.

    PubMed

    Griffith, G R; Chandler, J L; Gholson, R K

    1975-05-01

    Quinolinic acid (pyridine 2,3-dicarboxylic acid) which is an immediate precursor of the pyridine nucleotides, is synthesised from L-asparate and dihydroxyacetone phosphate in Escherichia coli. Extracts from certain nadB mutants complement the extracts prepared from all nadA mutants for the enzymic synthesis of quinolinate. Using the complementation assay, the quinolinate synthetase B protein has been purified more than 300-fold. The quinolinate synthetase B protein exists in all nadA and nadC mutants examined. The quinolinate synthetase A protein was present in all nadC mutants and most (but not all) nadB mutants. The facile separation of the wild-type quinolinate synthetase A and B proteins out of a nadC mutant suggests that quinolinate synthetase does not exists as a tightly bound complex. The partially purified quinolinate synthetase is inhibited by physiological concetrations of NAD and NADH but not by NADP or NADPH.

  8. Acetobixan, an inhibitor of cellulose synthesis identified by microbial bioprospecting.

    PubMed

    Xia, Ye; Lei, Lei; Brabham, Chad; Stork, Jozsef; Strickland, James; Ladak, Adam; Gu, Ying; Wallace, Ian; DeBolt, Seth

    2014-01-01

    In plants, cellulose biosynthesis is an essential process for anisotropic growth and therefore is an ideal target for inhibition. Based on the documented utility of small-molecule inhibitors to dissect complex cellular processes we identified a cellulose biosynthesis inhibitor (CBI), named acetobixan, by bio-prospecting among compounds secreted by endophytic microorganisms. Acetobixan was identified using a drug-gene interaction screen to sift through hundreds of endophytic microbial secretions for one that caused synergistic reduction in root expansion of the leaky AtcesA6prc1-1 mutant. We then mined this microbial secretion for compounds that were differentially abundant compared with Bacilli that failed to mimic CBI action to isolate a lead pharmacophore. Analogs of this lead compound were screened for CBI activity, and the most potent analog was named acetobixan. In living Arabidopsis cells visualized by confocal microscopy, acetobixan treatment caused CESA particles localized at the plasma membrane (PM) to rapidly re-localize to cytoplasmic vesicles. Acetobixan inhibited 14C-Glc uptake into crystalline cellulose. Moreover, cortical microtubule dynamics were not disrupted by acetobixan, suggesting specific activity towards cellulose synthesis. Previous CBI resistant mutants such as ixr1-2, ixr2-1 or aegeus were not cross resistant to acetobixan indicating that acetobixan targets a different aspect of cellulose biosynthesis.

  9. Acetobixan, an Inhibitor of Cellulose Synthesis Identified by Microbial Bioprospecting

    PubMed Central

    Xia, Ye; Lei, Lei; Brabham, Chad; Stork, Jozsef; Strickland, James; Ladak, Adam; Gu, Ying; Wallace, Ian; DeBolt, Seth

    2014-01-01

    In plants, cellulose biosynthesis is an essential process for anisotropic growth and therefore is an ideal target for inhibition. Based on the documented utility of small-molecule inhibitors to dissect complex cellular processes we identified a cellulose biosynthesis inhibitor (CBI), named acetobixan, by bio-prospecting among compounds secreted by endophytic microorganisms. Acetobixan was identified using a drug-gene interaction screen to sift through hundreds of endophytic microbial secretions for one that caused synergistic reduction in root expansion of the leaky AtcesA6prc1-1 mutant. We then mined this microbial secretion for compounds that were differentially abundant compared with Bacilli that failed to mimic CBI action to isolate a lead pharmacophore. Analogs of this lead compound were screened for CBI activity, and the most potent analog was named acetobixan. In living Arabidopsis cells visualized by confocal microscopy, acetobixan treatment caused CESA particles localized at the plasma membrane (PM) to rapidly re-localize to cytoplasmic vesicles. Acetobixan inhibited 14C-Glc uptake into crystalline cellulose. Moreover, cortical microtubule dynamics were not disrupted by acetobixan, suggesting specific activity towards cellulose synthesis. Previous CBI resistant mutants such as ixr1-2, ixr2-1 or aegeus were not cross resistant to acetobixan indicating that acetobixan targets a different aspect of cellulose biosynthesis. PMID:24748166

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

    PubMed

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

    2015-01-02

    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. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Coupling of NAD+ Biosynthesis and Nicotinamide Ribosyl Transport: Characterization of NadR Ribonucleotide Kinase Mutants of Haemophilus influenzae

    PubMed Central

    Merdanovic, Melisa; Sauer, Elizabeta; Reidl, Joachim

    2005-01-01

    Previously, we characterized a pathway necessary for the processing of NAD+ and for uptake of nicotinamide riboside (NR) in Haemophilus influenzae. Here we report on the role of NadR, which is essential for NAD+ utilization in this organism. Different NadR variants with a deleted ribonucleotide kinase domain or with a single amino acid change were characterized in vitro and in vivo with respect to cell viability, ribonucleotide kinase activity, and NR transport. The ribonucleotide kinase mutants were viable only in a nadV+ (nicotinamide phosphoribosyltransferase) background, indicating that the ribonucleotide kinase domain is essential for cell viability in H. influenzae. Mutations located in the Walker A and B motifs and the LID region resulted in deficiencies in both NR phosphorylation and NR uptake. The ribonucleotide kinase function of NadR was found to be feedback controlled by NAD+ under in vitro conditions and by NAD+ utilization in vivo. Taken together, our data demonstrate that the NR phosphorylation step is essential for both NR uptake across the inner membrane and NAD+ synthesis and is also involved in controlling the NAD+ biosynthesis rate. PMID:15968050

  12. Coupling of NAD+ biosynthesis and nicotinamide ribosyl transport: characterization of NadR ribonucleotide kinase mutants of Haemophilus influenzae.

    PubMed

    Merdanovic, Melisa; Sauer, Elizabeta; Reidl, Joachim

    2005-07-01

    Previously, we characterized a pathway necessary for the processing of NAD+ and for uptake of nicotinamide riboside (NR) in Haemophilus influenzae. Here we report on the role of NadR, which is essential for NAD+ utilization in this organism. Different NadR variants with a deleted ribonucleotide kinase domain or with a single amino acid change were characterized in vitro and in vivo with respect to cell viability, ribonucleotide kinase activity, and NR transport. The ribonucleotide kinase mutants were viable only in a nadV+ (nicotinamide phosphoribosyltransferase) background, indicating that the ribonucleotide kinase domain is essential for cell viability in H. influenzae. Mutations located in the Walker A and B motifs and the LID region resulted in deficiencies in both NR phosphorylation and NR uptake. The ribonucleotide kinase function of NadR was found to be feedback controlled by NAD+ under in vitro conditions and by NAD+ utilization in vivo. Taken together, our data demonstrate that the NR phosphorylation step is essential for both NR uptake across the inner membrane and NAD+ synthesis and is also involved in controlling the NAD+ biosynthesis rate.

  13. Subcellular Distribution of NAD+ between Cytosol and Mitochondria Determines the Metabolic Profile of Human Cells.

    PubMed

    VanLinden, Magali R; Dölle, Christian; Pettersen, Ina K N; Kulikova, Veronika A; Niere, Marc; Agrimi, Gennaro; Dyrstad, Sissel E; Palmieri, Ferdinando; Nikiforov, Andrey A; Tronstad, Karl Johan; Ziegler, Mathias

    2015-11-13

    The mitochondrial NAD pool is particularly important for the maintenance of vital cellular functions. Although at least in some fungi and plants, mitochondrial NAD is imported from the cytosol by carrier proteins, in mammals, the mechanism of how this organellar pool is generated has remained obscure. A transporter mediating NAD import into mammalian mitochondria has not been identified. In contrast, human recombinant NMNAT3 localizes to the mitochondrial matrix and is able to catalyze NAD(+) biosynthesis in vitro. However, whether the endogenous NMNAT3 protein is functionally effective at generating NAD(+) in mitochondria of intact human cells still remains to be demonstrated. To modulate mitochondrial NAD(+) content, we have expressed plant and yeast mitochondrial NAD(+) carriers in human cells and observed a profound increase in mitochondrial NAD(+). None of the closest human homologs of these carriers had any detectable effect on mitochondrial NAD(+) content. Surprisingly, constitutive redistribution of NAD(+) from the cytosol to the mitochondria by stable expression of the Arabidopsis thaliana mitochondrial NAD(+) transporter NDT2 in HEK293 cells resulted in dramatic growth retardation and a metabolic shift from oxidative phosphorylation to glycolysis, despite the elevated mitochondrial NAD(+) levels. These results suggest that a mitochondrial NAD(+) transporter, similar to the known one from A. thaliana, is likely absent and could even be harmful in human cells. We provide further support for the alternative possibility, namely intramitochondrial NAD(+) synthesis, by demonstrating the presence of endogenous NMNAT3 in the mitochondria of human cells. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. 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

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

    2016-09-01

    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.

  15. Benzylidenemalononitrile derivatives as substrates and inhibitors of a new NAD(P)H dehydrogenase of erythrocytes. Purification and crystallisation of two forms of the enzyme.

    PubMed

    Ueberschär, K H; Kille, S; Laule, G; Maurer, P; Wallenfels, K

    1979-10-01

    Using the powerful lachrymator (2-chlorobenzylidene)malononitrile as electron acceptor, two types of NAD(P)H dehydrogenases have been isolated from human blood. Crystallisation of the homogenous enzymes was performed in 50% polyethylene glycol solution. The enzymes (average molecular weight 18 000) are composed of only one polypeptide chain and have a very similar amino acid composition. B-side stereospecificity was determined with respect to the cofactor by gas chromatography-mass spectrometry for the reductase. Besides (2-chlorobenzylidene)malononitrile, 2,6-dichloroindophenol, methylene blue, 4-benzoquinone, FMN and FAD are also reduced using NADH or NADPH as hydrogen donor with the rates decreasing in the given order. Reduction of methemoglobin is observed only upon addition of methylene blue, FMN or FAD as carriers. (2-Chlorobenzylidene)malononitrile reduction is inhibited by most of the compounds known to be decouplers of oxidative phosphorylation.

  16. Quinazolinethiones and quinazolinediones, novel inhibitors of inosine monophosphate dehydrogenase: synthesis and initial structure-activity relationships.

    PubMed

    Buckley, George M; Davies, Natasha; Dyke, Hazel J; Gilbert, Philip J; Hannah, Duncan R; Haughan, Alan F; Hunt, Caroline A; Pitt, William R; Profit, Rachael H; Ray, Nicholas C; Richard, Marianna D; Sharpe, Andrew; Taylor, Alicia J; Whitworth, Justine M; Williams, Sophie C

    2005-02-01

    The development of a series of novel quinazolinethiones and quinazolinediones as inhibitors of inosine monophosphate dehydrogenase (IMPDH) is described. The synthesis, in vitro inhibitory values for IMPDH II and in vitro inhibitory value for PBMC proliferation are discussed.

  17. Senescence in isolated carnation petals : effects of indoleacetic Acid and inhibitors of protein synthesis.

    PubMed

    Wulster, G; Sacalis, J; Janes, H W

    1982-10-01

    Indoleacetic acid induces senescence in isolated carnation (Dianthus caryophyllus, cv. White Sim) petals, increasing the duration and amount of ethylene production. This effect is inhibited by Actinomycin D, an inhibitor of RNA synthesis, and cycloheximide, a translational inhibitor of protein synthesis. The ability of petals to respond to indoleacetic acid appears to be a function of physiological age. Indoleacetic acid is capable of enhancing ethylene evolution and senescence only in specific portions of the petal.

  18. Synthesis and characterization of Sant-75 derivatives as Hedgehog-pathway inhibitors.

    PubMed

    Che, Chao; Li, Song; Yang, Bo; Xin, Shengchang; Yu, Zhixiong; Shao, Taofeng; Tao, Chuanye; Lin, Shuo; Yang, Zhen

    2012-01-01

    Sant-75 is a newly identified potent inhibitor of the hedgehog pathway. We designed a diversity-oriented synthesis program, and synthesized a series of Sant-75 analogues, which lays the foundation for further investigation of the structure-activity relationship of this important class of hedgehog-pathway inhibitors.

  19. Nmnat3 Is Dispensable in Mitochondrial NAD Level Maintenance In Vivo.

    PubMed

    Yamamoto, Masashi; Hikosaka, Keisuke; Mahmood, Arshad; Tobe, Kazuyuki; Shojaku, Hideo; Inohara, Hidenori; Nakagawa, Takashi

    2016-01-01

    Nicotinamide adenine dinucleotide (NAD) is an essential co-enzyme mediating various enzymatic reactions. Mitochondrial NAD particularly occupies a considerable amount of total NAD in cells, and serves as a co-enzyme in tricarboxylic acid cycle (TCA cycle), β-oxidation, and oxidative phosphorylation. Despite the importance of mitochondrial NAD, its synthesis pathway remains unknown. It has been proposed that NAD synthesis enzyme, Nmnat3, was localized in mitochondria, but its physiological relevance to the metabolism in mitochondria was not fully elucidated. Previously, we have reported that murine Nmnat3 protein was strongly expressed in the cytoplasm of mature erythrocytes, in which mitochondria were absent, and Nmnat3-deficient mice (Nmnat3-KO mice) exhibited splenomegaly and hemolytic anemia due to reduced NAD levels in mature erythrocytes. These results challenged the role of Nmnat3 in mitochondrial NAD synthesis. In this study, we demonstrated that mitochondrial NAD levels in various tissues, except for red blood cells, were unchanged in Nmnat3-KO mice. We also analyzed the metabolites in glycolysis and TCA cycle and found that there were no differences between Nmnat3-KO and WT mice. In addition, the aged Nmnat3-KO mice had comparable NAD levels to that observed in WT mice. Our results indicated that Nmnat3 is dispensable in the maintenance of mitochondrial NAD levels, and that other NAD regulatory pathways may exist in mitochondria.

  20. 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

  1. Developing Inhibitors of Translesion DNA Synthesis as Therapeutic Agents against Lung Cancer

    DTIC Science & Technology

    2015-12-01

    AWARD NUMBER: W81XWH-13-1-0238 TITLE: Developing Inhibitors of Translesion DNA Synthesis as Therapeutic Agents against Lung Cancer PRINCIPAL...of Translesion DNA Synthesis as Therapeutic Agents against Lung Cancer 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S...Oxygen-rich environments can create pro-mutagenic DNA lesions such as 8-oxoguanine (8-oxo-G) that can be misreplicated during translesion DNA synthesis

  2. Concise and Practical Asymmetric Synthesis of a Challenging Atropisomeric HIV Integrase Inhibitor.

    PubMed

    Fandrick, Keith R; Li, Wenjie; Zhang, Yongda; Tang, Wenjun; Gao, Joe; Rodriguez, Sonia; Patel, Nitinchandra D; Reeves, Diana C; Wu, Jiang-Ping; Sanyal, Sanjit; Gonnella, Nina; Qu, Bo; Haddad, Nizar; Lorenz, Jon C; Sidhu, Kanwar; Wang, June; Ma, Shengli; Grinberg, Nelu; Lee, Heewon; Tsantrizos, Youla; Poupart, Marc-André; Busacca, Carl A; Yee, Nathan K; Lu, Bruce Z; Senanayake, Chris H

    2015-06-08

    A practical and efficient synthesis of a complex chiral atropisomeric HIV integrase inhibitor has been accomplished. The combination of a copper-catalyzed acylation along with the implementation of the BI-DIME ligands for a ligand-controlled Suzuki cross-coupling and an unprecedented bis(trifluoromethane)sulfonamide-catalyzed tert-butylation renders the synthesis of this complex molecule robust, safe, and economical. Furthermore, the overall synthesis was conducted in an asymmetric and diastereoselective fashion with respect to the imbedded atropisomer.

  3. 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

  4. NMNAT1 inhibits axon degeneration via blockade of SARM1-mediated NAD+ depletion

    PubMed Central

    Sasaki, Yo; Nakagawa, Takashi; Mao, Xianrong; DiAntonio, Aaron; Milbrandt, Jeffrey

    2016-01-01

    Overexpression of the NAD+ biosynthetic enzyme NMNAT1 leads to preservation of injured axons. While increased NAD+ or decreased NMN levels are thought to be critical to this process, the mechanism(s) of this axon protection remain obscure. Using steady-state and flux analysis of NAD+ metabolites in healthy and injured mouse dorsal root ganglion axons, we find that rather than altering NAD+ synthesis, NMNAT1 instead blocks the injury-induced, SARM1-dependent NAD+ consumption that is central to axon degeneration. DOI: http://dx.doi.org/10.7554/eLife.19749.001 PMID:27735788

  5. NadN and e (P4) are essential for utilization of NAD and nicotinamide mononucleotide but not nicotinamide riboside in Haemophilus influenzae.

    PubMed

    Kemmer, G; Reilly, T J; Schmidt-Brauns, J; Zlotnik, G W; Green, B A; Fiske, M J; Herbert, M; Kraiss, A; Schlör, S; Smith, A; Reidl, J

    2001-07-01

    Haemophilus influenzae has an absolute requirement for NAD (factor V) because it lacks almost all the biosynthetic enzymes necessary for the de novo synthesis of that cofactor. Factor V can be provided as either nicotinamide adenosine dinucleotide (NAD), nicotinamide mononucleotide (NMN), or nicotinamide riboside (NR) in vitro, but little is known about the source or the mechanism of uptake of these substrates in vivo. As shown by us earlier, at least two gene products are involved in the uptake of NAD, the outer membrane lipoprotein e (P4), which has phosphatase activity and is encoded by hel, and a periplasmic NAD nucleotidase, encoded by nadN. It has also been observed that the latter gene product is essential for H. influenzae growth on media supplemented with NAD. In this report, we describe the functions and substrates of these two proteins as they act together in an NAD utilization pathway. Data are provided which indicate that NadN harbors not only NAD pyrophosphatase but also NMN 5'-nucleotidase activity. The e (P4) protein is also shown to have NMN 5'-nucleotidase activity, recognizing NMN as a substrate and releasing NR as its product. Insertion mutants of nadN or deletion and site-directed mutants of hel had attenuated growth and a reduced uptake phenotype when NMN served as substrate. A hel and nadN double mutant was only able to grow in the presence of NR, whereas no uptake of NMN was observed.

  6. Synthesis and inhibitory activity of glycosidase inhibitors, glycosylamino-oxazolines.

    PubMed

    Uchida, C; Ogawa, S

    1996-02-01

    In connection with structural modification of the trehalase inhibitor trehazolin (1), as a new-type of glycohydrolase inhibitor, some glycosylamino-oxazolines were designed and synthesized. Among three oxazolines beta-galacto (3), beta-gluco (5) and alpha-manno-types (6) obtained in stable form, the latter 6 has been shown to possess a moderate inhibitory activity against alpha-mannosidase.

  7. Synthesis and screening of 3-MA derivatives for autophagy inhibitors.

    PubMed

    Wu, Yanyang; Wang, Xin; Guo, Haijing; Zhang, Bo; Zhang, Xiao-Bo; Shi, Zhang-Jie; Yu, Li

    2013-04-01

    Autophagy is a conserved degradation process, which plays important pathophysiological roles. The lack of effective inhibitors of autophagy has been an obstacle in both basic research and understanding the physiological role of autophagy in disease manifestation. The most widely used inhibitor, 3-methyladenine (3-MA), is poorly soluble at room temperature and is effective only at high concentrations. In this study, we synthesized a library of small compounds by chemically modifying 3-MA and screened this library for autophagy inhibitors. Three 3-MA derivatives generated through this approach showed improved solubility and effectiveness in inhibiting autophagy. We demonstrated that chemical modification of an existing autophagy inhibitor is an effective method to generate improved autophagy inhibitors.

  8. Synthesis and evaluation of indazole based analog sensitive Akt inhibitors.

    PubMed

    Okuzumi, Tatsuya; Ducker, Gregory S; Zhang, Chao; Aizenstein, Brian; Hoffman, Randy; Shokat, Kevan M

    2010-08-01

    The kinase Akt is a key signaling node in regulating cellular growth and survival. It is implicated in cancer by mutation and its role in the downstream transmission of aberrant PI3K signaling. For these reasons, Akt has become an increasingly important target of drug development efforts and several inhibitors are now reaching clinical trials. Paradoxically it has been observed that active site kinase inhibitors of Akt lead to hyperphosphorylation of Akt itself. To investigate this phenomenon we here describe the application of a chemical genetics strategy that replaces native Akt with a mutant version containing an active site substitution that allows for the binding of an engineered inhibitor. This analog sensitive strategy allows for the selective inhibition of a single kinase. In order to create the inhibitor selective for the analog sensitive kinase, a diversity of synthetic approaches was required, finally resulting in the compound PrINZ, a 7-substituted version of the Abbott Labs Akt inhibitor A-443654.

  9. 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

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

    PubMed

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

    2015-08-21

    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.

  11. Apoptosis induced by NAD depletion is inhibited by KN-93 in a CaMKII-independent manner.

    PubMed

    Takeuchi, Mikio; Yamamoto, Tomoko

    2015-07-01

    Nicotinamide phosphoribosyltransferase (NAMPT) is a key enzyme that catalyzes the synthesis of nicotinamide mononucleotide from nicotinamide (Nam) in the salvage pathway of mammalian NAD biosynthesis. Several potent NAMPT inhibitors have been identified and used to investigate the role of intracellular NAD and to develop therapeutics. NAD depletion induced by NAMPT inhibitors depolarizes mitochondrial membrane potential and causes apoptosis in a range of cell types. However, the mechanisms behind this depolarization have not been precisely elucidated. We observed that apoptosis of THP-1 cells in response to NAMPT inhibitors was reduced by the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN-93 via an unknown mechanism. The inactive analog of KN-93, KN-92, exhibited the same activity, but the CaMKII-inhibiting cell-permeable autocamtide-2-related inhibitory peptide II did not, indicating that the inhibition of THP-1 cell apoptosis was not dependent on CaMKII. In evaluating the mechanism of action, we confirmed that KN-93 did not inhibit decreases in NAD levels but did inhibit decreases in mitochondrial membrane potential, indicating that KN-93 exerts inhibition upstream of the mitochondrial pathway of apoptosis. Further, qPCR analysis of the Bcl-2 family of proteins showed that Bim is efficiently expressed following NAMPT inhibition and that KN-92 did not inhibit this expression. The L-type Ca(2+) channel blockers verapamil and nimodipine partially inhibited apoptosis, indicating that part of this effect is dependent on Ca(2+) channel inhibition, as both KN-93 and KN-92 are reported to inhibit L-type Ca(2+) channels. On the other hand, KN-93 and KN-92 did not markedly inhibit apoptosis induced by anti-cancer agents such as etoposide, actinomycin D, ABT-737, or TW-37, indicating that the mechanism of inhibition is specific to apoptosis induced by NAD depletion. These results demonstrate that NAD depletion induces a specific type of apoptosis that

  12. Scaffold oriented synthesis. Part 4: design, synthesis and biological evaluation of novel 5-substituted indazoles as potent and selective kinase inhibitors employing heterocycle forming and multicomponent reactions.

    PubMed

    Akritopoulou-Zanze, Irini; Wakefield, Brian D; Gasiecki, Alan; Kalvin, Douglas; Johnson, Eric F; Kovar, Peter; Djuric, Stevan W

    2011-03-01

    We report the synthesis and biological evaluation of 5-substituted indazoles as kinase inhibitors. The compounds were synthesized in a parallel synthesis fashion from readily available starting materials employing heterocycle forming and multicomponent reactions and were evaluated against a panel of kinase assays. Potent inhibitors were identified for Gsk3β, Rock2, and Egfr.

  13. Synthesis and biological evaluation of novel peptidomimetics as rhodesain inhibitors.

    PubMed

    Ettari, Roberta; Previti, Santo; Cosconati, Sandro; Kesselring, Jochen; Schirmeister, Tanja; Grasso, Silvana; Zappalà, Maria

    2016-12-01

    Novel rhodesain inhibitors were developed by combining an enantiomerically pure 3-bromoisoxazoline warhead with a 1,4-benzodiazepine scaffold as specific recognition moiety. All compounds were proven to inhibit rhodesain with Ki values in the low-micromolar range. Their activity towards rhodesain was found to be coupled to an in vitro antitrypanosomal activity, with IC50 values ranging from the mid-micromolar to a low-micromolar value for the most active rhodesain inhibitor (R,S,S)-3. All compounds showed a good selectivity against the target enzyme since all of them were proven to be poor inhibitors of human cathepsin L.

  14. Design and synthesis of a potent histone deacetylase inhibitor.

    PubMed

    Liu, Tao; Kapustin, Galina; Etzkorn, Felicia A

    2007-05-03

    Histone deacetylase (HDAC) inhibitors have potential for cancer therapy. An HDAC inhibitor based on a cyclic peptide mimic of known structure, linked by an aliphatic chain to a hydroxamic acid, was designed and synthesized. The chimeric compound showed potent competitive inhibition of nuclear HDACs, with an IC50 value of 46 nM and a Ki value of 13.7 nM. The designed inhibitor showed 4-fold selectivity for HDAC1 (57 nM) over HDAC8 (231 nM).

  15. Design, synthesis, and biological evaluation of novel histone deacetylase 1 inhibitors through click chemistry.

    PubMed

    Sun, Qiao; Yao, Yiwu; Liu, Chunping; Li, Hua; Yao, Hequan; Xue, Xiaowen; Liu, Jinsong; Tu, Zhengchao; Jiang, Sheng

    2013-06-01

    We report the design, synthesis, and biological evaluation of a new series of HDAC1 inhibitors using click chemistry. Compound 17 bearing a phenyl ring at meta-position was identified to show much better selectivity for HDAC1 over HDAC7 than SAHA. The compond 17 also showed better in vitro anticancer activities against several cancer cell lines than that of SAHA. This work could serve as a foundation for further exploration of selective HDAC inhibitors using the compound 17 molecular scaffold.

  16. Design, synthesis, and evaluation of aza inhibitors of chorismate mutase.

    PubMed

    Hediger, Mark E

    2004-09-15

    A series of aza inhibitors (4-9) of chorismate mutase (E.C. 5.4.99.5) was designed, prepared, and evaluated against the enzyme by monitoring the direct inhibition of the chorismate, 1, to prephenate, 2, conversion. None of these aza inhibitors displayed tighter binding to the enzyme than the native substrate chorismate or greater inhibitory action than the previously reported ether analogue, 3. Furthermore, no time-dependent loss of enzyme activity was observed in the presence of the two potentially reactive aza inhibitors (7 and 9). These results in conjunction with inhibition data from a broader series of chorismate mutase inhibitors allowed a novel proposal for the mechanistic role of chorismate mutase to be developed. This proposed mechanism was computationally verified and correlated with crystallographic studies of various chorismate mutases.

  17. Discovery and gram-scale synthesis of BMS-593214, a potent, selective FVIIa inhibitor

    SciTech Connect

    Priestley, E. Scott; De Lucca, Indawati; Zhou, Jinglan; Zhou, Jiacheng; Saiah, Eddine; Stanton, Robert; Robinson, Leslie; Luettgen, Joseph M.; Wei, Anzhi; Wen, Xiao; Knabb, Robert M.; Wong, Pancras C.; Wexler, Ruth R.

    2013-02-14

    A 6-amidinotetrahydroquinoline screening hit was driven to a structurally novel, potent, and selective FVIIa inhibitor through a combination of library synthesis and rational design. An efficient gram-scale synthesis of the active enantiomer BMS-593214 was developed, which required significant optimization of the key Povarov annulation. Importantly, BMS-593214 showed antithrombotic efficacy in a rabbit arterial thrombosis model. A crystal structure of BMS-593214 bound to FVIIa highlights key contacts with Asp 189, Lys 192, and the S2 pocket.

  18. Solid phase synthesis and biological evaluation of probestin as an angiogenesis inhibitor.

    PubMed

    Pathuri, Gopal; Thorpe, Jessica E; Disch, Bryan C; Bailey-Downs, Lora C; Ihnat, Michael A; Gali, Hariprasad

    2013-06-15

    Probestin is a potent aminopeptidase N (APN) inhibitor originally isolated from the bacterial culture broth. Here, we report probestin synthesis by solid phase peptide synthesis (SPPS) method and evaluated its activity to inhibit angiogenesis using a chicken embryo chorioallantoic membrane (CAM) assay and a CAM tumor xenograft model. Results from these studies demonstrate that probestin inhibits the angiogenic activity and tumor growth. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Inactivation of ribosomes by an inhibitor of protein synthesis from Salmonella enteritidis.

    PubMed

    Brigotti, M; Nanetti, A; Montanaro, L; Sperti, S

    1993-01-01

    Sonic extracts of Salmonella enteritidis contain a factor which inhibits protein synthesis in cell-free systems by irreversibly inactivating ribosomes. The extent of the inactivation of ribosomes depends on the system used to assay protein synthesis, natural mRNA translation being more strongly inhibited than poly(U) translation. The inhibitory power of the Salmonella factor is destroyed by trypsin and by 5% mercaptoethanol. Placental RNase inhibitor is unable to protect ribosomes from inactivation.

  20. Centrally administered lipopolysaccharide elicits sympathetic excitation via NAD(P)H oxidase-dependent mitogen-activated protein kinase signaling

    PubMed Central

    Zhang, Zhi-Hua; Yu, Yang; Wei, Shun-Guang; Felder, Robert B.

    2010-01-01

    Objective The mechanisms by which inflammation activates sympathetic drive in heart failure and hypertension remain ill-defined. In this study, an intracerebroventricular (ICV) injection of lipopolysaccharide (LPS) was used to induce the expression of cytokines and other inflammatory mediators in the brain, in the absence of other excitatory mediators, and the downstream signaling pathways leading to sympathetic activation were examined using ICV injections of blocking or inhibiting agents. Methods and Results In anesthetized rats, ICV injection of LPS (5 µg) increased (p<0.05) renal sympathetic nerve activity, blood pressure and heart rate. LPS increased (p<0.05) hypothalamic mRNA for NAD(P)H oxidase subunits p47 phox and gp91phox, NAD(P)H-oxidase-dependent superoxide generation, hypothalamic mRNA for tumor necrosis factor (TNF)-α, cyclooxygenase-2 (COX-2), and cerebrospinal fluid (CSF) levels of TNF-α and prostaglandin E2 (PGE2). In the paraventricular nucleus of hypothalamus, dihydroethidium staining for superoxide expression and c-Fos activity (indicating neuronal excitation) increased. The superoxide scavenger tempol significantly (p<0.05) diminished the expression of inflammatory mediators, as well as superoxide expression and neuronal excitation in paraventricular nucleus. SB203580 (p38 mitogen-activated protein kinase inhibitor) also reduced the expression of inflammatory mediators in hypothalamus and CSF. Tempol, apocynin (NAD(P)H oxidase inhibitor), SB203580 and NS398 (COX-2 inhibitor) all reduced CSF PGE2 and the sympatho-excitatory response to LPS. LPS also increased angiotensin II type 1 receptor mRNA, a response blocked by apocynin and tempol but not by SB203580. Conclusion These findings suggest that central inflammation in pathophysiological conditions activates the sympathetic nervous system via NAD(P)H-oxidase and p38 mitogen-activated protein kinase dependent synthesis of PGE2. PMID:20027123

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

    PubMed

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

    2008-05-01

    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.

  2. New Insights into the Roles of NAD+-Poly(ADP-ribose) Metabolism and Poly(ADP-ribose) Glycohydrolase.

    PubMed

    Tanuma, Seiichi; Sato, Akira; Oyama, Takahiro; Yoshimori, Atsushi; Abe, Hideaki; Uchiumi, Fumiaki

    2016-01-01

    Accumulating evidence has suggested the fundamental functions of NAD+-poly(ADP-ribose) metabolism in cellular and physiological processes, including energy homeostasis, signal transduction, DNA transaction, genomic stability and cell death or survival. The NAD+ biosynthesis and poly(ADP-ribose) [(ADP-R)n] turnover are tightly controlled by several key enzymes, such as nicotinamide phosphoribosyltransferase (NmPRT), nicotinamide mononucleotide adenylyltransferases (NMNATs), poly(ADP-ribose) polymerase (PARP), poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribose pyrophosphorylase (ADPRPPL). Many researches investigating the roles of these enzymes in cells have revealed the physiological and pathological importance, and thereby the therapeutical values. Among these enzymes, the polymer degrading enzyme PARG has not yet been intensively studied, because of the low cellular content, lack of cell-available PARG chemical inhibitors and PARG genetic models. So, the biological roles of (ADP-R)n catabolism by PARG are still being elucidated as compared to those of synthesis by PARP. However, recent studies delineate that PARG-dependent (ADP-R)n degradation is critical for many pathological conditions, and thus PARG is an important target for chemical therapeutics for several diseases. This review will present the recent progresses about the roles of NAD+-(ADP-R)n metabolism and the structures and functions of PARG, with a focus on its role in DNA repair and cell death by apoptosis in relation to central regulatory network, and the therapeutic potentials of PARG inhibitors in cancer chemotherapy.

  3. Synthesis of the novel PARP-1 inhibitor AG-690/11026014 and its protective effects on angiotensin II-induced mouse cardiac remodeling.

    PubMed

    Feng, Guo-Shuai; Zhu, Cui-Ge; Li, Zhuo-Ming; Wang, Pan-Xia; Huang, Yi; Liu, Min; He, Ping; Lou, Lan-Lan; Chen, Shao-Rui; Liu, Pei-Qing

    2017-02-27

    We previously identified AG-690/11026014 (6014) as a novel poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor that effectively prevented angiotensin II (Ang II)-induced cardiomyocyte hypertrophy. In the present study, we reported a new synthesis route for 6014, and investigated its protective effects on Ang II-induced cardiac remodeling and cardiac dysfunction and the underlying mechanisms in mice. We designed a new synthesis route to obtain a sufficient quantity of 6014 for this in vivo study. C57BL/6J mice were infused with Ang II and treated with 6014 (10, 30, 90 mg·kg(-1)·d(-1), ig) for 4 weeks. Then two-dimensional echocardiography was performed to assess the cardiac function and structure. Histological changes of the hearts were examined with HE staining and Masson's trichrome staining. The protein expression was evaluated by Western blot, immunohistochemistry and immunofluorescence assays. The activities of sirtuin-1 (SIRT-1) and the content of NAD+ were detected with the corresponding test kits. Treatment with 6014 dose-dependently improved cardiac function, including LVEF, CO and SV and reversed the changes of cardiac structure in Ang II-infused mice: it significantly ameliorated Ang II-induced cardiac hypertrophy evidenced by attenuating the enlargement of cardiomyocytes, decreased HW/BW and LVW/BW, and decreased expression of hypertrophic markers ANF, BNP and β-MHC; it also prevented Ang II-induced cardiac fibrosis, as implied by the decrease in excess accumulation of extracellular matrix (ECM) components collagen I, collagen III and FN. Further studies revealed that treatment with 6014 did not affect the expression levels of PARP-1, but dose-dependently inhibited the activity of PARP-1 and subsequently restored the activity of SIRT-1 in heart tissues due to the decreased consumption of NAD+ and attenuated Poly-ADP-ribosylation (PARylation) of SIRT-1. In conclusion, the novel PARP-1 inhibitor 6014 effectively protects mice against AngII-induced cardiac

  4. 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

  5. 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.

  6. Asymmetric Synthesis of the Aminocyclitol Pactamycin, a Universal Translocation Inhibitor

    PubMed Central

    Sharpe, Robert J.; Malinowski, Justin T.; Johnson, Jeffrey S.

    2014-01-01

    An asymmetric total synthesis of the aminocyclopentitol pactamycin is described, which delivers the title compound in 15 steps from 2,4-pentanedione. Critical to this approach was the exploitation of a complex symmetry-breaking reduction strategy to assemble the C1, C2, and C7 relative stereochemistry within the first four steps of the synthesis. Multiple iterations of this reduction strategy are described, and a thorough analysis of stereochemical outcomes is detailed. In the final case, an asymmetric Mannich reaction was developed to install a protected amine directly at the C2 position. Symmetry-breaking reduction of this material gave way to a remarkable series of stereochemical outcomes leading to the title compound without recourse to non-strategic downstream manipulations. This synthesis is immediately accommodating to the facile preparation of structural analogs. PMID:24245656

  7. The synthesis and biological evaluation of a novel series of phthalazine PDE4 inhibitors I.

    PubMed

    Napoletano, M; Norcini, G; Pellacini, F; Marchini, F; Morazzoni, G; Ferlenga, P; Pradella, L

    2000-10-02

    This communication describes the synthesis and in vitro evaluation of a novel and potent series of phosphodiesterase type IV (PDE4) inhibitors. The compounds described represent conformationally constrained analogues of RP 73401, Piclamilast. Preliminary evidences of reduced side effects of II compared to standards are also reported.

  8. The Antibiotic Micrococcin Is a Potent Inhibitor of Growth and Protein Synthesis in the Malaria Parasite

    PubMed Central

    Rogers, M. John; Cundliffe, Eric; McCutchan, Thomas F.

    1998-01-01

    The antibiotic micrococcin is a potent growth inhibitor of the human malaria parasite Plasmodium falciparum, with a 50% inhibitory concentration of 35 nM. This is comparable to or less than the corresponding levels of commonly used antimalarial drugs. Micrococcin, like thiostrepton, putatively targets protein synthesis in the plastid-like organelle of the parasite. PMID:9517961

  9. Histone Deacetylase Inhibitors: Synthesis of Cyclic Tetrapeptides and their Triazole Analogues

    PubMed Central

    Singh, Erinprit K.; Nazarova, Lidia A.; Lapera, Stephanie A.; Alexander, Leslie D.

    2010-01-01

    Synthesis of nine macrocyclic peptide HDAC inhibitors and three triazole derivatives are described. HDAC inhibitory activity of these compounds against HeLa cell lysate is evaluated. The biological data demonstrates that incorporation of a triazole unit improves the HDAC inhibitory activity. PMID:20865132

  10. Melanostatin, a new melanin synthesis inhibitor. Production, isolation, chemical properties, structure and biological activity.

    PubMed

    Ishihara, Y; Oka, M; Tsunakawa, M; Tomita, K; Hatori, M; Yamamoto, H; Kamei, H; Miyaki, T; Konishi, M; Oki, T

    1991-01-01

    Melanostatin, a new antibiotic with melanin synthesis inhibitor activity, was isolated from the fermentation broth of Streptomyces clavifer No. N924-2. Its structure was determined by spectral analysis and degradation experiments. Melanostatin strongly inhibited melanin formation in Streptomyces bikiniensis NRRL B-1049 and B16 melanoma cells.

  11. Synthesis of 5-ethynyl-2'-deoxyuridine-5'-boranomono phosphate as a potential thymidylate synthase inhibitor.

    PubMed

    Khan, Shoeb I; Dobrikov, Mikhail I; Shaw, Barbara Ramsay

    2005-01-01

    The 5-ethynyl-2'-deoxyuridine nucleoside and the 5'-boranomonophosphate nucleotide were synthesized as analogs of 5-fluoro-2'-deoxyuridine monophosphate (5-FdUMP), a widely used mechanism-based inhibitor of thymidylate synthase. Synthesis was carried out from protected 5-iodo-2'-deoxyuridine and trimethylsilylacetylene by Sonogashira palladium-catalyzed cross coupling reaction followed by selective phosphorylation and finally boronation.

  12. The antibiotic micrococcin is a potent inhibitor of growth and protein synthesis in the malaria parasite.

    PubMed

    Rogers, M J; Cundliffe, E; McCutchan, T F

    1998-03-01

    The antibiotic micrococcin is a potent growth inhibitor of the human malaria parasite Plasmodium falciparum, with a 50% inhibitory concentration of 35 nM. This is comparable to or less than the corresponding levels of commonly used antimalarial drugs. Micrococcin, like thiostrepton, putatively targets protein synthesis in the plastid-like organelle of the parasite.

  13. Discovery of protein phosphatase inhibitor classes by biology-oriented synthesis

    PubMed Central

    Nören-Müller, Andrea; Reis-Corrêa, Ivan; Prinz, Heino; Rosenbaum, Claudia; Saxena, Krishna; Schwalbe, Harald J.; Vestweber, Dietmar; Cagna, Guiseppe; Schunk, Stefan; Schwarz, Oliver; Schiewe, Hajo; Waldmann, Herbert

    2006-01-01

    Protein phosphatases have very recently emerged as important targets for chemical biology and medicinal chemistry research, and new phosphatase inhibitor classes are in high demand. The underlying frameworks of natural products represent the evolutionarily selected fractions of chemical space explored by nature so far and meet the criteria of relevance to nature and biological prevalidation most crucial to inhibitor development. We refer to synthesis efforts and compound collection development based on these criteria as biology-oriented synthesis. For the discovery of phosphatase inhibitor classes by means of this approach, four natural product-derived or -inspired medium-sized compound collections were synthesized and investigated for inhibition of the tyrosine phosphatases VE-PTP, Shp-2, PTP1B, MptpA, and MptpB and the dual-specificity phosphatases Cdc25A and VHR. The screen yielded four unprecedented and selective phosphatase inhibitor classes for four phosphatases with high hit rates. For VE-PTP and MptpB the first inhibitors were discovered. These results demonstrate that biology-oriented synthesis is an efficient approach to the discovery of new compound classes for medicinal chemistry and chemical biology research that opens up new opportunities for the study of phosphatases, which may lead to the development of new drug candidates. PMID:16809424

  14. Synthesis of a new class of HIV-1 inhibitors.

    PubMed

    Farese-Di Giorgio, A; Pairot, S; Patino, N; Condom, R; Di Giorgio, C; Aumelas, A; Aubertin, A M; Guedj, R

    1999-02-01

    A new family of molecules potentially inhibitors of the HIV-1 Tat-TAR complex was prepared. These compounds are constituted by dinucleotide analogs (PNA dimer) bound, through a linker, to an arginine residue. In this series, several molecules inhibit viral development in cell culture with a micromolar IC50 and without cellular toxicity until 200 microM concentration.

  15. Selective indole-based ECE inhibitors: synthesis and pharmacological evaluation.

    PubMed

    Brands, Michael; Ergüden, Jens-Kerim; Hashimoto, Kentaro; Heimbach, Dirk; Krahn, Thomas; Schröder, Christian; Siegel, Stephan; Stasch, Johannes-Peter; Tsujishita, Hideki; Weigand, Stefan; Yoshida, Nagahiro H

    2006-01-01

    Inhibition of the metalloprotease ECE-1 may be beneficial for the treatment of coronary heart disease, cancer, renal failure, and urological disorders. A novel class of indole-based ECE inhibitors was identified by high throughput screening. Optimization of the original screening lead structure 6 led to highly potent inhibitors such as 11, which bears a bisaryl amide moiety linked to the indole C2 position through an amide group. Docking of 11 into a model structure of ECE revealed a unique binding mode in which the Zn center of the enzyme is not directly addressed by the inhibitor, but key interactions are suggested for the central amide group. Testing of the lead compound 6 in hypertensive Dahl S rats resulted in a decrease in blood pressure after an initial period in which the blood pressure remained unchanged, most probably the result of ET-1 already present. Indole derivative 6 also displays a cardio-protective effect in a mouse model of acute myocardial infarction after oral administration. The more potent chloropyridine derivative 9 antagonizes big-ET-1-induced increase in blood pressure in rats at intravenous administration of 3 mg kg-1. All ECE inhibitors of the indole class showed high selectivity for ECE over related metalloproteases such as NEP and ACE. Therefore, these compounds might have further potential as drugs for the treatment of coronary heart diseases.

  16. 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

  17. 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.

  18. 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.

  19. Conditional modulation of NAD levels and metabolite profiles in Nicotiana sylvestris by mitochondrial electron transport and carbon/nitrogen supply.

    PubMed

    Hager, Jutta; Pellny, Till K; Mauve, Caroline; Lelarge-Trouverie, Caroline; De Paepe, Rosine; Foyer, Christine H; Noctor, Graham

    2010-04-01

    Environmental controls on leaf NAD status remain poorly understood. Here, we analyzed the effects of two key environmental variables, CO(2) and nitrogen, on leaf metabolite profiles, NAD status and the abundance of key transcripts involved in de novo NAD synthesis in wild-type (WT) Nicotiana sylvestris and the CMSII mutant that lacks respiratory complex I. High CO(2) and increased N supply both significantly enhanced NAD(+) and NADH pools in WT leaves. In nitrogen-sufficient conditions, CMSII leaves were enriched in NAD(+) and NADH compared to the WT, but the differences in NADH were smaller at high CO(2) than in air because high CO(2) increased WT NADH/NAD(+). The CMSII-linked increases in NAD(+) and NADH status were abolished by growth with limited nitrogen, which also depleted the nicotine and nicotinic acid pools in the CMSII leaves. Few statistically significant genotype and N-dependent differences were detected in NAD synthesis transcripts, with effects only on aspartate oxidase and NAD synthetase mRNAs. Non-targeted metabolite profiling as well as quantitative amine analysis showed that NAD(+) and NADH contents correlated tightly with leaf amino acid contents across all samples. The results reveal considerable genotype- and condition-dependent plasticity in leaf NAD(+) and NADH contents that is not linked to modified expression of NAD synthesis genes at the transcript level and show that NAD(+) and NADH contents are tightly integrated with nitrogen metabolism. A regulatory two-way feedback circuit between nitrogen and NAD in the regulation of N assimilation is proposed that potentially links the nutritional status to NAD-dependent signaling pathways.

  20. High-resolution structures of Thermus thermophilus enoyl-acyl carrier protein reductase in the apo form, in complex with NAD+ and in complex with NAD+ and triclosan

    PubMed Central

    Otero, José M.; Noël, Ann-Josée; Guardado-Calvo, Pablo; Llamas-Saiz, Antonio L.; Wende, Wolfgang; Schierling, Benno; Pingoud, Alfred; van Raaij, Mark J.

    2012-01-01

    Enoyl-acyl carrier protein reductase (ENR; the product of the fabI gene) is an important enzyme that is involved in the type II fatty-acid-synthesis pathway of bacteria, plants, apicomplexan protozoa and mitochondria. Harmful pathogens such as Mycobacterium tuberculosis and Plasmodium falciparum use the type II fatty-acid-synthesis system, but not mammals or fungi, which contain a type I fatty-acid-synthesis pathway consisting of one or two multifunctional enzymes. For this reason, specific inhibitors of ENR are attractive antibiotic candidates. Triclosan, a broad-range antibacterial agent, binds to ENR, inhibiting fatty-acid synthesis. As humans do not have an ENR enzyme, they are not affected. Here, high-resolution structures of Thermus thermophilus (Tth) ENR in the apo form, bound to NAD+ and bound to NAD+ plus triclosan are reported. Differences from and similarities to other known ENR structures are reported; in general, the structures are very similar. The cofactor-binding site is also very similar to those of other ENRs and, as reported for other species, triclosan leads to greater ordering of the loop that covers the cofactor-binding site, which, together with the presence of triclosan itself, presumably provides tight binding of the dinucleotide, preventing cycling of the cofactor. Differences between the structures of Tth ENR and other ENRs are the presence of an additional β-sheet at the N-terminus and a larger number of salt bridges and side-chain hydrogen bonds. These features may be related to the high thermal stability of Tth ENR. PMID:23027736

  1. Design, synthesis, and biological activity of urea derivatives as anaplastic lymphoma kinase inhibitors.

    PubMed

    af Gennäs, Gustav Boije; Mologni, Luca; Ahmed, Shaheen; Rajaratnam, Mohanathas; Marin, Oriano; Lindholm, Niko; Viltadi, Michela; Gambacorti-Passerini, Carlo; Scapozza, Leonardo; Yli-Kauhaluoma, Jari

    2011-09-05

    In anaplastic large-cell lymphomas, chromosomal translocations involving the kinase domain of anaplastic lymphoma kinase (ALK), generally fused to the 5' part of the nucleophosmin gene, produce highly oncogenic ALK fusion proteins that deregulate cell cycle, apoptosis, and differentiation in these cells. Other fusion oncoproteins involving ALK, such as echinoderm microtubule-associated protein-like 4-ALK, were recently found in patients with non-small-cell lung, breast, and colorectal cancers. Recent research has focused on the development of inhibitors for targeted therapy of these ALK-positive tumors. Because kinase inhibitors that target the inactive conformation are thought to be more specific than ATP-targeted inhibitors, we investigated the possibility of using two known inhibitors, doramapimod and sorafenib, which target inactive kinases, to design new urea derivatives as ALK inhibitors. We generated a homology model of ALK in its inactive conformation complexed with doramapimod or sorafenib in its active site. The results elucidated why doramapimod is a weak inhibitor and why sorafenib does not inhibit ALK. Virtual screening of commercially available compounds using the homology model of ALK yielded candidate inhibitors, which were tested using biochemical assays. Herein we present the design, synthesis, biological activity, and structure-activity relationships of a novel series of urea compounds as potent ALK inhibitors. Some compounds showed inhibition of purified ALK in the high nanomolar range and selective antiproliferative activity on ALK-positive cells.

  2. Synthesis of a selective HDAC6 inhibitor active in neuroblasts.

    PubMed

    Zwick, Vincent; Simões-Pires, Claudia A; Nurisso, Alessandra; Petit, Charlotte; Dos Santos Passos, Carolina; Randazzo, Giuseppe Marco; Martinet, Nadine; Bertrand, Philippe; Cuendet, Muriel

    2016-10-15

    In recent years, the role of HDAC6 in neurodegeneration has been partially elucidated, which led some authors to propose HDAC6 inhibitors as a therapeutic strategy to treat neurodegenerative diseases. In an effort to develop a selective HDAC6 inhibitor which can cross the blood brain barrier (BBB), a modified hydroxamate derivative (compound 3) was designed and synthetized. This compound was predicted to have potential for BBB penetration based on in silico and in vitro evaluation of passive permeability. When tested for its HDAC inhibitory activity, the IC50 value of compound 3 towards HDAC6 was in the nM range in both enzymatic and cell-based assays. Compound 3 showed a cell-based selectivity profile close to that of tubastatin A in SH-SY5Y human neuroblastoma cells, and a good BBB permeability profile.

  3. Bacterial transferase MraY inhibitors: synthesis and biological evaluation.

    PubMed

    Lecerclé, Delphine; Clouet, Anthony; Al-Dabbagh, Bayan; Crouvoisier, Muriel; Bouhss, Ahmed; Gravier-Pelletier, Christine; Le Merrer, Yves

    2010-06-15

    New inhibitors of the bacterial transferase MraY are described. Their structure is based on an aminoribosyl-O-uridine like scaffold, readily obtained in two key steps. The amino group can be coupled with proline or guanylated. Alternatively, these amino, prolinyl or guanidinyl groups can be introduced through a triazole linker. Biological evaluation of these compounds on MraY from Bacillus subtilis revealed interesting inhibitory activity for both amino compounds. Copyright 2010. Published by Elsevier Ltd.

  4. Inhibition of cholesterol synthesis by squalene synthase inhibitors does not induce myotoxicity in vitro.

    PubMed

    Flint, O P; Masters, B A; Gregg, R E; Durham, S K

    1997-07-01

    The cholesterol-lowering HMG CoA reductase inhibitors (HMGRI), pravastatin and lovastatin, have been associated with skeletal myopathy in humans and in rats. In a previous in vitro study, HMGRI-induced changes in neonatal rat skeletal muscle cells were characterized by reversible inhibition of protein synthesis and loss of differentiated myotubes at concentrations markedly lower than those inducing enzyme leakage. Myotoxicity was determined to be directly related to inhibition of HMG CoA reductase, since mevalonate, the immediate product of HMG CoA reductase metabolism, abrogated the drug-induced changes. Farnesol, geranylgeraniol, and squalene are metabolites of mevalonate. Squalene, formed from farnesol by squalene synthase, is the first metabolite solely committed to cholesterol synthesis. In contrast, geranylgeraniol, formed by the addition of an isoprene group to farnesol, is the first metabolite uncommitted to cholesterol synthesis. The objective of the present study was to determine the role of inhibition of cholesterol synthesis in HMGRI-induced in vitro myotoxicity. HMGRI-treated neonatal rat skeletal muscle cultures were supplemented with farnesol and geranylgeraniol, and in another study, muscle cultures were exposed to two squalene synthase inhibitors (SSI), BMS-187745 and its prodrug ester, BMS-188494. Endpoints evaluated for both studies included protein synthesis ([3H]leucine incorporation), total cellular protein (a measure of cell loss), intra- and extracellular lactate dehydrogenase activity (a measure of membrane integrity), cholesterol biosynthesis ([14C]acetate incorporation), and morphology. HMG CoA reductase inhibitor-induced morphologic changes and inhibition of protein synthesis were significantly ameliorated by supplementation with farnesol and geranylgeraniol. In contrast to HMGRI-induced in vitro myotoxicity, SSI induced an irreversible, minimal cytotoxicity at close to maximum soluble concentrations. These results indicate that

  5. Synthesis, structure-activity relationship and crystallographic studies of 3-substituted indolin-2-one RET inhibitors.

    PubMed

    Mologni, Luca; Rostagno, Roberta; Brussolo, Stefania; Knowles, Phillip P; Kjaer, Svend; Murray-Rust, Judith; Rosso, Enrico; Zambon, Alfonso; Scapozza, Leonardo; McDonald, Neil Q; Lucchini, Vittorio; Gambacorti-Passerini, Carlo

    2010-02-15

    The synthesis, structure-activity relationships (SAR) and structural data of a series of indolin-2-one inhibitors of RET tyrosine kinase are described. These compounds were designed to explore the available space around the indolinone scaffold within RET active site. Several substitutions at different positions were tested and biochemical data were used to draw a molecular model of steric and electrostatic interactions, which can be applied to design more potent and selective RET inhibitors. The crystal structures of RET kinase domain in complex with three inhibitors were solved. All three compounds bound in the ATP pocket and formed two hydrogen bonds with the kinase hinge region. Crystallographic analysis confirmed predictions from molecular modelling and helped refine SAR results. These data provide important information for the development of indolinone inhibitors for the treatment of RET-driven cancers. Copyright 2010 Elsevier Ltd. All rights reserved.

  6. 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.

  7. Variable effects of DNA-synthesis inhibitors upon DNA methylation in mammalian cells.

    PubMed Central

    Nyce, J; Liu, L; Jones, P A

    1986-01-01

    Post-synthetic enzymatic hypermethylation of DNA was induced in hamster fibrosarcoma cells by the DNA synthesis inhibitors cytosine arabinoside, hydroxyurea and aphidicolin. This effect required direct inhibition of DNA polymerase alpha or reduction in deoxynucleotide pools and was not specific to a single cell type. At equivalently reduced levels of DNA synthesis, neither cycloheximide, actinomycin D nor serum deprivation affected DNA methylation in this way. The topoisomerase inhibitors nalidixic acid and novobiocin caused significant hypomethylation indicating that increased 5-mCyt content was not a necessary consequence of DNA synthesis inhibition. The induced hypermethylation occurred predominantly in that fraction of the DNA synthesized in the presence of inhibitor; was stable in the absence of drug; was most prominent in low molecular weight DNA representing sites of initiated but incomplete DNA synthesis; and occurred primarily within CpG dinucleotides, although other dinucleotides were overmethylated as well. Drug-induced CpG hypermethylation may be capable of silencing genes, an effect which may be relevant to the aberrantly expressed genes characteristic of neoplastic cells. PMID:3086840

  8. Recent developments in the synthesis of acetylcholinesterase inhibitors.

    PubMed

    Marco, José L; Carreiras, M Carmo

    2003-09-01

    The acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory activities of a series of pyrano[2,3-b]quinolines (2, 3), [1,8]naphthyridines (5, 6), 4-amino-2,3-diaryl-5,6,7,8-tetrahydrofuro[2,3-b]quinolines (11-13)/ 4-amino-6,7,8,9-tetrahydro-2,3-diphenyl-5H-cyclohepta[e]furo[2,3-b]pyridine (14), 4-amino-5,6,7,8-tetrahydro-2,3-diphenylthieno[2,3-b]quinoline (15)/ 4-amino-6,7,8,9-tetrahydro-2,3-diphenyl-5H-cyclohepta[e]thieno[2,3-b]pyridine (16) are described. These compounds are tacrine analogues that have been prepared from readily available polyfunctionalized ethyl [6-amino-5-cyano-4H-pyran]-3-carboxylates (9, 10), ethyl [6-amino-5-cyanopyridine]-3-carboxylates (7, 8), 2-amino-3-cyano-4,5-diarylfurans (17-19) and 2-amino-3-cyano-4,5-diphenylthiophene (20) via Friedländer condensation with selected ketones. These compounds are competitive and, in a few cases, non-competitive inhibitors for AChE, the most potent being compound (14), though three-fold less active than tacrine. The BuChE inhibitory activity is only significant in compounds 11 and 14, ten-fold less active than tacrine. Furthermore, the products 12 and 13 are selective and moderate AChE inhibitors.

  9. 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.

  10. Synthesis, biological characterization and molecular modeling insights of spirochromanes as potent HDAC inhibitors.

    PubMed

    Thaler, Florian; Moretti, Loris; Amici, Raffaella; Abate, Agnese; Colombo, Andrea; Carenzi, Giacomo; Fulco, Maria Carmela; Boggio, Roberto; Dondio, Giulio; Gagliardi, Stefania; Minucci, Saverio; Sartori, Luca; Varasi, Mario; Mercurio, Ciro

    2016-01-27

    In the last decades, inhibitors of histone deacetylases (HDAC) have become an important class of anti-cancer agents. In a previous study we described the synthesis of spiro[chromane-2,4'-piperidine]hydroxamic acid derivatives able to inhibit histone deacetylase enzymes. Herein, we present our exploration for new derivatives by replacing the piperidine moiety with various cycloamines. The goal was to obtain highly potent compounds with a good in vitro ADME profile. In addition, molecular modeling studies unravelled the binding mode of these inhibitors.

  11. Synthesis and biological evaluation of histone deacetylase inhibitors that are based on FR235222

    PubMed Central

    Singh, Erinprit K.; Ravula, Suchitra; Pan, Chung-Mao; Pan, Po-Shen; Vasko, Robert C.; Lapera, Stephanie A.; Weerasinghe, Sujith V. W.; Pflum, Mary Kay H.; McAlpine, Shelli R.

    2008-01-01

    We outline the synthesis of six novel derivatives that are based on a recently discovered HDAC inhibitor FR235222. Our work is the first report utilizing a novel binding element, guanidine, as metal coordinators in HDAC inhibitors. Further, we demonstrate that these compounds show cytotoxicity that parallels their ability to inhibit deacetylase activity, and that the most potent compounds maintain an l-Phe at position 1, and a d-Pro at position 4. Both inhibition of HDAC activity and cytotoxicity against the pancreatic cancer cell line BxPC3 are exhibited by these compounds, establishing that a guanidine unit can be utilized successfully to inhibit HDAC activity. PMID:18381239

  12. Synthesis and SAR of Benzisothiazole- and Indolizine-β-d-glucopyranoside Inhibitors of SGLT2

    PubMed Central

    2010-01-01

    A series of benzisothiazole- and indolizine-β-d-glucopyranoside inhibitors of human SGLT2 are described. The synthesis of the C-linked heterocyclic glucosides took advantage of a palladium-catalyzed cross-coupling reaction between a glucal boronate and the corresponding bromo heterocycle. The compounds have been evaluated for their human SGLT2 inhibition potential using cell-based functional transporter assays, and their structure−activity relationships have been described. Benzisothiazole-C-glucoside 16d was found to be an inhibitor of SGLT2 with an IC50 of 10 nM. PMID:24900169

  13. Synthesis and evaluation of aminopyridine derivatives as potential BACE1 inhibitors.

    PubMed

    Konno, Hiroyuki; Sato, Taki; Saito, Yugo; Sakamoto, Iori; Akaji, Kenichi

    2015-11-15

    To identify a new non-peptidyl BACE1 inhibitor, we focused on the aminopyridine structure, which binds to the active sites of BACE1. Synthesis of aminopyridine derivatives and evaluation of inhibitory activity against rBACE1 are described. The 2-aminopyridine moiety and/or 3-methoxybenzaldehyde could be converted to terminal acetylene derivatives by the Sonogashira method. Sonogashira or Glaser cross-coupling reactions with the corresponding derivatives followed by hydrogenation could derive the designed compounds. Although inhibitory activities of the synthetic compounds against rBACE1 were weak, the aminopyridine motif has potential as a BACE1 inhibitor.

  14. Design, synthesis, and evaluation of benzophenone derivatives as novel acetylcholinesterase inhibitors.

    PubMed

    Belluti, Federica; Piazzi, Lorna; Bisi, Alessandra; Gobbi, Silvia; Bartolini, Manuela; Cavalli, Andrea; Valenti, Piero; Rampa, Angela

    2009-03-01

    Starting from a structure-based drug design, new acetylcholinesterase inhibitors were designed and synthesized as analogues of donepezil. The compounds were composed by an aromatic function and a tertiary amino moiety connected by a suitable spacer. In particular, the benzophenone nucleus and the N,N-benzylmethylamine function were selected. The easily accessible three-step synthesis of these compounds resulted to be significantly less difficult and expensive than that of donepezil. Several compounds possess anti-cholinesterase activity in the order of micro and sub-micromolar. Particularly, compounds 1 and 10 were the most potent inhibitors of the series.

  15. Synthesis and SAR of Benzisothiazole- and Indolizine-β-d-glucopyranoside Inhibitors of SGLT2.

    PubMed

    Zhou, Huiqiang; Danger, Dana P; Dock, Steven T; Hawley, Lora; Roller, Shane G; Smith, Chari D; Handlon, Anthony L

    2010-04-08

    A series of benzisothiazole- and indolizine-β-d-glucopyranoside inhibitors of human SGLT2 are described. The synthesis of the C-linked heterocyclic glucosides took advantage of a palladium-catalyzed cross-coupling reaction between a glucal boronate and the corresponding bromo heterocycle. The compounds have been evaluated for their human SGLT2 inhibition potential using cell-based functional transporter assays, and their structure-activity relationships have been described. Benzisothiazole-C-glucoside 16d was found to be an inhibitor of SGLT2 with an IC50 of 10 nM.

  16. Boosting NAD to spare hearing.

    PubMed

    Brenner, Charles

    2014-12-02

    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.

  17. Synthesis of aminoalkyl-substituted coumarin derivatives as acetylcholinesterase inhibitors.

    PubMed

    Nam, Seung Ok; Park, Dong Hyun; Lee, Young Hun; Ryu, Jong Hoon; Lee, Yong Sup

    2014-02-15

    Alzheimer's disease, one of the most common forms of dementia, is a progressive neurodegenerative disorder symptomatically characterized by declines in memory and cognitive abilities. To date, the successful therapeutic strategy to treat AD is maintaining levels of acetylcholine by inhibiting acetylcholinesterase (AChE). In the present study, coumarin derivatives were designed and synthesized as AChE inhibitors based on the lead structure of scopoletin. Of those synthesized, pyrrolidine-substituted coumarins 3b and 3f showed ca. 160-fold higher AChE inhibitory activities than scopoletin. These compounds also ameliorated scopolamine-induced memory deficit in mice when administered orally at the dose of 1 and 2 mg/kg. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Design and Synthesis of Novel Macrocyclic Mer Tyrosine Kinase Inhibitors.

    PubMed

    Wang, Xiaodong; Liu, Jing; Zhang, Weihe; Stashko, Michael A; Nichols, James; Miley, Michael J; Norris-Drouin, Jacqueline; Chen, Zhilong; Machius, Mischa; DeRyckere, Deborah; Wood, Edgar; Graham, Douglas K; Earp, H Shelton; Kireev, Dmitri; Frye, Stephen V

    2016-12-08

    Mer tyrosine kinase (MerTK) is aberrantly elevated in various tumor cells and has a normal anti-inflammatory role in the innate immune system. Inhibition of MerTK may provide dual effects against these MerTK-expressing tumors through reducing cancer cell survival and redirecting the innate immune response. Recently, we have designed novel and potent macrocyclic pyrrolopyrimidines as MerTK inhibitors using a structure-based approach. The most active macrocycles had an EC50 below 40 nM in a cell-based MerTK phosphor-protein ELISA assay. The X-ray structure of macrocyclic analogue 3 complexed with MerTK was also resolved and demonstrated macrocycles binding in the ATP binding pocket of the MerTK protein as anticipated. In addition, the lead compound 16 (UNC3133) had a 1.6 h half-life and 16% oral bioavailability in a mouse PK study.

  19. 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

  20. 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.

  1. Scaffold oriented synthesis. Part 3: design, synthesis and biological evaluation of novel 5-substituted indazoles as potent and selective kinase inhibitors employing [2+3] cycloadditions.

    PubMed

    Akritopoulou-Zanze, Irini; Wakefield, Brian D; Gasiecki, Alan; Kalvin, Douglas; Johnson, Eric F; Kovar, Peter; Djuric, Stevan W

    2011-03-01

    We report the synthesis and biological evaluation of 5-substituted indazoles and amino indazoles as kinase inhibitors. The compounds were synthesized in a parallel synthesis fashion from readily available starting materials employing [2+3] cycloaddition reactions and were evaluated against a panel of kinase assays. Potent inhibitors were identified for numerous kinases such as Rock2, Gsk3β, Aurora2 and Jak2.

  2. Diversity-oriented synthesis yields novel multistage antimalarial inhibitors.

    PubMed

    Kato, Nobutaka; Comer, Eamon; Sakata-Kato, Tomoyo; Sharma, Arvind; Sharma, Manmohan; Maetani, Micah; Bastien, Jessica; Brancucci, Nicolas M; Bittker, Joshua A; Corey, Victoria; Clarke, David; Derbyshire, Emily R; Dornan, Gillian L; Duffy, Sandra; Eckley, Sean; Itoe, Maurice A; Koolen, Karin M J; Lewis, Timothy A; Lui, Ping S; Lukens, Amanda K; Lund, Emily; March, Sandra; Meibalan, Elamaran; Meier, Bennett C; McPhail, Jacob A; Mitasev, Branko; Moss, Eli L; Sayes, Morgane; Van Gessel, Yvonne; Wawer, Mathias J; Yoshinaga, Takashi; Zeeman, Anne-Marie; Avery, Vicky M; Bhatia, Sangeeta N; Burke, John E; Catteruccia, Flaminia; Clardy, Jon C; Clemons, Paul A; Dechering, Koen J; Duvall, Jeremy R; Foley, Michael A; Gusovsky, Fabian; Kocken, Clemens H M; Marti, Matthias; Morningstar, Marshall L; Munoz, Benito; Neafsey, Daniel E; Sharma, Amit; Winzeler, Elizabeth A; Wirth, Dyann F; Scherer, Christina A; Schreiber, Stuart L

    2016-10-20

    Antimalarial drugs have thus far been chiefly derived from two sources-natural products and synthetic drug-like compounds. Here we investigate whether antimalarial agents with novel mechanisms of action could be discovered using a diverse collection of synthetic compounds that have three-dimensional features reminiscent of natural products and are underrepresented in typical screening collections. We report the identification of such compounds with both previously reported and undescribed mechanisms of action, including a series of bicyclic azetidines that inhibit a new antimalarial target, phenylalanyl-tRNA synthetase. These molecules are curative in mice at a single, low dose and show activity against all parasite life stages in multiple in vivo efficacy models. Our findings identify bicyclic azetidines with the potential to both cure and prevent transmission of the disease as well as protect at-risk populations with a single oral dose, highlighting the strength of diversity-oriented synthesis in revealing promising therapeutic targets.

  3. Synthetic silvestrol analogues as potent and selective protein synthesis inhibitors.

    PubMed

    Liu, Tao; Nair, Somarajan J; Lescarbeau, André; Belani, Jitendra; Peluso, Stéphane; Conley, James; Tillotson, Bonnie; O'Hearn, Patrick; Smith, Sherri; Slocum, Kelly; West, Kip; Helble, Joseph; Douglas, Mark; Bahadoor, Adilah; Ali, Janid; McGovern, Karen; Fritz, Christian; Palombella, Vito J; Wylie, Andrew; Castro, Alfredo C; Tremblay, Martin R

    2012-10-25

    Misregulation of protein translation plays a critical role in human cancer pathogenesis at many levels. Silvestrol, a cyclopenta[b]benzofuran natural product, blocks translation at the initiation step by interfering with assembly of the eIF4F translation complex. Silvestrol has a complex chemical structure whose functional group requirements have not been systematically investigated. Moreover, silvestrol has limited development potential due to poor druglike properties. Herein, we sought to develop a practical synthesis of key intermediates of silvestrol and explore structure-activity relationships around the C6 position. The ability of silvestrol and analogues to selectively inhibit the translation of proteins with high requirement on the translation-initiation machinery (i.e., complex 5'-untranslated region UTR) relative to simple 5'UTR was determined by a cellular reporter assay. Simplified analogues of silvestrol such as compounds 74 and 76 were shown to have similar cytotoxic potency and better ADME characteristics relative to those of silvestrol.

  4. Facile synthesis of chrysin-derivatives with promising activities as aromatase inhibitors.

    PubMed

    Mohammed, Hamdoon A; Ba, Lalla A; Burkholz, Torsten; Schumann, Elena; Diesel, Britta; Zapp, Josef; Kiemer, Alexandra K; Ries, Christina; Hartmann, Rolf W; Hosny, Mohammed; Jacob, Claus

    2011-01-01

    Flavones such as chrysin show structural similarities to androgens, the substrates of human aromatase, which converts androgens to estrogens. Aromatase is a key target in the treatment of hormone-dependent tumors, including breast cancer. Flavone-based aromatase inhibitors are of growing interest, and chrysin in particular provides a (natural) lead structure. This paper reports multicomponent synthesis as a means for facile modification of the chrysin core structure in order to add functional elements. A Mannich-type reaction was used to synthesize a range of mono- and disubstituted chrysin derivatives, some of which are more effective aromatase inhibitors than the benchmark compound, aminoglutethimide. Similarly, the reaction of chrysin with various isonitriles and acetylene dicarboxylates results in a new class of flavone derivatives, tricyclic pyrano-flavones which also inhibit human aromatase. Multicomponent reactions involving flavones therefore enable the synthesis of a variety of derivatives, some of which may be useful as anticancer agents.

  5. A Protecting Group-Free Synthesis of Deazathiamine: A Step Toward Inhibitor Design

    PubMed Central

    Zhao, Hong; de Carvalho, Luiz Pedro S.; Nathan, Carl

    2010-01-01

    The discovery of 3-deazathiamine diphosphate (deazaThDP) as a potent inhibitor analog of the cofactor thiamine diphosphate (ThDP) has highlighted the need for an efficient and scalable synthesis of deazaThDP. Such a method would facilitate development of analogs with the ability to inhibit individual ThDP-dependent enzymes selectively. Toward the goal of developing selective inhibitors of the mycobacterial enzyme 2-hydroxy-3-oxoadipate synthase (HOAS), we report an improved synthesis of deazaThDP without use of protecting groups. Tribromo-3-methylthiophene served as a versatile starting material whose selective functionalization permitted access to deazaThDP in five steps, with potential to make other analogs accessible in substantial amounts. PMID:20943392

  6. Discovery of a Specific Inhibitor of Pyomelanin Synthesis in Legionella pneumophila.

    PubMed

    Aubi, Oscar; Flydal, Marte I; Zheng, Huaixin; Skjærven, Lars; Rekand, Illimar; Leiros, Hanna-Kirsti S; Haug, Bengt Erik; Cianciotto, Nicholas P; Martinez, Aurora; Underhaug, Jarl

    2015-11-12

    Phenylalanine hydroxylase catalyzes the first step in the synthesis of pyomelanin, a pigment that aids in the acquisition of essential iron in certain bacteria. In this work, we present the development and application of a drug discovery protocol by targeting this enzyme in Legionella pneumophila, the major causative agent of Legionnaires' disease. We employ a combination of high-throughput screening to identify small-molecule binders, enzymatic activity measurements to identify inhibitors in vitro, and the verification of the inhibitory effect in vivo. The most potent inhibitor shows an IC50 value in the low micromolar range and successfully abolishes the synthesis of pyomelanin in L. pneumophila cultures at 10 μM. Thus, this compound represents a novel and effective tool for investigating the role of pyomelanin in the biology and pathogenicity of this organism. Altogether, the results demonstrate a successful pathway for drug development focusing on binding specificity in the initial high-throughput screening steps.

  7. Vibrio Phage KVP40 Encodes a Functional NAD(+) Salvage Pathway.

    PubMed

    Lee, Jae Yun; Li, Zhiqun; Miller, Eric S

    2017-05-01

    The genome of T4-type Vibrio bacteriophage KVP40 has five genes predicted to encode proteins of pyridine nucleotide metabolism, of which two, nadV and natV, would suffice for an NAD(+) salvage pathway. NadV is an apparent nicotinamide phosphoribosyltransferase (NAmPRTase), and NatV is an apparent bifunctional nicotinamide mononucleotide adenylyltransferase (NMNATase) and nicotinamide-adenine dinucleotide pyrophosphatase (Nudix hydrolase). Genes encoding the predicted salvage pathway were cloned and expressed in Escherichia coli, the proteins were purified, and their enzymatic properties were examined. KVP40 NadV NAmPRTase is active in vitro, and a clone complements a Salmonella mutant defective in both the bacterial de novo and salvage pathways. Similar to other NAmPRTases, the KVP40 enzyme displayed ATPase activity indicative of energy coupling in the reaction mechanism. The NatV NMNATase activity was measured in a coupled reaction system demonstrating NAD(+) biosynthesis from nicotinamide, phosphoribosyl pyrophosphate, and ATP. The NatV Nudix hydrolase domain was also shown to be active, with preferred substrates of ADP-ribose, NAD(+), and NADH. Expression analysis using reverse transcription-quantitative PCR (qRT-PCR) and enzyme assays of infected Vibrio parahaemolyticus cells demonstrated nadV and natV transcription during the early and delayed-early periods of infection when other KVP40 genes of nucleotide precursor metabolism are expressed. The distribution and phylogeny of NadV and NatV proteins among several large double-stranded DNA (dsDNA) myophages, and also those from some very large siphophages, suggest broad relevance of pyridine nucleotide scavenging in virus-infected cells. NAD(+) biosynthesis presents another important metabolic resource control point by large, rapidly replicating dsDNA bacteriophages.IMPORTANCE T4-type bacteriophages enhance DNA precursor synthesis through reductive reactions that use NADH/NADPH as the electron donor and NAD

  8. New aromatase inhibitors. Synthesis and biological activity of aryl-substituted pyrrolizine and indolizine derivatives.

    PubMed

    Sonnet, P; Dallemagne, P; Guillon, J; Enguehard, C; Stiebing, S; Tanguy, J; Bureau, R; Rault, S; Auvray, P; Moslemi, S; Sourdaine, P; Séralini, G E

    2000-05-01

    We report herein the design and the synthesis of some aryl-substituted pyrrolizine and indolizine derivatives, on the basis of a hypothetical pharmacophore structure designed to fit the catalytic site of the human cytochrome P450 aromatase. The in vitro biological evaluation of these compounds allowed us to point out two new potent non-steroidal aromatase inhibitors, MR 20494 and MR 20492, with IC50 values in the range of 0.1 microM.

  9. Novel leucine ureido derivatives as aminopeptidase N inhibitors. Design, synthesis and activity evaluation.

    PubMed

    Ma, Chunhua; Cao, Jiangying; Liang, Xuewu; Huang, Yongxue; Wu, Ping; Li, Yingxia; Xu, Wenfang; Zhang, Yingjie

    2016-01-27

    Aminopeptidase N (APN/CD13) over-expressed on tumor cells and tumor microenvironment, plays critical roles in tumor invasion, metastasis and angiogenesis. Here we described the design, synthesis and preliminary activity studies of novel leucine ureido derivatives as aminopeptidase N (APN/CD13) inhibitors. The results showed that compound 7a had the most potent inhibitory activity against APN with the IC50 value of 20 nM, which could be used for further anticancer agent research.

  10. DNA gyrase (GyrB)/topoisomerase IV (ParE) inhibitors: synthesis and antibacterial activity.

    PubMed

    East, Stephen P; White, Clara Bantry; Barker, Oliver; Barker, Stephanie; Bennett, James; Brown, David; Boyd, E Andrew; Brennan, Christopher; Chowdhury, Chandana; Collins, Ian; Convers-Reignier, Emmanuelle; Dymock, Brian W; Fletcher, Rowena; Haydon, David J; Gardiner, Mihaly; Hatcher, Stuart; Ingram, Peter; Lancett, Paul; Mortenson, Paul; Papadopoulos, Konstantinos; Smee, Carol; Thomaides-Brears, Helena B; Tye, Heather; Workman, James; Czaplewski, Lloyd G

    2009-02-01

    The synthesis and antibacterial activities of three chemotypes of DNA supercoiling inhibitors based on imidazolo[1,2-a]pyridine and [1,2,4]triazolo[1,5-a]pyridine scaffolds that target the ATPase subunits of DNA gyrase and topoisomerase IV (GyrB/ParE) is reported. The most potent scaffold was selected for optimization leading to a series with potent Gram-positive antibacterial activity and a low resistance frequency.

  11. Novel lavendamycin analogues as antitumor agents: synthesis, in vitro cytotoxicity, structure-metabolism, and computational molecular modeling studies with NAD(P)H:quinone oxidoreductase 1.

    PubMed

    Hassani, Mary; Cai, Wen; Holley, David C; Lineswala, Jayana P; Maharjan, Babu R; Ebrahimian, G Reza; Seradj, Hassan; Stocksdale, Mark G; Mohammadi, Farahnaz; Marvin, Christopher C; Gerdes, John M; Beall, Howard D; Behforouz, Mohammad

    2005-12-01

    Novel lavendamycin analogues with various substituents were synthesized and evaluated as potential NAD(P)H:quinone oxidoreductase (NQO1)-directed antitumor agents. Pictet-Spengler condensation of quinoline- or quninoline-5,8-dione aldehydes with tryptamine or tryptophans yielded the lavendamycins. Metabolism studies with recombinant human NQO1 revealed that addition of NH2 and CH2OH groups at the quinolinedione-7-position and indolopyridine-2'-position had the greatest positive impact on substrate specificity. The best and poorest substrates were 37 (2'-CH2OH-7-NH2 derivative) and 31 (2'-CONH2-7-NHCOC3H7-n derivative) with reduction rates of 263 +/- 30 and 0.1 +/- 0.1 micromol/min/mg NQO1, respectively. Cytotoxicity toward human colon adenocarcinoma cells was determined for the lavendamycins. The best substrates for NQO1 were also the most selectively toxic to the NQO1-rich BE-NQ cells compared to NQO1-deficient BE-WT cells with 37 as the most selective. Molecular docking supported a model in which the best substrates were capable of efficient hydrogen-bonding interactions with key residues of the active site along with hydride ion reception.

  12. Synthesis and application of water-soluble macromolecular derivatives of the redox coenzymes NAD(H), NADP(H) and FAD.

    PubMed

    Bückmann, A F; Carrea, G

    1989-01-01

    During the past 15 years, the development of strategies to apply the catalytic potential of redox coenzyme-requiring enzymes has been a subject of intensive study; the main purpose of which has been to cut the cost of coenzyme to an economically acceptable level. One approach has been the utilization of isolated coenzyme-dependent enzyme systems with simultaneous enzymatic coenzyme regeneration (recycling). This has been used in conjugation with ultrafiltration reactor technology (enzyme membrane reactor), with coenzyme concentration being kept at a catalytic level. The concept implies confinement (immobilization) and practically 100% retention of both enzymes and coenzymes being dissolved in homogeneous solution within the reactor space that is closed off by an ultrafiltration membrane through which low-molecular-weight reactants (substrates and products) can freely pass. Since the problem of retaining nearly 100% native coenzymes of relatively low molecular weight by ultrafiltration membranes has not been satisfactorily solved, active macromolecular coenzyme derivatives are required. In this review, the syntheses, properties and merits of water-soluble macromolecular derivatives of NAD(H), NADP(H) and FAD are considered with respect to their biotechnological application.

  13. Design and synthesis of new piperidone grafted acetylcholinesterase inhibitors.

    PubMed

    Basiri, Alireza; Xiao, Michelle; McCarthy, Alec; Dutta, Debashis; Byrareddy, Siddappa N; Conda-Sheridan, Martin

    2017-01-15

    Alzheimer's disease (AD) is a neurodegenerative disorder affecting 35million people worldwide. A common strategy to improve the well-being of AD patients consists on the inhibition of acetylcholinesterase with the concomitant increase of the neurotransmitter acetylcholine at cholinergic synapses. Two series of unreported N-benzylpiperidines 5(a-h) and thiazolopyrimidines 9(a-q) molecules were synthesized and evaluated in vitro for their acetylcholinesterase (AChE) inhibitory activities. Among the newly synthesized compounds, 5h, 9h, 9j, and 9p displayed higher AChE enzyme inhibitory activities than the standard drug, galantamine, with IC50 values of 0.83, 0.98, and 0.73μM, respectively. Cytotoxicity studies of 5h, 9h, 9j, 9n and 9p on human neuroblastoma cells SH-SY5Y, showed no toxicity up to 40μM concentration. Molecular docking simulations of the active compounds 5h and 9p disclosed the crucial role of π-π-stacking in their binding interaction to the active site AChE enzyme. The presented compounds have potential as AChE inhibitors and potential AD drugs. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Synthesis of indole analogs as potent β-glucuronidase inhibitors.

    PubMed

    Baharudin, Mohd Syukri; Taha, Muhammad; Imran, Syahrul; Ismail, Nor Hadiani; Rahim, Fazal; Javid, Muhammad Tariq; Khan, Khalid Mohammed; Ali, Muhammad

    2017-06-01

    Natural products are the main source of motivation to design and synthesize new molecules for drug development. Designing new molecules against β-glucuronidase inhibitory is utmost essential. In this study indole analogs (1-35) were synthesized, characterized using various spectroscopic techniques including (1)H NMR and EI-MS and evaluated for their β-glucuronidase inhibitory activity. Most compounds were identified as potent inhibitors for the enzyme with IC50 values ranging between 0.50 and 53.40μM, with reference to standard d-saccharic acid 1,4-lactone (IC50=48.4±1.25μM). Structure-activity relationship had been also established. The results obtained from docking studies for the most active compound 10 showed that hydrogen bond donor features as well as hydrogen bonding with (Oε1) of nucleophilic residue Glu540 is believed to be the most importance interaction in the inhibition activity. It was also observed that hydroxyl at fourth position of benzylidene ring acts as a hydrogen bond donor and interacts with hydroxyl (OH) on the side chain of catalysis residue Tyr508. The enzyme-ligand complexed were being stabilized through electrostatic π-anion interaction with acid-base catalyst Glu451 (3.96Å) and thus preventing Glu451 from functioning as proton donor residue. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Synthesis of antimicrobial glucosamides as bacterial quorum sensing mechanism inhibitors.

    PubMed

    Biswas, Nripendra N; Yu, Tsz Tin; Kimyon, Önder; Nizalapur, Shashidhar; Gardner, Christopher R; Manefield, Mike; Griffith, Renate; Black, David StC; Kumar, Naresh

    2017-02-01

    Bacteria communicate with one another and regulate their pathogenicity through a phenomenon known as quorum sensing (QS). When the bacterial colony reaches a threshold density, the QS system induces the production of virulence factors and the formation of biofilms, a powerful defence system against the host's immune responses. The glucosamine monomer has been shown to disrupt the bacterial QS system by inhibiting autoinducer (AI) signalling molecules such as the acyl-homoserine lactones (AHLs). In this study, the synthesis of acetoxy-glucosamides 8, hydroxy-glucosamides 9 and 3-oxo-glucosamides 12 was performed via the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and N,N'-dicyclohexylcarbodiimide (DCC) coupling methods. All of the synthesized compounds were tested against two bacterial strains, P. aeruginosa MH602 (LasI/R-type QS) and E. coli MT102 (LuxI/R-type QS), for QS inhibitory activity. The most active compound 9b showed 79.1% QS inhibition against P. aeruginosa MH602 and 98.4% against E. coli MT102, while compound 12b showed 64.5% inhibition against P. aeruginosa MH602 and 88.1% against E. coli MT102 strain at 2mM concentration. The ability of the compounds to inhibit the production of the virulence factor pyocyanin and biofilm formation in the P. aeruginosa (PA14) strain was also examined. Finally, computational docking studies were performed with the LasR receptor protein.

  16. Synthesis of quaternary α-amino acid-based arginase inhibitors via the Ugi reaction.

    PubMed

    Golebiowski, Adam; Whitehouse, Darren; Beckett, R Paul; Van Zandt, Michael; Ji, Min Koo; Ryder, Todd R; Jagdmann, Erik; Andreoli, Monica; Lee, Yung; Sheeler, Ryan; Conway, Bruce; Olczak, Jacek; Mazur, Marzena; Czestkowski, Wojciech; Piotrowska, Wieslawa; Cousido-Siah, Alexandra; Ruiz, Francesc X; Mitschler, Andre; Podjarny, Alberto; Schroeter, Hagen

    2013-09-01

    The Ugi reaction has been successfully applied to the synthesis of novel arginase inhibitors. In an effort to decrease conformational flexibility of the previously reported series of 2-amino-6-boronohexanoic acid (ABH) analogs 1, we designed and synthesized a series of compounds, 2, in which a piperidine ring is linked directly to a quaternary amino acid center. Further improvement of in vitro activity was achieved by adding two carbon bridge in the piperidine ring, that is, tropane analogs 11. These improvements in activity are rationalized by X-ray crystallography analysis, which show that the tropane ring nitrogen atom moves into direct contact with Asp202 (arginase II numbering). The synthetic routes described here enabled the design of novel arginase inhibitors with improved potency and markedly different physico-chemical properties compared to ABH. Compound 11c represents the most in vitro active arginase inhibitor reported to date.

  17. 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.

  18. Effect of a cyclooxygenase-2 inhibitor on postexercise muscle protein synthesis in humans

    PubMed Central

    Burd, Nicholas A.; Dickinson, Jared M.; LeMoine, Jennifer K.; Carroll, Chad C.; Sullivan, Bridget E.; Haus, Jacob M.; Jemiolo, Bozena; Trappe, Scott W.; Hughes, Gordon M.; Sanders, Charles E.

    2010-01-01

    Nonselective blockade of the cyclooxygenase (COX) enzymes in skeletal muscle eliminates the normal increase in muscle protein synthesis following resistance exercise. The current study tested the hypothesis that this COX-mediated increase in postexercise muscle protein synthesis is regulated specifically by the COX-2 isoform. Sixteen males (23 ± 1 yr) were randomly assigned to one of two groups that received three doses of either a selective COX-2 inhibitor (celecoxib; 200 mg/dose, 600 mg total) or a placebo in double-blind fashion during the 24 h following a single bout of knee extensor resistance exercise. At rest and 24 h postexercise, skeletal muscle protein fractional synthesis rate (FSR) was measured using a primed constant infusion of [2H5]phenylalanine coupled with muscle biopsies of the vastus lateralis, and measurements were made of mRNA and protein expression of COX-1 and COX-2. Mixed muscle protein FSR in response to exercise (P < 0.05) was not suppressed by the COX-2 inhibitor (0.056 ± 0.004 to 0.108 ± 0.014%/h) compared with placebo (0.074 ± 0.004 to 0.091 ± 0.005%/h), nor was there any difference (P > 0.05) between the placebo and COX-2 inhibitor postexercise when controlling for resting FSR. The COX-2 inhibitor did not influence COX-1 mRNA, COX-1 protein, or COX-2 protein levels, whereas it did increase (P < 0.05) COX-2 mRNA (3.0 ± 0.9-fold) compared with placebo (1.3 ± 0.3-fold). It appears that the elimination of the postexercise muscle protein synthesis response by nonselective COX inhibitors is not solely due to COX-2 isoform blockade. Furthermore, the current data suggest that the COX-1 enzyme is likely the main isoform responsible for the COX-mediated increase in muscle protein synthesis following resistance exercise in humans. PMID:19934404

  19. Suppression of the biosynthesis of guanosine triphosphate by protein synthesis inhibitors

    SciTech Connect

    Volkin, E.; Boling, M.E.; Jones, M.H.; Lee, W.H.; Pike, L.M.

    1980-10-10

    In a prior report it was observed that CTP synthesis and concomitant incorporation of CMP into RNA and dCMP into DNA were markedly reduced in cells cultured in the presence of cycloheximide and puromycin. Experiments described here with Novikoff hepatoma cells reveal that the purine biosynthetic pathway is similarly affected. When the cells are subjected to cycloheximide (30 or 60 ..mu..g/ml) or puromycin (100 ..mu..g/ml), there is a substantial reduction in the bioconversion of hypoxanthine, adenosine, and deoxyadenosine into guanylate compared to untreated cultures. Whereas synthesis (counts per min/nmol) of pool ATP was 70 to 100% of controls, that of pool GTP was 20 to 35% of controls. Incorporation of AMP into RNA was 40 to 60% of controls, but that of GMP was only 10 to 25% of controls. Incorporation of dAMP into DNA averaged 10% of controls, but that of dGMP was only 4% of controls. Synthesis of guanylates from formate by the de novo pathway was similarly reduced, but incorporation of guanosine, which enters via kinase action alone, was not disproportionately lowered. These results suggest that protein synthesis inhibitors cause a severely reduced availability of newly synthesized GTP and CTP as well as their deoxy counterparts, dGTP and dCTP, the proximal precursors for the synthesis of RNA and DNA. However, the nanomolar levels of all nucleoside triphosphates remain high, probably as a result of recycling of nucleic acid breakdown products. Thus, reduced synthesis of these compounds may restrict nucleic acid synthesis only of some sort of compartmentation leads to a limitation of these precursors at the site(s) of nucleic acid synthesis.

  20. 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.

  1. Nutritional energy stimulates NAD+ production to promote tankyrase-mediated PARsylation in insulinoma cells.

    PubMed

    Zhong, Linlin; Yeh, Tsung-Yin J; Hao, Jun; Pourtabatabaei, Nasim; Mahata, Sushil K; Shao, Jianhua; Chessler, Steven D; Chi, Nai-Wen

    2015-01-01

    The poly-ADP-ribosylation (PARsylation) activity of tankyrase (TNKS) regulates diverse physiological processes including energy metabolism and wnt/β-catenin signaling. This TNKS activity uses NAD+ as a co-substrate to post-translationally modify various acceptor proteins including TNKS itself. PARsylation by TNKS often tags the acceptors for ubiquitination and proteasomal degradation. Whether this TNKS activity is regulated by physiological changes in NAD+ levels or, more broadly, in cellular energy charge has not been investigated. Because the NAD+ biosynthetic enzyme nicotinamide phosphoribosyltransferase (NAMPT) in vitro is robustly potentiated by ATP, we hypothesized that nutritional energy might stimulate cellular NAMPT to produce NAD+ and thereby augment TNKS catalysis. Using insulin-secreting cells as a model, we showed that glucose indeed stimulates the autoPARsylation of TNKS and consequently its turnover by the ubiquitin-proteasomal system. This glucose effect on TNKS is mediated primarily by NAD+ since it is mirrored by the NAD+ precursor nicotinamide mononucleotide (NMN), and is blunted by the NAMPT inhibitor FK866. The TNKS-destabilizing effect of glucose is shared by other metabolic fuels including pyruvate and amino acids. NAD+ flux analysis showed that glucose and nutrients, by increasing ATP, stimulate NAMPT-mediated NAD+ production to expand NAD+ stores. Collectively our data uncover a metabolic pathway whereby nutritional energy augments NAD+ production to drive the PARsylating activity of TNKS, leading to autoPARsylation-dependent degradation of the TNKS protein. The modulation of TNKS catalytic activity and protein abundance by cellular energy charge could potentially impose a nutritional control on the many processes that TNKS regulates through PARsylation. More broadly, the stimulation of NAD+ production by ATP suggests that nutritional energy may enhance the functions of other NAD+-driven enzymes including sirtuins.

  2. Review of synthesis, biological assay and QSAR studies of β-secretase inhibitors.

    PubMed

    Niño, Helena; García-Pintos, Isela; Rodríguez-Borges, José E; Escobar-Cubiella, Manolo; García-Mera, Xerardo; Prado-Prado, Francisco

    2011-12-01

    Alzheimer's disease (AD) is highly complex. While several pathologies characterize this disease, amyloid plaques, composed of the β-amyloid peptide, are hallmark neuropathological lesions in Alzheimer's disease brain. Indeed, a wealth of evidence suggests that β-amyloid is central to the pathophysiology of AD and is likely to play an early role in this intractable neurodegenerative disorder. The BACE-1 enzyme is essential for the generation of β-amyloid. BACE-1 knockout mice do not produce β-amyloid and are free from Alzheimer's associated pathologies, including neuronal loss and certain memory deficits. The fact that BACE-1 initiates the formation of β-amyloid, and the observation that BACE-1 levels are elevated in this disease provide direct and compelling reasons to develop therapies directed at BACE-1 inhibition, thus reducing β-amyloid and its associated toxicities. In this sense, quantitative structure-activity relationships (QSAR) could play an important role in studying these β-secretase inhibitors. QSAR models are necessary in order to guide the β-secretase synthesis. This work is aimed at reviewing different design and synthesis and computational studies for a very large and heterogeneous series of β-secretase inhibitors. First, we review design, synthesis, and Biological assay of β-secretase inhibitors. Next, we review 2D QSAR, 3D QSAR, CoMFA, CoMSIA and Docking with different compounds to find out the structural requirements. Next, we review QSAR studies using the method of Linear Discriminant Analysis (LDA) in order to understand the essential structural requirement for receptor binding for β- secretase inhibitors.

  3. 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

  4. Synthesis, cytotoxicity and molecular modelling studies of new phenylcinnamide derivatives as potent inhibitors of cholinesterases.

    PubMed

    Saeed, Aamer; Mahesar, Parvez Ali; Zaib, Sumera; Khan, Muhammad Siraj; Matin, Abdul; Shahid, Mohammad; Iqbal, Jamshed

    2014-05-06

    The present study reports the synthesis of cinnamide derivatives and their biological activity as inhibitors of both cholinesterases and anticancer agents. Controlled inhibition of brain acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) may slow neurodegeneration in Alzheimer's diseases (AD). The anticholinesterase activity of phenylcinnamide derivatives was determined against Electric Eel acetylcholinesterase (EeAChE) and horse serum butyrylcholinesterase (hBChE) and some of the compounds appeared as moderately potent inhibitors of EeAChE and hBChE. The compound 3-(2-(Benzyloxy)phenyl)-N-(3,4,5-trimethoxyphenyl)acrylamide (3i) showed maximum activity against EeAChE with an IC50 0.29 ± 0.21 μM whereas 3-(2-chloro-6-nitrophenyl)-N-(3,4,5-trimethoxyphenyl)acrylamide (3k) was proved to be the most potent inhibitor of hBChE having IC50 1.18 ± 1.31 μM. To better understand the enzyme-inhibitor interaction of the most active compounds toward cholinesterases, molecular modelling studies were carried out on high-resolution crystallographic structures. The anticancer effects of synthesized compounds were also evaluated against cancer cell line (lung carcinoma). The compounds may be useful leads for the design of a new class of anticancer drugs for the treatment of cancer and cholinesterase inhibitors for Alzheimer's disease (AD). Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  5. Design, synthesis and characterization of a highly effective inhibitor for analog-sensitive (as) kinases.

    PubMed

    Klein, Michael; Morillas, Montse; Vendrell, Alexandre; Brive, Lars; Gebbia, Marinella; Wallace, Iain M; Giaever, Guri; Nislow, Corey; Posas, Francesc; Grøtli, Morten

    2011-01-01

    Highly selective, cell-permeable and fast-acting inhibitors of individual kinases are sought-after as tools for studying the cellular function of kinases in real time. A combination of small molecule synthesis and protein mutagenesis, identified a highly potent inhibitor (1-Isopropyl-3-(phenylethynyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine) of a rationally engineered Hog1 serine/threonine kinase (Hog1(T100G)). This inhibitor has been successfully used to study various aspects of Hog1 signaling, including a transient cell cycle arrest and gene expression changes mediated by Hog1 in response to stress. This study also underscores that the general applicability of this approach depends, in part, on the selectivity of the designed the inhibitor with respect to activity versus the engineered and wild type kinases. To explore this specificity in detail, we used a validated chemogenetic assay to assess the effect of this inhibitor on all gene products in yeast in parallel. The results from this screen emphasize the need for caution and for case-by-case assessment when using the Analog-Sensitive Kinase Allele technology to assess the physiological roles of kinases.

  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.

  7. 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.

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

    PubMed

    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

    2015-05-18

    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.

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

    PubMed

    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

    2015-05-18

    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. © 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

  10. 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

  11. The identification of translesion DNA synthesis regulators: Inhibitors in the spotlight.

    PubMed

    Bertolin, A P; Mansilla, S F; Gottifredi, V

    2015-08-01

    Over the past half-century, we have become increasingly aware of the ubiquity of DNA damage. Under the constant exposure to exogenous and endogenous genomic stress, cells must attempt to replicate damaged DNA. The encounter of replication forks with DNA lesions triggers several cellular responses, including the activation of translesion DNA synthesis (TLS), which largely depends upon specialized DNA polymerases with flexible active sites capable of accommodating bulky DNA lesions. A detrimental aspect of TLS is its intrinsic mutagenic nature, and thus the activity of the TLS polymerases must ideally be restricted to synthesis on damaged DNA templates. Despite their potential clinical importance in chemotherapy, TLS inhibitors have been difficult to identify since a direct assay designed to quantify genomic TLS events is still unavailable. Herein we discuss the methods that have been used to validate TLS inhibitors such as USP1, p21 and Spartan, highlighting research that has revealed their contribution to the control of DNA synthesis on damaged and undamaged templates. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. The identification of translesion DNA synthesis regulators: inhibitors in the spotlight

    PubMed Central

    Bertolin, AP; Mansilla, SF; Gottifredi, V

    2015-01-01

    Over the past half-century, we have become increasingly aware of the ubiquity of DNA damage. Under the constant exposure to exogenous and endogenous genomic stress, cells must attempt to replicate damaged DNA. The encounter of replication forks with DNA lesions triggers several cellular responses, including the activation of translesion DNA synthesis (TLS), which largely depends upon specialized DNA polymerases with flexible active sites capable of accommodating bulky DNA lesions. A detrimental aspect of TLS is its intrinsic mutagenic nature, and thus the activity of the TLS polymerases must ideally be restricted to synthesis on damaged DNA templates. Despite their potential clinical importance in chemotherapy, TLS inhibitors have been difficult to identify since a direct assay designed to quantify genomic TLS events is still unavailable. Herein we discuss the methods that have been used to validate TLS inhibitors such as USP1, p21 and Spartan, highlighting research that has revealed their contribution to the control of DNA synthesis on damaged and undamaged templates. PMID:26002196

  13. Steroid synthesis by Taenia crassiceps WFU cysticerci is regulated by enzyme inhibitors.

    PubMed

    Aceves-Ramos, A; Valdez, R A; Gaona, B; Willms, K; Romano, M C

    2013-07-01

    Cysticerci and tapeworms from Taenia crassiceps WFU, ORF and Taenia solium synthesize sex-steroid hormones in vitro. Corticosteroids increase the 17β-estradiol synthesis by T. crassiceps cysticerci. T. crassiceps WFU cysticerci synthesize corticosteroids, mainly 11-deoxycorticosterone (DOC). The aim of this work was to investigate whether classical steroidogenic inhibitors modify the capacity of T. crassiceps WFU cysticerci to synthesize corticosteroids and sex steroid hormones. For this purpose, T. crassiceps WFU cysticerci were obtained from the abdominal cavity of mice, pre-cultured for 24h in DMEM+antibiotics/antimycotics and cultured in the presence of tritiated progesterone ((3)H-P4), androstendione ((3)H-A4), or dehydroepiandrosterone ((3)H-DHEA) plus different doses of the corresponding inhibitors, for different periods. Blanks with the culture media adding the tritiated precursors were simultaneously incubated. At the end of the incubation period, parasites were separated and media extracted with ether. The resulting steroids were separated by thin layer chromatography (TLC). Data were expressed as percent transformation of the tritiated precursors. Results showed that after 2h of exposure of the cysticerci to 100 μM formestane, the (3)H-17β-estradiol synthesis from tritiated androstenedione was significantly inhibited. The incubation of cysticerci in the presence of (3)H-DHEA and danazol (100 nM) resulted in (3)H-androstenediol accumulation and a significant reduction of the 17β-estradiol synthesis. The cysticerci (3)H-DOC synthesis was significantly inhibited when the parasites were cultured in the presence of different ketoconazole dosis. The drug treatments did not affect parasite's viability. The results of this study showed that corticosteroid and sex steroid synthesis in T. crassiceps WFU cysticerci can be modified by steroidogenic enzyme inhibitors. As was shown previously by our laboratory and others, parasite survival and development depends

  14. 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.

  15. 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.

  16. 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. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

    PubMed

    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; 8.7 μM) 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.

  18. 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

  19. 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.

  20. Synthesis and Evaluation of Quinazolines as Inhibitors of the Bacterial Cell Division Protein FtsZ.

    PubMed

    Nepomuceno, Gabriella M; Chan, Katie M; Huynh, Valerie; Martin, Kevin S; Moore, Jared T; O'Brien, Terrence E; Pollo, Luiz A E; Sarabia, Francisco J; Tadeus, Clarissa; Yao, Zi; Anderson, David E; Ames, James B; Shaw, Jared T

    2015-03-12

    The bacterial cell division protein FtsZ is one of many potential targets for the development of novel antibiotics. Recently, zantrin Z3 was shown to be a cross-species inhibitor of FtsZ; however, its specific interactions with the protein are still unknown. Herein we report the synthesis of analogues that contain a more tractable core structure and an analogue with single-digit micromolar inhibition of FtsZ's GTPase activity, which represents the most potent inhibitor of Escherichia coli FtsZ reported to date. In addition, the zantrin Z3 core has been converted to two potential photo-cross-linking reagents for proteomic studies that could shed light on the molecular interactions between FtsZ and molecules related to zantrin Z3.

  1. 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

  2. Synthesis and Evaluation of Quinazolines as Inhibitors of the Bacterial Cell Division Protein FtsZ

    PubMed Central

    2015-01-01

    The bacterial cell division protein FtsZ is one of many potential targets for the development of novel antibiotics. Recently, zantrin Z3 was shown to be a cross-species inhibitor of FtsZ; however, its specific interactions with the protein are still unknown. Herein we report the synthesis of analogues that contain a more tractable core structure and an analogue with single-digit micromolar inhibition of FtsZ’s GTPase activity, which represents the most potent inhibitor of Escherichia coli FtsZ reported to date. In addition, the zantrin Z3 core has been converted to two potential photo-cross-linking reagents for proteomic studies that could shed light on the molecular interactions between FtsZ and molecules related to zantrin Z3. PMID:25815151

  3. Design, synthesis, and 3D QSAR of novel potent and selective aromatase inhibitors.

    PubMed

    Leonetti, Francesco; Favia, Angelo; Rao, Angela; Aliano, Rosaria; Paluszcak, Anja; Hartmann, Rolf W; Carotti, Angelo

    2004-12-30

    The design, synthesis, and biological evaluation of a series of new aromatase inhibitors bearing an imidazole or triazole ring linked to a fluorene (A), indenodiazine (B), or coumarin scaffold (C) are reported. Properly substituted coumarin derivatives displayed the highest aromatase inhibitory potency and selectivity over 17-alpha-hydroxylase/17-20 lyase. The modeling of the aromatase inhibition data by Comparative Molecular Field Analysis (CoMFA/GOLPE 3D QSAR approach) led to the development of a PLS model with good fitting and predictive powers (n = 22, ONC = 3, r(2) = 0.949, s = 0.216, and q(2) = 0.715). The relationship between aromatase inhibition and the steric and electrostatic fields generated by the examined azole inhibitors enables a clear understanding of the nature and spatial location of the main interactions modulating the aromatase inhibitory potency.

  4. Identification of polymerase and processivity inhibitors of vaccinia DNA synthesis using a stepwise screening approach

    PubMed Central

    Silverman, Janice Elaine Y.; Ciustea, Mihai; Shudofsky, Abigail M. Druck; Bender, Florent; Shoemaker, Robert H.; Ricciardi, Robert P.

    2008-01-01

    Nearly all DNA polymerases require processivity factors to ensure continuous incorporation of nucleotides. Processivity factors are specific for their cognate DNA polymerases. For this reason, the vaccinia DNA polymerase (E9) and the proteins associated with processivity (A20 and D4) are excellent therapeutic targets. In this study, we show the utility of stepwise rapid plate assays that i) screen for compounds that block vaccinia DNA synthesis, ii) eliminate trivial inhibitors, e.g. DNA intercalators, and iii) distinguish whether inhibitors are specific for blocking DNA polymerase activity or processivity. The sequential plate screening of 2,222 compounds from the NCI Diversity Set library yielded a DNA polymerase inhibitor (NSC 55636) and a processivity inhibitor (NSC 123526) that were capable of reducing vaccinia viral plaques with minimal cellular cytotoxicity. These compounds are predicted to block cellular infection by the smallpox virus, variola, based on the very high sequence identity between A20, D4 and E9 of vaccinia and the corresponding proteins of variola. PMID:18621425

  5. 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.

  6. Synthesis, in vitro and in vivo activity of thiamine antagonist transketolase inhibitors.

    PubMed

    Thomas, Allen A; Le Huerou, Y; De Meese, J; Gunawardana, Indrani; Kaplan, Tomas; Romoff, Todd T; Gonzales, Stephen S; Condroski, Kevin; Boyd, Steven A; Ballard, Josh; Bernat, Bryan; DeWolf, Walter; Han, May; Lee, Patrice; Lemieux, Christine; Pedersen, Robin; Pheneger, Jed; Poch, Greg; Smith, Darin; Sullivan, Francis; Weiler, Solly; Wright, S Kirk; Lin, Jie; Brandhuber, Barb; Vigers, Guy

    2008-03-15

    Tumor cells extensively utilize the pentose phosphate pathway for the synthesis of ribose. Transketolase is a key enzyme in this pathway and has been suggested as a target for inhibition in the treatment of cancer. In a pharmacodynamic study, nude mice with xenografted HCT-116 tumors were dosed with 1 ('N3'-pyridyl thiamine'; 3-(6-methyl-2-amino-pyridin-3-ylmethyl)-5-(2-hydroxy-ethyl)-4-methyl-thiazol-3-ium chloride hydrochloride), an analog of thiamine, the co-factor of transketolase. Transketolase activity was almost completely suppressed in blood, spleen, and tumor cells, but there was little effect on the activity of the other thiamine-utilizing enzymes alpha-ketoglutarate dehydrogenase or glucose-6-phosphate dehydrogenase. Synthesis and SAR of transketolase inhibitors is described.

  7. 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

  8. Diels-Alder cycloaddition in the synthesis of 1-azafagomine, analogs, and derivatives as glycosidase inhibitors.

    PubMed

    Salgueiro, Daniela A L; Sousa, Cristina E A; Fortes, A Gil; Alves, M José

    2012-12-01

    This comprehensive review deals with the synthesis of 1-azafagomine, analogs, and derivatives having the Diels-Alder cycloaddition as the key step. Most of the compounds referred are racemic or have been resolved by lipase transesterification. There are two asymmetric cycloadditions leading to 1-azafagomine or to an analog. In one case both enantiomers of 1-azafagomine were prepared together with a pair of derivatives. The study comprises glycosidase inhibition studies of the target compounds to a set of glycosidic enzymes, and evidenced molecular features that enhance or diminish their activity as glycosidase inhibitors.

  9. Microwave assisted organic synthesis (MAOS) of small molecules as potential HIV-1 integrase inhibitors.

    PubMed

    Ferro, Stefania; Grazia, Sara De; De Luca, Laura; Gitto, Rosaria; Faliti, Caterina Elisa; Debyzer, Zeger; Chimirri, Alba

    2011-08-11

    Integrase (IN) represents a clinically validated target for the development of antivirals against human immunodeficiency virus (HIV). In recent years our research group has been engaged in the stucture-function study of this enzyme and in the development of some three-dimensional pharmacophore models which have led to the identification of a large series of potent HIV-1 integrase strand-transfer inhibitors (INSTIs) bearing an indole core. To gain a better understanding of the structure-activity relationships (SARs), herein we report the design and microwave-assisted synthesis of a novel series of 1-H-benzylindole derivatives.

  10. A practical total synthesis of the microbial alkaline proteinase inhibitor (MAPI).

    PubMed

    Haebich, Dieter; Hillisch, Alexander; El Sheikh, Sherif

    2009-12-01

    Diverse serine and cysteine proteases as well as alkaline proteinases and elastases play a crucial role in numerous biological processes. Natural peptide aldehydes such as the "microbial alkaline proteinase inhibitor" (MAPI, 1) are valuable tools to characterize novel enzymes and to study their function in nature. Within a drug discovery program we wanted to design and explore non-natural MAPI congeners with novel biological profiles. To that end we devised a simple, practical, and scalable synthesis of MAPI 1 from readily available amino acid building blocks. The modular nature of our approach allows convenient structural modification of the MAPI backbone.

  11. Chemical and functional properties of mutastein, an inhibitor of insoluble glucan synthesis by Streptococcus sobrinus.

    PubMed

    Hayashida, O; Hasumi, K; Endo, A

    1997-04-01

    Mutastein, a potent inhibitor of insoluble glucan synthesis by Streptococcus sobrinus, is a protein with a molecular weight of approximately 2 x 10(6). Amino acid and ELISA analyses suggested that mutastein is a mixture of heterogenous polymers of alpha-casein contained in the culture medium of the producing strain, Aspergillus terreus M3328. Mutastein strongly inhibited the primer-dependent insoluble glucan synthase of S. sobrinus B13. The primer-independent soluble glucan synthase was not affected by mutastein while primer-dependent soluble glucan synthase was slightly activated.

  12. Increased sensitivity to protein synthesis inhibitors in cells lacking tmRNA.

    PubMed Central

    de la Cruz, J; Vioque, A

    2001-01-01

    tmRNA (also known as SsrA or 10Sa RNA) is involved in a trans-translation reaction that contributes to the recycling of stalled ribosomes at the 3' end of an mRNA lacking a stop codon or at an internal mRNA cluster of rare codons. Inactivation of the ssrA gene in most bacteria results in viable cells bearing subtle phenotypes, such as temperature-sensitive growth. Herein, we report on the functional characterization of the ssrA gene in the cyanobacterium Synechocystis sp. strain PCC6803. Deletion of the ssrA gene in Synechocystis resulted in viable cells with a growth rate identical to wild-type cells. However, null ssrA cells (deltassrA) were not viable in the presence of the protein synthesis inhibitors chloramphenicol, lincomycin, spiramycin, tylosin, erythromycin, and spectinomycin at low doses that do not significantly affect the growth of wild-type cells. Sensitivity of deltassrA cells similar to wild-type cells was observed with kasugamycin, fusidic acid, thiostrepton, and puromycin. Antibiotics unrelated to protein synthesis, such as ampicillin or rifampicin, had no differential effect on the deltassrA strain. Furthermore, deletion of the ssrA gene is sufficient to impair global protein synthesis when chloramphenicol is added at sublethal concentrations for the wild-type strain. These results indicate that ribosomes stalled by some protein synthesis inhibitors can be recycled by tmRNA. In addition, this suggests that the first elongation cycle with tmRNA, which incorporates a noncoded alanine on the growing peptide chain, may have mechanistic differences with the normal elongation cycles that bypasses the block produced by these specific antibiotics. tmRNA inactivation could be an useful therapeutic target to increase the sensitivity of pathogenic bacteria against antibiotics. PMID:11780628

  13. Salmonella typhimurium mutants lacking NAD pyrophosphatase.

    PubMed Central

    Park, U E; Roth, J R; Olivera, B M

    1988-01-01

    NAD can serve as both a purine and a pyridine source for Salmonella typhimurium. Exogenous NAD is rapidly broken down into nicotinamide mononucleotide and AMP by an NAD pyrophosphatase, the first step in the pathway for the assimilation of exogenous NAD. We isolated and characterized mutants of S. typhimurium lacking NAD pyrophosphatase activity; such mutants were identified by their failure to use exogenous NAD as a purine source. These mutants carry mutations that map at a new locus, designated pnuE, between 86 and 87 min on the Salmonella chromosome. PMID:2841298

  14. Effect of NAD on PARP-mediated insulin sensitivity in oleic acid treated hepatocytes.

    PubMed

    Pang, Jing; Cui, Ju; Gong, Huan; Xi, Chao; Zhang, Tie-Mei

    2015-07-01

    High serum free fatty acids levels are associated with the development of insulin resistance in type 2 diabetes; however, the precise mechanisms underlying this lipid toxicity are unclear. To investigate whether PARP1 activation and NAD depletion are involved in the impairment of insulin sensitivity associated with lipotoxicity, HepG2 cells were cultured with 500 μM oleic acid for 48 h. Oleic acid-treated cells exhibited increased ROS generation, lipid accumulation and PARP1 activation. Treatment with the PARP1 inhibitor PJ34 and transfection with PARP1 small interfering RNA both prevented the oleic acid-induced impairment of the insulin signaling pathway. Furthermore, treatment with PJ34 reversed the oleic acid-induced decrease in intracellular NAD concentration, while exogenous NAD protected cells against oleic acid-induced insulin insensitivity. Combined NAD and PJ34 administration did not enhance the effects obtained by treatment with either NAD or PJ34 alone. Interestingly, when cells were treated with the SIRT1 inhibitor EX527, the protective effects of PJ34 and NAD treatment were diminished. Taken together, these data suggest that NAD depletion by PARP1 activation is essential for the modulation of insulin sensitivity in oleic acid-induced lipotoxicity.

  15. Independent AMP and NAD signaling regulates C2C12 differentiation and metabolic adaptation.

    PubMed

    Hsu, Chia George; Burkholder, Thomas J

    2016-12-01

    The balance of ATP production and consumption is reflected in adenosine monophosphate (AMP) and nicotinamide adenine dinucleotide (NAD) content and has been associated with phenotypic plasticity in striated muscle. Some studies have suggested that AMPK-dependent plasticity may be an indirect consequence of increased NAD synthesis and SIRT1 activity. The primary goal of this study was to assess the interaction of AMP- and NAD-dependent signaling in adaptation of C2C12 myotubes. Changes in myotube developmental and metabolic gene expression were compared following incubation with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and nicotinamide mononucleotide (NMN) to activate AMPK- and NAD-related signaling. AICAR showed no effect on NAD pool or nampt expression but significantly reduced histone H3 acetylation and GLUT1, cytochrome C oxidase subunit 2 (COX2), and MYH3 expression. In contrast, NMN supplementation for 24 h increased NAD pool by 45 % but did not reduce histone H3 acetylation nor promote mitochondrial gene expression. The combination of AMP and NAD signaling did not induce further metabolic adaptation, but NMN ameliorated AICAR-induced myotube reduction. We interpret these results as indication that AMP and NAD contribute to C2C12 differentiation and metabolic adaptation independently.

  16. NAMPT regulates mitochondria biogenesis via NAD metabolism and calcium binding proteins during skeletal muscle contraction.

    PubMed

    Kim, Jeong Seok; Yoon, Chung-Su; Park, Dae Ryoung

    2014-09-01

    The purpose of this study was to investigate the effect that muscle contraction induced NAD metabolism via NAMPT has on mitochondrial biogenesis. Primary skeletal muscle cells were isolated from the gastrocnemius in C57BL/6 mice. The muscle cells were stimulated by electrical current at 1Hz for 3 minutes in conditions of normal or NAD metabolism related inhibitor treatment. NAD/NADH level, Sirt1 and mitochondria biogenesis related signal factor's changes were examined in normal or NAD metabolism related inhibitor treated cells. Electrical stimulation (ES) induced muscle contractions significantly increased NAD/NADH levels, NAMPT inhibitor FK-866 inhibited ES-induced NAD formation, which caused SIRT1 expression and PGC-1α deacetylation to decrease. Moreover, NAMPT inhibition decreased mitochondrial biogenesis related mRNA, COX-1 and Tfam levels. Along with AMPK inhibitor, compound C decreases SIRT1 expression, PGC-1α deacetylation and muscle contraction induced mitochondrial biogenesis related mRNA increment. These results indicated that the AMPK-NAMPT signal is a key player for muscle contraction induced SIRT1 expression and PGC-1α deacetylation, which influences mitochondrial biogenesis. Inhibition of the AMPK upregulator, Camkkβ, STO-609 decreased AMPK phosphorylation and SIRT1 expression but did not decrease PGC-1α deacetylation. However, CAMKII inhibition via AIP decreased PGC-1α deacetylation. In conclusion, the results indicate that NAMPT plays an important role in NAD metabolism and mitochondrial biogenesis. However, mitochondrial biogenesis is also controlled by different calcium binding protein signals including Camkkβ and CAMKII. [Keyword] Muscle contraction, NAD metabolism, SIRT1, PGC-1 α, mitochondria biogenesis.

  17. 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

  18. Chitin synthesis inhibitor effect on Aedes aegypti populations susceptible and resistant to organophosphate temephos.

    PubMed

    Martins, Ademir Jesus; Belinato, Thiago Affonso; Lima, José Bento Pereira; Valle, Denise

    2008-06-01

    In Brazil, dengue vector control is hampered by the resistance of Aedes aegypti L. populations to organophosphates (OPs). Insect growth regulators (IGRs) are a promising alternative, as their mechanisms of action are different from those of conventional insecticides. The authors analysed the effect of the IGR triflumuron, a chitin synthesis inhibitor, on the Ae. aegypti insecticide-susceptible strain Rockefeller, as well as on field populations both susceptible (TemS) and resistant (TemR) to the OP temephos. Triflumuron arrested development and inhibited adult emergence of the Rockefeller strain in a dose-dependent way (EI(50) and EI(90) of 0.8 and 1.8 microg L(-1) respectively). A direct relationship between triflumuron concentration and the precocity of its effects was evident. TemS and TemR temephos resistance ratios (RR(90)) were 4.5 and 13.8, triflumuron RR(90) being 1.0 and 1.3 respectively. The IGR triflumuron exhibited a dose-dependent effect against the reference Ae. aegypti Rockefeller strain. It was also effective against two field populations, regardless of their OP resistance status. The present results are discussed in the context of utilization of chitin synthesis inhibitors as potential alternatives in the control of Ae. aegypti in Brazil. Copyright (c) 2008 Society of Chemical Industry.

  19. 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.

  20. Total synthesis and structure-activity relationship studies of a series of selective G protein inhibitors

    NASA Astrophysics Data System (ADS)

    Xiong, Xiao-Feng; Zhang, Hang; Underwood, Christina R.; Harpsøe, Kasper; Gardella, Thomas J.; Wöldike, Mie F.; Mannstadt, Michael; Gloriam, David E.; Bräuner-Osborne, Hans; Strømgaard, Kristian

    2016-11-01

    G proteins are key mediators of G protein-coupled receptor signalling, which facilitates a plethora of important physiological processes. The cyclic depsipeptides YM-254890 and FR900359 are the only known specific inhibitors of the Gq subfamily of G proteins; however, no synthetic route has been reported previously for these complex natural products and they are not easily isolated from natural sources. Here we report the first total synthesis of YM-254890 and FR900359, as well as of two known analogues, YM-385780 and YM-385781. The versatility of the synthetic approach also enabled the design and synthesis of ten analogues, which provided the first structure-activity relationship study for this class of compounds. Pharmacological characterization of all the compounds at Gq-, Gi- and Gs-mediated signalling provided succinct information on the structural requirements for inhibition, and demonstrated that both YM-254890 and FR900359 are highly potent inhibitors of Gq signalling, with FR900359 being the most potent. These natural products and their analogues represent unique tools for explorative studies of G protein inhibition.

  1. 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.

  2. The nadA gene of Pseudomonas fluorescens PGPR strain 267.1.

    PubMed

    Marek-Kozaczuk, Monika; Rogalski, Jerzy; Skorupska, Anna

    2005-08-01

    An insertion mutant of Pseudomonas fluorescens PGPR strain 267.1 was found to be auxotrophic for niacin (nicotinic acid) and could not synthesize quinolinic acid. The Tn5 interrupted gene was cloned and sequenced. The cloned fragment contained an open reading frame, nadA, capable of encoding a 359-amino-acid protein (39.0 kDa) with substantial identity to various bacterial quinolinate synthetases. The nadA gene complemented quinolinic acid synthesis deficiency and niacin auxotrophy of the P. fluorescens 106 P nadA mutant.

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

    PubMed

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

    2015-07-02

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

  4. Protein synthesis inhibitors enhance the expression of mRNAs for early inducible inflammatory genes via mRNA stabilization.

    PubMed

    Yamazaki, Soh; Takeshige, Koichiro

    2008-02-01

    Expression of inflammatory genes is regulated at multiple steps, including transcriptional activation and mRNA stabilization. During an investigation into the requirement of de novo protein synthesis for the induction of inflammatory genes, it was revealed that protein synthesis inhibitors unexpectedly potentiated the induction of mRNAs for primary response genes, while the inhibitors suppressed the induction of secondary inducible genes as previously described. Stimulus-induced nuclear translocation and promoter recruitment of NF-kappaB, which is responsible for the transcriptional activation of many inflammatory genes, were largely unaffected by the inhibitors. Instead, these inhibitors prolonged the half-lives of all of the primary inducible mRNAs tested. Thus, these findings emphasize the important contribution of regulated mRNA longevity to gene expression induced by pro-inflammatory stimulation.

  5. Synthesis and structure-activity relationships of selective norepinephrine reuptake inhibitors (sNRI) with improved pharmaceutical characteristics.

    PubMed

    Pontillo, Joseph; Wu, Dongpei; Ching, Brett; Hudson, Sarah; Genicot, Marc J; Gao, Yinghong; Ewing, Todd; Fleck, Beth A; Gogas, Kathleen; Aparicio, Anna; Wang, Hua; Wen, Jenny; Wade, Warren S

    2008-12-01

    The design synthesis and SAR of a series of chiral ring-constrained norepinephrine reuptake inhibitors with improved physicochemical properties is described. Typical compounds are potent (IC(50)s<10 nM), selective against the other monoamine transporters, weak CYP2D6 inhibitors (IC(50)s>1 microM) and stable to oxidation by human liver microsomes. In addition, the compounds exhibit a favorable polarity profile.

  6. NAD+ and Sirtuins in Aging and Disease

    PubMed Central

    Imai, Shin-ichiro; Guarente, Leonard

    2014-01-01

    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 anti-aging intervention, providing hope to aging societies worldwide. PMID:24786309

  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.

  8. Novel Inhibitors of Rad6 Ubiquitin Conjugating Enzyme: Design, Synthesis, Identification, and Functional Characterization

    PubMed Central

    Nangia-Makker, Pratima; Balan, Vitaly; Morelli, Matteo; Kothayer, Hend; Westwell, Andrew D.; Shekhar, Malathy P.V.

    2013-01-01

    Protein ubiquitination is important for cell signaling, DNA repair, and proteasomal degradation, and it is not surprising that alterations in ubiquitination occur frequently in cancer. Ubiquitin-conjugating enzymes (E2) mediate ubiquitination by selective interactions with ubiquitin-activating (E1) and ubiquitin ligase (E3) enzymes, and thus selective E2 small molecule inhibitor (SMI) will provide specificity unattainable with proteasome inhibitors. Here we describe synthesis and functional characterization of the first SMIs of human E2 Rad6B, a fundamental component of translesion synthesis DNA repair. A pharmacophore model for consensus E2 ubiquitin-binding sites was generated for virtual screening to identify E2 inhibitor candidates. Twelve triazine (TZ) analogs screened in silico by molecular docking to the Rad6B X-ray structure were verified by their effect on Rad6B ubiquitination of histone H2A. TZs #8 and 9 docked to the Rad6B catalytic site with highest complementarity. TZs #1, 2, 8, and 9 inhibited Rad6B-ubiquitin thioester formation and subsequent ubiquitin transfer to histone H2A. SMI #9 inhibition of Rad6 was selective as BCA2 ubiquitination by E2 UbcH5 was unaffected by SMI #9. SMI #9 more potently inhibited proliferation, colony formation, and migration than SMI #8, and induced MDA-MB-231 breast cancer cell G2–M arrest and apoptosis. Ubiquitination assays using Rad6 immunoprecipitated from SMI #8- or 9-treated cells confirmed inhibition of endogenous Rad6 activity. Consistent with our previous data showing Rad6B-mediated polyubiquitination stabilizes β-catenin, MDAMB-231 treatment with SMIs #8 or 9 decreased β-catenin protein levels. Together these results describe identification of the first Rad6 SMIs. PMID:23339190

  9. Novel inhibitors of Rad6 ubiquitin conjugating enzyme: design, synthesis, identification, and functional characterization.

    PubMed

    Sanders, Matthew A; Brahemi, Ghali; Nangia-Makker, Pratima; Balan, Vitaly; Morelli, Matteo; Kothayer, Hend; Westwell, Andrew D; Shekhar, Malathy P V

    2013-04-01

    Protein ubiquitination is important for cell signaling, DNA repair, and proteasomal degradation, and it is not surprising that alterations in ubiquitination occur frequently in cancer. Ubiquitin-conjugating enzymes (E2) mediate ubiquitination by selective interactions with ubiquitin-activating (E1) and ubiquitin ligase (E3) enzymes, and thus selective E2 small molecule inhibitor (SMI) will provide specificity unattainable with proteasome inhibitors. Here we describe synthesis and functional characterization of the first SMIs of human E2 Rad6B, a fundamental component of translesion synthesis DNA repair. A pharmacophore model for consensus E2 ubiquitin-binding sites was generated for virtual screening to identify E2 inhibitor candidates. Twelve triazine (TZ) analogs screened in silico by molecular docking to the Rad6B X-ray structure were verified by their effect on Rad6B ubiquitination of histone H2A. TZs #8 and 9 docked to the Rad6B catalytic site with highest complementarity. TZs #1, 2, 8, and 9 inhibited Rad6B-ubiquitin thioester formation and subsequent ubiquitin transfer to histone H2A. SMI #9 inhibition of Rad6 was selective as BCA2 ubiquitination by E2 UbcH5 was unaffected by SMI #9. SMI #9 more potently inhibited proliferation, colony formation, and migration than SMI #8, and induced MDA-MB-231 breast cancer cell G2-M arrest and apoptosis. Ubiquitination assays using Rad6 immunoprecipitated from SMI #8- or 9-treated cells confirmed inhibition of endogenous Rad6 activity. Consistent with our previous data showing Rad6B-mediated polyubiquitination stabilizes β-catenin, MDA-MB-231 treatment with SMIs #8 or 9 decreased β-catenin protein levels. Together these results describe identification of the first Rad6 SMIs.

  10. 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.

  11. Deprotonation of the horse liver alcohol dehydrogenase-NAD+ complex controls formation of the ternary complexes.

    PubMed

    Kovaleva, Elena G; Plapp, Bryce V

    2005-09-27

    Binding of NAD+ to wild-type horse liver alcohol dehydrogenase is strongly pH-dependent and is limited by a unimolecular step, which may be related to a conformational change of the enzyme-NAD+ complex. Deprotonation during binding of NAD+ and inhibitors that trap the enzyme-NAD+ complex was examined by transient kinetics with pH indicators, and formation of complexes was monitored by absorbance and protein fluorescence. Reactions with pyrazole and trifluoroethanol had biphasic proton release, whereas reaction with caprate showed proton release followed by proton uptake. Proton release (200-550 s(-1)) is a common step that precedes binding of all inhibitors. At all pH values studied, the rate constants for proton release or uptake matched those for formation of ternary complexes, and the most significant quenching of protein fluorescence (or perturbation of adenine absorbance at 280 nm) was observed for enzyme species involved in deprotonation steps. Kinetic simulations of the combined transient data for the multiple signals indicate that all inhibitors bind faster and tighter to the unprotonated enzyme-NAD+ complex, which has a pK of about 7.3. The results suggest that rate-limiting deprotonation of the enzyme-NAD+ complex is coupled to the conformational change and controls the formation of ternary complexes.

  12. Elevation of cellular NAD levels by nicotinic acid and involvement of nicotinic acid phosphoribosyltransferase in human cells.

    PubMed

    Hara, Nobumasa; Yamada, Kazuo; Shibata, Tomoko; Osago, Harumi; Hashimoto, Tatsuya; Tsuchiya, Mikako

    2007-08-24

    NAD plays critical roles in various biological processes through the function of SIRT1. Although classical studies in mammals showed that nicotinic acid (NA) is a better precursor than nicotinamide (Nam) in elevating tissue NAD levels, molecular details of NAD synthesis from NA remain largely unknown. We here identified NA phosphoribosyltransferase (NAPRT) in humans and provided direct evidence of tight link between NAPRT and the increase in cellular NAD levels. The enzyme was abundantly expressed in the small intestine, liver, and kidney in mice and mediated [(14)C]NAD synthesis from [(14)C]NA in human cells. In cells expressing endogenous NAPRT, the addition of NA but not Nam almost doubled cellular NAD contents and decreased cytotoxicity by H(2)O(2). Both effects were reversed by knockdown of NAPRT expression. These results indicate that NAPRT is essential for NA to increase cellular NAD levels and, thus, to prevent oxidative stress of the cells. Kinetic analyses revealed that NAPRT, but not Nam phosphoribosyltransferase (NamPRT, also known as pre-B-cell colony-enhancing factor or visfatin), is insensitive to the physiological concentration of NAD. Together, we conclude that NA elevates cellular NAD levels through NAPRT function and, thus, protects the cells against stress, partly due to lack of feedback inhibition of NAPRT but not NamPRT by NAD. The ability of NA to increase cellular NAD contents may account for some of the clinically observed effects of the vitamin and further implies a novel application of the vitamin to treat diseases such as those associated with the depletion of cellular NAD pools.

  13. Effects of prostaglandins and prostaglandin synthesis inhibitors on sexual behavior in boars.

    PubMed

    Estienne, Mark J; Harper, Allen F; Beal, Wilfred E; Crawford, Russell J

    2007-07-01

    Experiments were conducted investigating the effects of prostaglandins and prostaglandin synthesis inhibitors on libido in boars. In Experiment 1, two prostaglandin products were compared with regard to expediting the training of boars for semen collection. On each of five consecutive days, boars received i.m. treatment with saline, dinoprost tromethamine or cloprostenol sodium (n=12/group). On each of day 1 (p=0.06), day 2 (p<0.05), and day 3 (p<0.05), but not on day 4 or 5 (p>0.1), the percentage of boars collected after dinoprost tromethamine, but not cloprostenol sodium, was greater than controls. In Experiments 2 and 3, libido in boars that were trained previously for semen collection was assessed after treatment with prostaglandin synthesis inhibitors, testing the hypothesis that endogenous release of prostaglandin is necessary for expression of sexual behaviors. In Experiment 2, boars treated with flunixin meglumine (n=12) had suppressed (p<0.01) levels of 15-ketodihydro-prostaglandin-F(2) (PGFM) in serum but characteristics of libido were similar (p>0.1) to controls (n=12). In Experiment 3, boars were administered indomethacin orally (n=12) or served as untreated controls (n=12). Indomethacin decreased (p<0.01) serum levels of PGFM, increased (p<0.05) the number of false mounts (mounting artificial sow but dismounting before an ejaculate was collected), and tended (p=0.09) to lengthen the interval between entering the collection pen and the start of ejaculation. These results suggest that prostaglandin synthesis and release is necessary for the complete display of normal sexual behaviors in boars.

  14. Synthesis of Bi-substrate State Mimics of Dihydropteroate Synthase as Potential Inhibitors and Molecular Probes

    PubMed Central

    Qi, Jianjun; Virga, Kristopher G.; Das, Sourav; Zhao, Ying; Yun, Mi-Kyung; White, Stephen W.; Lee, Richard E.

    2010-01-01

    The increasing emergence of resistant bacteria drives us to design and develop new antimicrobial agents. Pursuant to that goal, a new targeting approach of the dihydropteroate synthase enzyme, which serves as the site of action for the sulfonamide class of antimicrobial agents, is being explored. Using structural information, a new class of transition state mimics has been designed and synthesized that have the capacity to bind to the pterin, phosphate and para-amino binding sites. The design, synthesis and evaluation of these compounds as inhibitors of Bacillus anthracis dihydropteroate synthase is described herein. Outcomes from this work have identified the first trivalent inhibitors of dihydropteroate synthase whose activity displayed slow binding inhibition. The most active compounds in this series contained an oxidized pterin ring. The binding of these inhibitors was modeled into the dihydropteroate synthase active site and demonstrated a good correlation with the observed bioassay data, as well as provided important insight for the future design of higher affinity transition state mimics. PMID:21216602

  15. 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

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

    SciTech Connect

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

    2015-02-20

    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. We also show 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.

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

    DOE PAGES

    Dziedzic, Pawel; Cisneros, José A.; Robertson, Michael J.; ...

    2015-02-20

    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. Activitymore » 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. We also show 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.« less

  18. 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.

  19. Unscheduled synthesis of DNA and poly(ADP-ribose) in human fibroblasts following DNA damage

    SciTech Connect

    McCurry, L.S.; Jacobson, M.K.

    1981-01-01

    Unscheduled DNA synthesis has been measured in human fibroblasts under conditions of reduced rates of conversion of NAD to poly)ADP-ribose). Cells heterozygous for the xeroderma pigmentosum genotype showed normal rates of uv induced unscheduled DNA synthesis under conditions in which the rate of poly(ADP-ribose) synthesis was one-half the rate of normal cells. The addition of theophylline, a potent inhibitor of poly(ADP-ribose) polymerase, to the culture medium of normal cells blocked over 90% of the conversion of NAD to poly(ADP-ribose) following treatment with uv or N-methyl-N'-nitro-N-nitro-soguanidine but did not affect the rate of unscheduled DNA synthesis.

  20. NAD+ as a signaling molecule modulating metabolism

    PubMed Central

    Cantó, Carles; Auwerx, Johan

    2013-01-01

    The ability of NAD+ to act as a metabolic cofactor and as a rate-limiting cosubstrate for many enzymes, particularly the sirtuins, has led to the identification of a pivotal role of NAD+ levels in the control of whole body metabolic homeostasis. Bioavailability and compartmentalization of NAD+ have become highly relevant issues that we need to understand in order to elucidate how NAD+ acts both as a readout of the metabolic milieu and as an effector triggering appropriate cellular adaptations. PMID:22345172

  1. Inhibitors of Copi and Copii Do Not Block PEX3-Mediated Peroxisome Synthesis

    PubMed Central

    South, Sarah T.; Sacksteder, Katherine A.; Li, Xiaoling; Liu, Yifei; Gould, Stephen J.

    2000-01-01

    In humans, defects in peroxisome biogenesis are the cause of lethal diseases typified by Zellweger syndrome. Here, we show that inactivating mutations in human PEX3 cause Zellweger syndrome, abrogate peroxisome membrane synthesis, and result in reduced abundance of peroxisomal membrane proteins (PMPs) and/or mislocalization of PMPs to the mitochondria. Previous studies have suggested that PEX3 may traffic through the ER en route to the peroxisome, that the COPI inhibitor, brefeldin A, leads to accumulation of PEX3 in the ER, and that PEX3 overexpression alters the morphology of the ER. However, we were unable to detect PEX3 in the ER at early times after expression. Furthermore, we find that inhibition of COPI function by brefeldin A has no effect on trafficking of PEX3 to peroxisomes and does not inhibit PEX3-mediated peroxisome biogenesis. We also find that inhibition of COPII-dependent membrane traffic by a dominant negative SAR1 mutant fails to block PEX3 transport to peroxisomes and PEX3-mediated peroxisome synthesis. Based on these results, we propose that PEX3 targeting to peroxisomes and PEX3-mediated peroxisome membrane synthesis may occur independently of COPI- and COPII-dependent membrane traffic. PMID:10871277

  2. Amnesia produced by altered release of neurotransmitters after intraamygdala injections of a protein synthesis inhibitor.

    PubMed

    Canal, Clinton E; Chang, Qing; Gold, Paul E

    2007-07-24

    Amnesia produced by protein synthesis inhibitors such as anisomycin provides major support for the prevalent view that the formation of long-lasting memories requires de novo protein synthesis. However, inhibition of protein synthesis might disrupt other neural functions to interfere with memory formation. Intraamygdala injections of anisomycin before inhibitory avoidance training impaired memory in rats tested 48 h later. Release of norepinephrine (NE), dopamine (DA), and serotonin, measured at the site of anisomycin infusions, increased quickly by approximately 1,000-17,000%, far above the levels seen under normal conditions. NE and DA release later decreased far below baseline for several hours before recovering at 48 h. Intraamygdala injections of a beta-adrenergic receptor antagonist or agonist, each timed to blunt effects of increases and decreases in NE release after anisomycin, attenuated anisomycin-induced amnesia. In addition, similar to the effects on memory seen with anisomycin, intraamygdala injections of a high dose of NE before training impaired memory tested at 48 h after training. These findings suggest that altered release of neurotransmitters may mediate amnesia produced by anisomycin and, further, raise important questions about the empirical bases for many molecular theories of memory formation.

  3. Amnesia produced by altered release of neurotransmitters after intraamygdala injections of a protein synthesis inhibitor

    PubMed Central

    Canal, Clinton E.; Chang, Qing; Gold, Paul E.

    2007-01-01

    Amnesia produced by protein synthesis inhibitors such as anisomycin provides major support for the prevalent view that the formation of long-lasting memories requires de novo protein synthesis. However, inhibition of protein synthesis might disrupt other neural functions to interfere with memory formation. Intraamygdala injections of anisomycin before inhibitory avoidance training impaired memory in rats tested 48 h later. Release of norepinephrine (NE), dopamine (DA), and serotonin, measured at the site of anisomycin infusions, increased quickly by ≈1,000–17,000%, far above the levels seen under normal conditions. NE and DA release later decreased far below baseline for several hours before recovering at 48 h. Intraamygdala injections of a β-adrenergic receptor antagonist or agonist, each timed to blunt effects of increases and decreases in NE release after anisomycin, attenuated anisomycin-induced amnesia. In addition, similar to the effects on memory seen with anisomycin, intraamygdala injections of a high dose of NE before training impaired memory tested at 48 h after training. These findings suggest that altered release of neurotransmitters may mediate amnesia produced by anisomycin and, further, raise important questions about the empirical bases for many molecular theories of memory formation. PMID:17640910

  4. 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.

  5. 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. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. The design and synthesis of novel N-hydroxyformamide inhibitors of ADAM-TS4 for the treatment of osteoarthritis.

    PubMed

    De Savi, Chris; Pape, Andrew; Cumming, John G; Ting, Attilla; Smith, Peter D; Burrows, Jeremy N; Mills, Mark; Davies, Chris; Lamont, Scott; Milne, David; Cook, Calum; Moore, Peter; Sawyer, Yvonne; Gerhardt, Stefan

    2011-03-01

    Two series of N-hydroxyformamide inhibitors of ADAM-TS4 were identified from screening compounds previously synthesised as inhibitors of matrix metalloproteinase-13 (collagenase-3). Understanding of the binding mode of this class of compound using ADAM-TS1 as a structural surrogate has led to the discovery of potent and very selective inhibitors with favourable DMPK properties. Synthesis, structure-activity relationships, and strategies to improve selectivity and lower in vivo metabolic clearance are described. Copyright © 2011 Elsevier Ltd. All rights reserved.

  7. Image-Guided Synthesis Reveals Potent Blood-Brain Barrier Permeable Histone Deacetylase Inhibitors

    PubMed Central

    2014-01-01

    Recent studies have revealed that several histone deacetylase (HDAC) inhibitors, which are used to study/treat brain diseases, show low blood-brain barrier (BBB) penetration. In addition to low HDAC potency and selectivity observed, poor brain penetrance may account for the high doses needed to achieve therapeutic efficacy. Here we report the development and evaluation of highly potent and blood-brain barrier permeable HDAC inhibitors for CNS applications based on an image-guided approach involving the parallel synthesis and radiolabeling of a series of compounds based on the benzamide HDAC inhibitor, MS-275 as a template. BBB penetration was optimized by rapid carbon-11 labeling and PET imaging in the baboon model and using the imaging derived data on BBB penetration from each compound to feed back into the design process. A total of 17 compounds were evaluated, revealing molecules with both high binding affinity and BBB permeability. A key element conferring BBB penetration in this benzamide series was a basic benzylic amine. These derivatives exhibited 1–100 nM inhibitory activity against recombinant human HDAC1 and HDAC2. Three of the carbon-11 labeled aminomethyl benzamide derivatives showed high BBB penetration (∼0.015%ID/cc) and regional binding heterogeneity in the brain (high in thalamus and cerebellum). Taken together this approach has afforded a strategy and a predictive model for developing highly potent and BBB permeable HDAC inhibitors for CNS applications and for the discovery of novel candidate molecules for small molecule probes and drugs. PMID:24780082

  8. 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.

  9. 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

  10. In vitro synthesis of pre-proteins of vacuolar compartmented proteinase inhibitors that accumulate in leaves of wounded tomato plants.

    PubMed

    Nelson, C E; Ryan, C A

    1980-04-01

    Two proteinase inhibitor proteins that are compartmented in leaf vacuoles (lysosomes) were synthesized in vitro. mRNA was isolated from 17-day-old expanding tomato leaves by extraction with chaotropic buffers followed by chromatography on oligo(dT)-cellulose and was translated with a rabbit reticulocyte lysate system. Preparations of mRNA from leaves of both wounded plants and unwounded plants directed the incorporation of equivalent amounts of label into trichloroacetic acid-precipitable proteins. Only mRNA from leaves of wounded plants directed label into proteins that could be immunoprecipitated with rabbit IgG specific for either inhibitor I or inhibitor II. These results indicate that the wound-induced accumulation of proteinase inhibitors I and II in leaf vacuoles is a result of the presence of translatable mRNA species not present in leaves of unwounded plants. Gel electrophoresis of the immunoprecipitates in NaDodSO(4)/urea/polyacrylamide gels revealed that inhibitors I and II were translated in vitro as precursors about 2000 daltons larger than the inhibitors found in leaves. The presence of the additional polypeptide sequences in the newly synthesized inhibitors indicates that the inhibitors are processed either during or after synthesis, and the presequences may be signal peptides that are part of the process of inhibitor transport into the vacuolar compartments of tomato leaf cells.

  11. Halofuginone--an inhibitor of collagen type I synthesis--prevents postoperative formation of abdominal adhesions.

    PubMed Central

    Nagler, A; Rivkind, A I; Raphael, J; Levi-Schaffer, F; Genina, O; Lavelin, I; Pines, M

    1998-01-01

    OBJECTIVE: To evaluate the effects of halofuginone, a specific inhibitor of collagen type I synthesis, on the postoperative formation of abdominal adhesions in rats. SUMMARY BACKGROUND DATA: Postoperative adhesions remain the leading cause of small bowel obstruction in the Western world. Surgical trauma causes the release of a serosanguineous exudate that forms a fibrinous bridge between two organs. This becomes ingrown with fibroblasts, and subsequent collagen deposition leads to the formation of a permanent adhesion. Most of the drugs used have been clinically ineffective, and none has been specific to a particular extracellular matrix molecule. Therefore, there are serious concerns about the toxic consequences of interfering with the biosynthesis of other collagens, other matrix proteins, or vital collagen-like molecules. METHODS: Adhesions were induced by scraping the cecum until capillary bleeding occurred. The adhesions were scored 21 days later. Halofuginone was either injected intraperitoneally (1 microg/25 g body weight) every day, starting on the day of operation, or added orally at concentrations of 5 or 10 mg/kg, starting 4 days before the operation. Collagen alpha1(I) gene expression was evaluated by in situ hybridization, total collagen was estimated by Sirius red staining, and collagen type III was detected by immunohistochemistry. RESULTS: The adhesions formed between the intestinal walls were composed of collagen and were populated with cells expressing the collagen alpha1(I) gene. Regardless of the administration procedure, halofuginone significantly reduced the number and severity of the adhesions. Halofuginone prevented the increase in collagen alpha1(I) gene expression observed in the operated rats, thus reducing collagen content to the control level. In fibroblasts derived from abdominal adhesions, halofuginone induced dose-dependent inhibition of collagen alpha1(I) gene expression and collagen synthesis. Collagen type III levels were not

  12. Infusion of protein synthesis inhibitors in the entorhinal cortex blocks consolidation but not reconsolidation of object recognition memory.

    PubMed

    Lima, Ramón H; Rossato, Janine I; Furini, Cristiane R; Bevilaqua, Lia R; Izquierdo, Iván; Cammarota, Martín

    2009-05-01

    Memory consolidation and reconsolidation require the induction of protein synthesis in some areas of the brain. Here, we show that infusion of the protein synthesis inhibitors anisomycin, emetine and cycloheximide in the entorhinal cortex immediately but not 180 min or 360 min after training in an object recognition learning task hinders long-term memory retention without affecting short-term memory or behavioral performance. Inhibition of protein synthesis in the entorhinal cortex after memory reactivation involving either a combination of familiar and novel objects or two familiar objects does not affect retention. Our data suggest that protein synthesis in the entorhinal cortex is necessary early after training for consolidation of object recognition memory. However, inhibition of protein synthesis in this cortical region after memory retrieval does not seem to affect the stability of the recognition trace.

  13. Phthalazine PDE4 inhibitors. Part 3: the synthesis and in vitro evaluation of derivatives with a hydrogen bond acceptor.

    PubMed

    Napoletano, Mauro; Norcini, Gabriele; Pellacini, Franco; Marchini, Francesco; Morazzoni, Gabriele; Fattori, Raimondo; Ferlenga, Pierpaolo; Pradella, Lorenzo

    2002-01-07

    This communication describes the synthesis and in vitro evaluation of a novel and potent series of phthalazine phosphodiesterase type (IV) (PDE4) inhibitors. The interaction with two distinct polar binding sites allowed us to eliminate the cyclopentyloxy substitution from rolipram-like analogues.

  14. Phthalazine PDE4 inhibitors. Part 2: the synthesis and biological evaluation of 6-methoxy-1,4-disubstituted derivatives.

    PubMed

    Napoletano, M; Norcini, G; Pellacini, F; Marchini, F; Morazzoni, G; Ferlenga, P; Pradella, L

    2001-01-08

    This communication describes the synthesis and in vitro evaluation of a novel and potent series of phosphodiesterase type IV (PDE4) inhibitors. The compounds described present substituents in position 4 of the phthalazine ring to replace the commonly observed cyclopentyloxy moiety of rolipram analogues. Preliminary evidences of reduced side effects compared to standards and improved pharmacokinetic properties for selected derivatives are also reported.

  15. Effects of a Number of Classes of 50S Inhibitors on Stop Codon Readthrough during Protein Synthesis

    PubMed Central

    Thompson, Jill; Pratt, Catherine A.; Dahlberg, Albert E.

    2004-01-01

    The effect of a number of antibiotics on stop codon readthrough during protein synthesis in Escherichia coli was examined. Inhibitors which bind close to the entrance of the peptide exit tunnel on the 50S ribosomal subunit promote substantial levels of readthrough, presumably by disrupting the mechanism of peptide release. PMID:15561874

  16. 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.

  17. 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.

  18. 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

  19. Novel peptidomimetics as BACE-1 inhibitors: synthesis, molecular modeling, and biological studies.

    PubMed

    Butini, Stefania; Gabellieri, Emanuele; Brindisi, Margherita; Casagni, Alice; Guarino, Egeria; Huleatt, Paul B; Relitti, Nicola; La Pietra, Valeria; Marinelli, Luciana; Giustiniano, Mariateresa; Novellino, Ettore; Campiani, Giuseppe; Gemma, Sandra

    2013-01-01

    Aiming at identifying new scaffolds for BACE-1 inhibition devoid of the pharmacokinetic drawbacks of peptide-like structures, we investigated a series of novel peptidomimetics based on a 1,4-benzodiazepine (BDZ) core 1a-h and their seco-analogues 2a-d. We herein discuss synthesis, molecular modeling and in vitro studies which, starting from 1a, led to the seco-analogues (R)-2c and (S)-2d endowed with BACE-1 inhibition properties in the micromolar range both on the isolated enzyme and in cellular studies. These data can encourage to pursue these analogues as hits for the development of a new series of BACE-1 inhibitors active on whole-cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

  20. Synthesis, kinetic studies and molecular modeling of novel tacrine dimers as cholinesterase inhibitors.

    PubMed

    de Aquino, Roney Anderson Nascimento; Modolo, Luzia Valentina; Alves, Rosemeire Brondi; de Fátima, Ângelo

    2013-12-28

    This study presents the synthesis of 15 new tacrine dimers as well as the Ki and IC50 results, studies of the kinetic mechanism, and molecular docking analysis of the dimers in relation to the cholinesterases hAChE, hBChE, EeAChE and eqBChE. In addition to spectroscopic characterization, X-ray structure determination was performed for two of the new compounds. These new dimers were found to be mixed nanomolar inhibitors of the evaluated targets with a broad and significant selectivity profile, and these properties are dependent on both the type of the linker and the volume of the hydroacridine alicyclic ring. The results indicate that the aromatic linkers play a significant role in generating specific interactions with the half-gorge region of the catalytic center. Thus, these types of linkers can positively modulate the electronic properties of the tacrine dimers studied with an improvement of their cholinesterase inhibition activity.

  1. Synthesis and evaluation of fluorobenzoylated di- and tripeptides as inhibitors of cyclooxygenase-2 (COX-2).

    PubMed

    Sharma, Sai Kiran; Al-Hourani, Baker Jawabrah; Wuest, Melinda; Mane, Jonathan Y; Tuszynski, Jack; Baracos, Vickie; Suresh, Mavanur; Wuest, Frank

    2012-04-01

    A series of fluorobenzoylated di- and tripeptides as potential leads for the development of molecular probes for imaging of COX-2 expression was prepared according to standard Fmoc-based solid-phase peptide synthesis. All peptides were assessed for their COX-2 inhibitory potency and selectivity profile in a fluorescence-based COX binding assay. Within the series of 15 peptides tested, cysteine-containing peptides numbered 7, 8, 11 and 12, respectively, were the most potent COX-2 inhibitors possessing IC(50) values ranging from 5 to 85 μM. Fluorobenzoylated tripeptides 7 and 8 displayed some COX-2 selectivity (COX-2 selectivity index 2.1 and 1.6), whereas fluorobenzoylated dipeptides 11 and 12 were shown not to be COX-2 selective. Fluorbenzoylated tripeptide FB-Phe-Cys-Ser-OH was further used in molecular modeling docking studies to determine the binding mode within the active site of the COX-2 enzyme.

  2. Synthesis and structural characterisation of selective non-carbohydrate-based inhibitors of bacterial sialidases.

    PubMed

    Brear, Paul; Telford, Judith; Taylor, Garry L; Westwood, Nicholas J

    2012-11-05

    The major human pathogen Streptococcus pneumoniae plays a key role in several disease states including septicaemia, meningitis and community-acquired pneumonia. Although vaccines against S. pneumoniae are available as prophylactics, there remains a need to identify and characterise novel chemical entities that can treat the diseases caused by this pathogen. S. pneumoniae expresses three sialidases, enzymes that cleave sialic acid from carbohydrate-based surface molecules. Two of these enzymes, NanA and NanB, have been implicated in the pathogenesis of S. pneumoniae and are considered to be validated drug targets. Here we report our studies on the synthesis and structural characterisation of novel NanB-selective inhibitors that are inspired by the β-amino-sulfonic acid family of buffers.

  3. Synthesis and Characterisation of Substrate-Based Peptides as Inhibitors of Histone Demethylase KDM4C.

    PubMed

    Nielsen, Simon D; Leurs, Ulrike; Bergner, Magnus; Barris, Silvia A; Devkota, Kanchan; Meyer, Kamilla; Iaria, Daniella; McCaughan, Jack; Lohse, Brian; Kristensen, Jesper L; Clausen, Rasmus P

    2016-01-01

    The design and synthesis of modified pentapeptides based on a truncated version of the substrate for KDM4C, a histone lysine demethylase (KDM), and investigation of their inhibitory activity at KDM4C is reported. By modifying the lysine residue corresponding to lysine 9 at histone 3 (H3K9), three different series of peptides were designed and synthesized. One series contained N-acylated H3K9 and two series introduced triazoles in this position via click chemistry to enable facile variation of headgroups. The click reaction is compatible with free amino acids and this was performed on an azido containing deprotected pentapeptide demonstrating a highly facile and convergent synthetic strategy for making substrate-based inhibitors. One of the 14 peptides showed inhibitory activity at KDM4C demonstrating the need for an iron chelator in the pentapeptide series.

  4. Action of protein synthesis inhibitors in blocking electrogenic H/sup +/ efflux from corn roots

    SciTech Connect

    Chastain, C.J.; LaFayette, P.R.; Hanson, J.B.

    1981-04-01

    The block in the electrogenic H/sup +/ 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/sup +/ efflux is inhibition of K/sup +/ influx and the growth of the root tip; fusicoccin partially relieves these inhibitions, but nigericin does not. The inhibition of H/sup +/ 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.

  5. Visualization and quantification of NAD(H) in brain sections by a novel histo-enzymatic nitrotetrazolium blue staining technique

    PubMed Central

    Balan, Irina S.; Fiskum, Gary; Kristian, Tibor

    2010-01-01

    A histo-enzymatic technique for visualizing and quantifying endogenous NAD(H) in brain tissue was developed, based on coupled enzymatic cycling reactions that reduce nitrotetrazolium blue chloride to produce formazan. Conditions were used where the endogenous level of nicotinamide adenine dinucleotides (NAD(H)) was the rate limiting factor for formazan production. Spontaneous degradation of NAD+ that occurs during incubation of thawed tissue was minimized by the addition of nicotinamide mononucleotide, an inhibitor of NAD+ glycohydrolases. Cryostat sections of brains obtained from rats immediately after decapitation and at 30 min later were used to determine the effects of ischemia alone on brain NAD(H) levels and neuroanatomic distribution. The ischemic insult resulted in a greater than 50% decline in the rate of formazan generation in the CA1 pyramidal neuronal layer of the hippocampus and in the parietal cortex and striatum, but not in the CA3 and dentate gyrus (DG) subregions of the hippocampus. The ischemia-induced changes in NAD(H) levels were confirmed by utilizing spectrofluorimetric measurements of NAD(H) present in perchloric acid extracts of brain samples. This new histo-enzymatic technique is suitable for visualizing and quantifying relative NAD(H) levels in the brain. This assay could prove useful in identifying region-selective NAD(H) catabolism that may contribute to neurodegeneration. PMID:20036220

  6. Visualization and quantification of NAD(H) in brain sections by a novel histo-enzymatic nitrotetrazolium blue staining technique.

    PubMed

    Balan, Irina S; Fiskum, Gary; Kristian, Tibor

    2010-02-26

    A histo-enzymatic technique for visualizing and quantifying endogenous NAD(H) in brain tissue was developed, based on coupled enzymatic cycling reactions that reduce nitrotetrazolium blue chloride to produce formazan. Conditions were used where the endogenous level of nicotinamide adenine dinucleotides (NAD(H)) was the rate limiting factor for formazan production. Spontaneous degradation of NAD(+) that occurs during incubation of thawed tissue was minimized by the addition of nicotinamide mononucleotide, an inhibitor of NAD(+) glycohydrolases. Cryostat sections of brains obtained from rats immediately after decapitation and 30 min later were used to determine the effects of ischemia alone on brain NAD(H) levels and neuroanatomic distribution. The ischemic insult resulted in a greater than 50% decline in the rate of formazan generation in the CA1 pyramidal neuronal layer of the hippocampus and in the parietal cortex and striatum, but not in the CA3 and dentate gyrus (DG) subregions of the hippocampus. The ischemia-induced changes in NAD(H) levels were confirmed by utilizing spectrofluorimetric measurements of NAD(H) present in perchloric acid extracts of brain samples. This new histo-enzymatic technique is suitable for visualizing and quantifying relative NAD(H) levels in the brain. This assay could prove useful in identifying region-selective NAD(H) catabolism that may contribute to neurodegeneration.

  7. 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.

  8. 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.

  9. 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. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  10. Mechanism of triclosan inhibition of bacterial fatty acid synthesis.

    PubMed

    Heath, R J; Rubin, J R; Holland, D R; Zhang, E; Snow, M E; Rock, C O

    1999-04-16

    Triclosan is a broad-spectrum antibacterial agent that inhibits bacterial fatty acid synthesis at the enoyl-acyl carrier protein reductase (FabI) step. Resistance to triclosan in Escherichia coli is acquired through a missense mutation in the fabI gene that leads to the expression of FabI[G93V]. The specific activity and substrate affinities of FabI[G93V] are similar to FabI. Two different binding assays establish that triclosan dramatically increases the affinity of FabI for NAD+. In contrast, triclosan does not increase the binding of NAD+ to FabI[G93V]. The x-ray crystal structure of the FabI-NAD+-triclosan complex confirms that hydrogen bonds and hydrophobic interactions between triclosan and both the protein and the NAD+ cofactor contribute to the formation of a stable ternary complex, with the drug binding at the enoyl substrate site. These data show that the formation of a noncovalent "bi-substrate" complex accounts for the effectiveness of triclosan as a FabI inhibitor and illustrates that mutations in the FabI active site that interfere with the formation of a stable FabI-NAD+-triclosan ternary complex acquire resistance to the drug.

  11. Induction of human choriogonadotropin in HeLa-cell cultures by aliphatic monocarboxylates and inhibitors of deoxyribonucleic acid synthesis

    PubMed Central

    Ghosh, Nimai K.; Rukenstein, Adriana; Cox, Rody P.

    1977-01-01

    The ectopic production of the glycopeptide hormone human placental choriogonadotropin by HeLa65 cells was measured by radioimmunoassay with antiserum against the β-subunit of choriogonadotropin and with the 125I-labelled β-subunit as a tracer antigen. Choriogonadotropin synthesis was markedly (500-fold) stimulated by sodium butyrate. Kinetic studies and the use of an inhibitor of protein synthesis, cycloheximide, indicated that protein synthesis was required for this induction. Investigation of the efficiency of 22 aliphatic short-chain fatty acids and derivatives in causing increased choriogonadotropin synthesis by HeLa cells showed stringent structural requirements. Induction of choriogonadotropin synthesis in HeLa cells was not restricted to butyrate. Other aliphatic acids (propionate, isobutyrate, valerate and hexanoate) were also capable of inducing choriogonadotropin synthesis at 10–50% of the efficiency of butyrate. Hydroxy derivatives of monocarboxylate inducers, related mono- and di-carboxylic acids, alcohols, amines, ketones, esters and sulphoxide were ineffective in increasing choriogonadotropin production by HeLa cells. A saturated C4 straight-chain acid without substituent hydroxyl groups but with a methyl group at one end and a carboxyl moiety at the other appeared to be most efficient in activating choriogonadotropin production. A second clonal line of HeLa cells, HeLa71, showed a higher constitutive synthesis of choriogonadotropin than HeLa65 cells, which was also markedly increased by butyrate. Butyrate and other aliphatic monocarboxylate inducers of choriogonadotropin synthesis inhibited HeLa-cell growth and DNA synthesis. This inhibition of DNA replication may be related to the mechanism of choriogonadotropin synthesis, since two well-characterized anti-neoplastic inhibitors of DNA synthesis, hydroxyurea and 1-β-d-arabinofuranosylcytosine, also stimulated a 300-fold increase in choriogonadotropin synthesis in HeLa cells and were synergistic

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

    PubMed

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

    2016-01-13

    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.

  13. 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

  14. 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.

  15. Synthesis and biological evaluation of geminal disulfones as HIV-1 integrase inhibitors.

    PubMed

    Meadows, D Christopher; Mathews, Timothy B; North, Thomas W; Hadd, Michael J; Kuo, Chih Lin; Neamati, Nouri; Gervay-Hague, Jacquelyn

    2005-07-14

    Integration of HIV-1 viral DNA into the host genome is carried out by HIV-integrase (IN) and is a critical step in viral replication. Although several classes of compounds have been reported to inhibit IN in enzymatic assays, inhibition is not always correlated with antiviral activity. Moreover, potent antiviral IN inhibitors such as the chicoric acids do not act upon the intended enzymatic target but behave as entry inhibitors instead. The charged nature of the chicoric acids contributes to poor cellular uptake, and these compounds are further plagued by rapid ester hydrolysis in vivo. To address these critical deficiencies, we designed neutral, nonhydrolyzable analogues of the chicoric acids. Herein, we report the synthesis, enzyme inhibition studies, and cellular antiviral data for a series of geminal disulfones. Of the 10 compounds evaluated, 8 showed moderate to high inhibition of IN in purified enzyme assays. The purified enzyme data correlated with antiviral assays for all but two compounds, suggesting alternative modes of inhibition. Time-of-addition studies were performed on these analogues, and the results indicate that they inhibit an early stage in the replication process, perhaps entry. In contrast, the most potent member of the correlative group shows behavior consistent with IN being the cellular target.

  16. Synthesis and evaluation of inhibitors of bacterial drug efflux pumps of the major facilitator superfamily.

    PubMed

    Okandeji, Babajide O; Greenwald, Daniel M; Wroten, Jessica; Sello, Jason K

    2011-12-15

    Inhibitors of drug efflux pumps have great potential as pharmacological agents that restore the drug susceptibility of multidrug resistant bacterial pathogens. Most attention has been focused on the discovery of small molecules that inhibit the resistance nodulation division (RND) family drug efflux pumps in Gram-negative bacteria. The prototypical inhibitor of RND-family efflux pumps in Gram-negative bacteria is MC-207,110 (Phe-Arg-β-naphthylamide), a C-capped dipeptide. Here, we report that C-capped dipeptides inhibit two chloramphenicol-specific efflux pumps in Streptomyces coelicolor, a Gram-positive bacterium that is a relative of the human pathogen Mycobacterium tuberculosis. Diversity-oriented synthesis of a library of structurally related C-capped dipeptides via an Ugi four component reaction and screening of the resulting compounds resulted in the discovery of a compound that is threefold more potent as a suppressor of chloramphenicol resistance in S. coelicolor than MC-207,110. Since chloramphenicol resistance in S. coelicolor is mediated by major facilitator superfamily drug efflux pumps, our findings provide the first evidence that C-capped dipeptides can inhibit drug efflux pumps outside of the RND superfamily.

  17. Synthesis and biological evaluation of di-aryl urea derivatives as c-Kit inhibitors.

    PubMed

    Ravez, Séverine; Arsenlis, Stéphane; Barczyk, Amélie; Dupont, Anthony; Frédérick, Raphaël; Hesse, Stéphanie; Kirsch, Gilbert; Depreux, Patrick; Goossens, Laurence

    2015-11-15

    Inhibition of receptor tyrosine kinases (RTKs) continued to be a successful approach for the treatment of many types of human cancers and many potent small molecules kinase inhibitors have been discovered the last decade. In the present study, we describe the synthesis of thienopyrimidine derivatives and their pharmacological evaluation against nine kinases (EGFR, PDGFR-ß, c-Kit, c-Met, Src, Raf, VEGFR-1, -2 and -3). Most of the synthesized compounds showed from moderate to potent activities against c-Kit with IC50 values in the nanomolar range. Among them, 4-anilino(urea)thienopyrimidine analogs showed selectivity and potent c-Kit inhibition with IC50 values less than 6 nM. Docking simulation was performed for the most promising compound 9 into the c-Kit active site to determine the potential binding mode. This study reveal that the 4-anilino(urea)thienopyrimidine is an interesting scaffold to design novel potent and selective c-Kit inhibitors which may make promising candidates for cancers where c-Kit receptors are overexpressed.

  18. Review of synthesis, assay, and prediction of β and γ-secretase inhibitors.

    PubMed

    Niño, Helena; Rodríguez-Borges, José Enrique; García-Mera, Xerardo; Prado-Prado, Francisco

    2012-01-01

    Alzheimer's disease (AD) is characterize with several pathologies this disease, amyloid plaques, composed of the β-amyloid peptide and β-amyloid peptide are hallmark neuropathological lesions in Alzheimer's disease brain. Indeed, a wealth of evidence suggests that β-amyloid is central to the pathophysiology of AD and is likely to play an early role in this intractable neurodegenerative disorder. AD is the most prevalent form of dementia, and current indications show that twenty-nine million people live with AD worldwide, a figure expected rise exponentially over the coming decades. Clearly, blocking disease progression or, in the best-case scenario, preventing AD altogether would be of benefit in both social and economic terms. However, current AD therapies are merely palliative and only temporarily slow cognitive decline, and treatments that address the underlying pathologic mechanisms of AD are completely lacking. While familial AD (FAD) is caused by autosomal dominant mutations in either amyloid precursor protein (APP) or the presenilin (PS1, PS2) genes. First, we revised Desing, synthesis, and Biological assay of β and γ-secretase inhibitors. Next, we review 2D QSAR, 3D QSAR, CoMFA, CoMSIA and Docking with different compound to find out the structural requirements. Next, we revised QSAR studies using method of Artificial Neural Network (ANN) in order to understand the essential structural requirement for binding with receptor for β and γ-secretase inhibitors.

  19. Microwave-assisted synthesis of novel purine nucleosides as selective cholinesterase inhibitors.

    PubMed

    Schwarz, S; Csuk, R; Rauter, A P

    2014-04-21

    Alzheimer's disease (AD), the most common form of senile dementia, is characterized by high butyrylcholinesterase (BChE) levels in the brain in later AD stages, for which no treatment is available. Pursuing our studies on selective BChE inhibitors, that may contribute to understand the role of this enzyme in disease progression, we present now microwave-assisted synthesis and anticholinesterase activity of a new nucleoside series embodying 6-chloropurine or 2-acetamido-6-chloropurine linked to D-glucosyl, D-galactosyl and D-mannosyl residues. It was designed to assess the contribution of sugar stereochemistry, purine structure and linkage to the sugar for cholinesterase inhibition efficiency and selectivity. Compounds were subjected to Ellman's assay and their inhibition constants determined. The α-anomers were the most active compounds, while selectivity for BChE or acetylcholinesterase (AChE) inhibition could be tuned by the purine base, by the glycosyl moiety and by N(7)-ligation. Some of the nucleosides were far more potent than the drug galantamine, and the most promising competitive and selective BChE inhibitor, the N(7)-linked 2-acetamido-α-D-mannosylpurine, showed a Ki of 50 nM and a selectivity factor of 340 fold for BChE over AChE.

  20. 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.

  1. Design, synthesis, and structure-activity relationship studies of a potent PACE4 inhibitor.

    PubMed

    Kwiatkowska, Anna; Couture, Frédéric; Levesque, Christine; Ly, Kévin; Desjardins, Roxane; Beauchemin, Sophie; Prahl, Adam; Lammek, Bernard; Neugebauer, Witold; Dory, Yves L; Day, Robert

    2014-01-09

    PACE4 plays an important role in the progression of prostate cancer and is an attractive target for the development of novel inhibitor-based tumor therapies. We previously reported the design and synthesis of a novel, potent, and relatively selective PACE4 inhibitor known as a Multi-Leu (ML) peptide. In the present work, we examined the ML peptide through detailed structure-activity relationship studies. A variety of ML-peptide analogues modified at the P8-P5 positions with leucine isomers (Nle, DLeu, and DNle) or substituted at the P1 position with arginine mimetics were tested for their inhibitory activity, specificity, stability, and antiproliferative effect. By incorporating d isomers at the P8 position or a decarboxylated arginine mimetic, we obtained analogues with an improved stability profile and excellent antiproliferative properties. The DLeu or DNle residue also has improved specificity toward PACE4, whereas specificity was reduced for a peptide modified with the arginine mimetic, such as 4-amidinobenzylamide.

  2. Synthesis of novel polybrominated benzimidazole derivatives-potential CK2 inhibitors with anticancer and proapoptotic activity.

    PubMed

    Łukowska-Chojnacka, Edyta; Wińska, Patrycja; Wielechowska, Monika; Poprzeczko, Martyna; Bretner, Maria

    2016-02-15

    The efficient method for the synthesis of novel cell permeable inhibitors of protein kinase CK2 with anticancer and proapoptotic activity has been developed. A series of polybrominated benzimiadazole derivatives substituted by various cyanoalkyl groups have been synthesized. Cyanoethyl derivatives were obtained by Michael type addition of 4,5,6,7-tetrabromo-1H-benzimidazole (TBBi) and 4,5,6,7-tetrabromo-2-methyl-1H-benzimidazole to acrylonitrile, whilst cyanomethyl, cyanopropyl and cyanobutyl analogs by N-alkylation of 4,5,6,7-tetrabromo-1H-benzimidazole and 4,5,6,7-tetrabromo-2-methyl-1H-benzimidazole with appropriate cyanoalkyl halides. The inhibitory activity against protein kinase rhCK2α catalytic subunit and cytotoxicity against two human cancer cell lines: acute lymphocytic leukemia (CCRF-CEM) and breast (MCF-7) were evaluated for all newly synthesized compounds. Additionally, the proapoptotic activity toward leukemia cells and intracellular inhibition of CK2 for the most cytotoxic derivatives have been performed, demonstrating 4,5,6,7-tetrabromo-2-methyl-1H-benzimidazole as a new selective inhibitor of rhCK2 with twenty-fold better proapoptotic activity than parental compound (TBBi).

  3. Design, synthesis, in vitro Evaluation and docking studies on dihydropyrimidine-based urease inhibitors.

    PubMed

    Iftikhar, Fatima; Ali, Yousaf; Ahmad Kiani, Farooq; Fahad Hassan, Syed; Fatima, Tabeer; Khan, Ajmal; Niaz, Basit; Hassan, Abbas; Latif Ansari, Farzana; Rashid, Umer

    2017-10-01

    In our previous report, we have identified 3,4-dihydropyrimidine scaffold as promising class of urease inhibitor in a structure based virtual screen (SBVS) experiment. In present study, we attempted to optimize the scaffold by varying C-5 substituent. The elongation of the C-5 chain was achieved by the reaction of C-5 ester with hydrazine leading to C-5 carbohydrazides which were further used as building blocks for the synthesis of fifteen new compounds having diverse moieties. A significantly higher in vitro urease inhibitory activity with IC50 values in submicromolar range was observed for semithiocarbazide derivatives (4a-c, 0.58-0.79µM) and isatin Schiff base derivative 5a (0.23µM). Docking analysis suggests that the synthesized compounds were anchored well in the catalytic site and extending to the entrance of binding pocket and thus restrict the mobility of the flap by interacting with its key amino acid residues. The overall results of urease inhibition have shown that these compounds can be further optimized and developed as lead urease inhibitors. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Rational design of nitrofuran derivatives: Synthesis and valuation as inhibitors of Trypanosoma cruzi trypanothione reductase.

    PubMed

    Arias, D G; Herrera, F E; Garay, A S; Rodrigues, D; Forastieri, P S; Luna, L E; Bürgi, M D L M; Prieto, C; Iglesias, A A; Cravero, R M; Guerrero, S A

    2017-01-05

    The rational design and synthesis of a series of 5-nitro-2-furoic acid analogues are presented. The trypanocidal activity against epimastigote forms of Trypanosoma cruzi and the toxic effects on human HeLa cells were tested. Between all synthetic compounds, three of thirteen had an IC50 value in the range of Nfx, but compound 13 exhibited an improved effect with an IC50 of 1.0 ± 0.1 μM and a selective index of 70 in its toxicity against HeLa cells. We analyzed the activity of compounds 8, 12 and 13 to interfere in the central redox metabolic pathway in trypanosomatids, which is dependent of reduced trypanothione as the major pivotal thiol. The three compounds behaved as better inhibitors of trypanothione reductase than Nfx (Ki values of 118 μM, 61 μM and 68 μM for 8, 12 and 13, respectively, compared with 245 μM for Nfx), all following an uncompetitive enzyme inhibition pattern. Docking analysis predicted a binding of inhibitors to the enzyme-substrate complex with binding energy calculated in-silico that supports such molecular interaction.

  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.

  6. 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.

  7. Facile dimethyl amino group triggered cyclic sulfonamides synthesis and evaluation as alkaline phosphatase inhibitors.

    PubMed

    Bhatti, Huma Aslam; Khatoon, Memoona; Al-Rashida, Mariya; Bano, Huma; Iqbal, Nafees; Zaib-Un-Nisa; Yousuf, Sammer; Khan, Khalid Mohammed; Hameed, Abdul; Iqbal, Jamshed

    2017-04-01

    Owing to the biological importance of cyclic sulfonamides (sultams), herein we report a new, facile and cost-effective method for the synthesis of sultams that makes use of a reaction between dansyl amide and easily accessible benzaldehydes under mildly acidic conditions. All compounds were obtained in good yields (69-96%). Consequently a series of cyclic sulfonamides (7a-7n) was synthesized and characterized using FTIR, MS and NMR spectroscopy, crystal structure of compound 7b has also been determined. All compounds were evaluated for their potential to inhibit alkaline phosphatase (bTNAP and bIAP). All compounds were found to be excellent inhibitors of bTNAP with IC50 values in lower micro-molar range (0.11-6.63μM). Most of the compounds were selective inhibitors of bTNAP over bIAP. Only six compounds were found to be active against bIAP (IC50 values in the range 0.38-3.48μM). Molecular docking studies were carried out to identify and rationalize the structural elements necessary for efficient AP inhibition. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. The use of ascorbate as an oxidation inhibitor in prebiotic amino acid synthesis: a cautionary note.

    PubMed

    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 CO(2)-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 CO(2) 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 CO(2)-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 CO(2)-rich atmosphere under the conditions studied.

  9. Design, Synthesis and Evaluation of Marinopyrrole Derivatives as Selective Inhibitors of Mcl-1 Binding to Pro-apoptotic Bim and Dual Mcl-1/Bcl-xL Inhibitors

    PubMed Central

    Li, Rongshi; Daniel, Kenyon G.; Li, Jerry; Qin, Yong; Gavathiotis, Evripidis; Sebti, Said M.

    2015-01-01

    Inhibition of anti-apoptotic Mcl-1 is a promising anticancer strategy to overcome the survival and chemoresistance of a broad spectrum of human cancers. We previously reported on the identification of a natural product marinopyrrole A (1) that induces apoptosis in Mcl-1-dependent cells through Mcl-1 degradation. Here, we report the design and synthesis of novel marinopyrrole-based analogues and their evaluation as selective inhibitors of Mcl-1 as well as dual Mcl-1/Bcl-xL inhibitors. The most selective Mcl-1 antagonists were 34, 36 and 37 with 16-, 13- and 9-fold more selectivity for disrupting Mcl-1/Bim over Bcl-xL/Bim binding, respectively. Among the most potent dual inhibitors is 42 which inhibited Mcl-1/Bim and Bcl-xL/Bim binding 15-fold (IC50 = 600 nM) and 33-fold (500 nM) more potently than (±)-marinopyrrole A (1), respectively. Fluorescence quenching, NMR analysis and molecular docking indicated binding of marinopyrroles to the BH3 binding site of Mcl-1. Several marinopyrroles potently decreased Mcl-1 cellular levels and induced caspase 3 activation in human breast cancer cells. Our studies provide novel “lead” marinopyrroles for further optimization as selective Mcl-1 inhibitors and dual Mcl-1 and Bcl-xL inhibitors. PMID:25437618

  10. First-In-Class Inhibitor of Ribosomal RNA Synthesis with Antimicrobial Activity against Staphylococcus aureus.

    PubMed

    Yang, Xiao; Luo, Ming Jing; Yeung, Apple C M; Lewis, Peter J; Chan, Paul K S; Ip, Margaret; Ma, Cong

    2017-09-26

    We report the discovery of the first bacterial ribosomal RNA (rRNA) synthesis inhibitor that has specific antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA). A pharmacophore model was constructed on the basis of the protein-protein interaction between essential bacterial rRNA transcription factors NusB and NusE and employed for an in silico screen to identify potential leads. One compound, (E)-2-{[(3-ethynylphenyl)imino]methyl}-4-nitrophenol (MC4), demonstrated antimicrobial activity against a panel of S. aureus strains, including MRSA, without significant toxicity to mammalian cells. MC4 resulted in a decrease in the rRNA level in bacteria, and the target specificity of MC4 was confirmed at the molecular level. Results obtained from this work validated the bacterial rRNA transcription machinery as a novel antimicrobial target. This approach may be extended to other factors in rRNA transcription, and MC4 could be applied as a chemical probe to dissect the relationship among MRSA infection, MRSA growth rate, and rRNA synthesis, in addition to its therapeutic potential.

  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. Synthesis and NMR structure of p41icf, a potent inhibitor of human cathepsin L.

    PubMed

    Chiva, Cristina; Barthe, Philippe; Codina, Anna; Gairí, Margarida; Molina, Franck; Granier, Claude; Pugnière, Martine; Inui, Tatsuya; Nishio, Hideki; Nishiuchi, Yuji; Kimura, Terutoshi; Sakakibara, Shumpei; Albericio, Fernando; Giralt, Ernest

    2003-02-12

    The total synthesis and structural characterization of the MHCII-associated p41 invariant chain fragment (P41icf) is described. P41icf plays a crucial role in the maturation of MHC class II molecules and antigen processing, acting as a highly selective cathepsin L inhibitor. P41icf synthesis was achieved using a combined solid-phase/solution approach. The entire molecule (65 residues, 7246 Da unprotected) was assembled in solution from fully protected peptides in the size range of 10 residues. After deprotection, oxidative folding in carefully adjusted experimental conditions led to the completely folded and functional P41icf with a disulfide pairing identical to that of native P41icf. CD, NMR, and surface plasmon resonance (SPR) were used for the structural and functional characterization of synthetic P41icf. CD thermal denaturation showed clear cooperative behavior. Tight cathepsin L binding was demonstrated by SPR. (1)H NMR spectroscopy at 800 MHz of unlabeled P41icf was used to solve the three-dimensional structure of the molecule. P41icf behaves as a well-folded protein domain with a topology very close to the crystallographic cathepsin L-bound form.

  13. Design and synthesis of novel distamycin-modified nucleoside analogues as HIV-1 reverse transcriptase inhibitors.

    PubMed

    Li, Chao; Ma, Chunying; Zhang, Jin; Qian, Ning; Ding, Jingjing; Qiao, Renzhong; Zhao, Yufen

    2014-02-01

    Design and synthesis of nucleoside analogues have persistently attracted extensive interest because of their potential application in the field of antiviral therapy, and its study also receives additional impetus for improvement in the ProTide technology. Previous studies have made great strides in the design and discovery of monophosphorylated nucleoside analogues as potential kinase-independent antiretrovirals. In this work, a series of nucleoside phosphoramidates modified by distamycin analogues was synthesized and evaluated as nucleoside reverse transcriptase inhibitors (NRTIs) in HIV-1-infected MT-4 and CEM cells, including variations in nucleoside, alkyl moiety, and the structure of distamycin analogues. These compounds exhibited modest potency with the EC50 value in the range of 1.3- to 6.5-fold lower than their corresponding parent drugs in MT-4 cells, which may be attributed to increasing intracellular availability due to the existence of distamycin analogue with favorable hydrophilic-lipophilic equilibrium. Meanwhile, the length of distamycin analogue was considered and assessed as an important factor that could affect antiviral activity and cytotoxicity. Enzymatic and metabolic stability studies have been performed in order to better understand the antiviral behavior of these compounds. The present work revealed the compounds to have a favorable and selective anti-HIV-1 activity in MT-4 and CEM cells, and helped to develop strategies for design and synthesis of effective monophosphorylated nucleoside analogues, which may be applied to antiretroviral research as NRTIs. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. Metabolic basis for the differential susceptibility of Gram-positive pathogens to fatty acid synthesis inhibitors

    PubMed Central

    Parsons, Joshua B.; Frank, Matthew W.; Subramanian, Chitra; Saenkham, Panatda; Rock, Charles O.

    2011-01-01

    The rationale for the pursuit of bacterial type 2 fatty acid synthesis (FASII) as a target for antibacterial drug discovery in Gram-positive organisms is being debated vigorously based on their ability to incorporate extracellular fatty acids. The regulation of FASII by extracellular fatty acids was examined in Staphylococcus aureus and Streptococcus pneumoniae, representing two important groups of pathogens. Both bacteria use the same enzymatic tool kit for the conversion of extracellular fatty acids to acyl-acyl carrier protein, elongation, and incorporation into phospholipids. Exogenous fatty acids completely replace the endogenous fatty acids in S. pneumoniae but support only 50% of phospholipid synthesis in S. aureus. Fatty acids overcame FASII inhibition in S. pneumoniae but not in S. aureus. Extracellular fatty acids strongly suppress malonyl-CoA levels in S. pneumoniae but not in S. aureus, showing a feedback regulatory system in S. pneumoniae that is absent in S. aureus. Fatty acids overcame either a biochemical or a genetic block at acetyl-CoA carboxylase (ACC) in S. aureus, confirming that regulation at the ACC step is the key difference between these two species. Bacteria that possess a stringent biochemical feedback inhibition of ACC and malonyl-CoA formation triggered by environmental fatty acids are able to circumvent FASII inhibition. However, if exogenous fatty acids do not suppress malonyl-CoA formation, FASII inhibitors remain effective in the presence of fatty acid supplements. PMID:21876172

  15. Relaxin secretion by porcine large luteal cells: effect of protein synthesis inhibitors.

    PubMed

    Taylor, M J; Clark, C L

    1993-02-01

    The purpose of the experiments reported herein was to investigate the relative importance of new hormone synthesis to basal and prostaglandin E2-stimulated rates of relaxin release. A relaxin-reverse hemolytic plaque assay was used to monitor relaxin release from individual large luteal cells (LLC) in which new protein synthesis was inhibited by cycloheximide or actinomycin D. These treatments significantly decreased the rate of relaxin release. In addition, cycloheximide reduced the total fraction of LLC possessing the ability to form plaques by about 10%, suggesting complete suppression of relaxin from this subset of cells. Exposure of inhibitor-treated LLC to prostaglandin E2 (a relaxin stimulatory secretagogue) enhanced relaxin release, and restored suppressed LLC back into the secretory population. Taken overall, these results demonstrate that the majority of relaxin-releasing LLC exploit a mixture of newly synthesized and older, stored hormone to achieve basal secretion. A minority of relaxin-releasing LLC, however, appear to depend wholly on newly synthesized hormone for basal secretion. The differential activity (and interaction) of these pathways in individual LLC may provide a potential explanation for the markedly heterogenous manner of hormone release observed in this (and other) cell types, and for the action of relaxin secretagogues.

  16. 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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. A bionanohybrid ZnAl-NADS ecological pesticide as a treatment for soft rot disease in potato (Solanum tuberosum L.).

    PubMed

    Morales-Irigoyen, Erika Elizabeth; de Las Mercedes Gómez-Y-Gómez, Yolanda; Flores-Moreno, Jorge Luis; Franco-Hernández, Marina Olivia

    2017-09-18

    Pectobacterium carotovorum (Pc) is a phytopathogenic strain that causes soft rot disease in potato (Solanum tuberosum L.), resulting in postharvest losses. Chemical control is effective for managing this disease, but overdoses cause adverse effects. Because farmers insist on using chemical agents for crop protection, it is necessary to develop more effective pesticides in which the active compound released can be regulated. In this context, we proposed the synthesis of ZnAl-NADS, in which nalidixic acid sodium salt (NADS) is linked to a ZnAl-NO3 layered double hydroxide (LDH) host as a nanocarrier. XRD, FT-IR, and SEM analyses confirmed the successful intercalation of NADS into the interplanar LDH space. The drug release profile indicated that the maximum release was completed in 70 or 170 min for free NADS (alone) or for NADS released from ZnAl-NADS, respectively. This slow release was attributed to strong electrostatic interactions between the drug and the anion exchanger. A modulated release is preferable to the action of the bulk NADS, showing increased effectiveness and minimizing the amount of the chemical available to pollute the soil and the water. The fitting data from modified Freundlich and parabolic diffusion models explain the release behavior of the NADS, suggesting that the drug released from ZnAl-NADS bionanohybrid was carried out from the interlamellar sites, according to the ion exchange diffusion process also involving intraparticle diffusion (coeffect). ZnAl-NADS was tested in vitro against Escherichia coli (Ec) and Pc and exhibited bacteriostatic and biocidal effects at 0.025 and 0.075 mg mL(-1), respectively. ZnAl-NADS was also tested in vivo as an ecological pesticide for combating potato soft rot and was found to delay typical disease symptoms. In conclusion, ZnAl-NADS can potentially be used to control pests, infestation, and plant disease.

  18. NAD(+), sirtuins, and cardiovascular disease.

    PubMed

    Borradaile, Nica M; Pickering, J Geoffrey

    2009-01-01

    Cardiovascular disease (CVD) is the most prevalent disease worldwide and there is intense interest in pharmaceutical approaches to reduce the burden of this chronic, aging-related condition. The sirtuin (SIRT) family of NAD(+)-dependent protein deacetylases and ADP-ribosyltransferases have emerged as exciting targets for CVD management that can impact the cardiovascular system both directly and indirectly, the latter by modulating whole body metabolism. SIRT1-4 regulate the activities of a variety of transcription factors, coregulators, and enzymes that improve metabolic control in adipose tissue, liver, skeletal muscle, and pancreas, particularly during obesity and aging. SIRT1 and 7 can control myocardial development and resist stress- and aging-associated myocardial dysfunction through the deacetylation of p53 and forkhead box O1 (FoxO1). By modulating the activity of endothelial nitric oxide synthase (eNOS), FoxO1, and p53, and the expression of angiotensin II type 1 receptor (AT1R), SIRT1 also promotes vasodilatory and regenerative functions in endothelial and smooth muscle cells of the vascular wall. Given the array of potentially beneficial effects of SIRT activation on cardiovascular health, interest in developing specific SIRT agonists is well-substantiated. Because SIRT activity depends on cellular NAD+ availability, enzymes involved in NAD+ biosynthesis, including nicotinamide phosphoribosyltransferase (Nampt), may also be valuable pharmaceutical targets for managing CVD. Herein we review the actions of the SIRT proteins on the cardiovascular system and consider the potential of modulating SIRT activity and NAD+ availability to control CVD.

  19. Recent advances in inhibitors of bacterial fatty acid synthesis type II (FASII) system enzymes as potential antibacterial agents.

    PubMed

    Wang, Yi; Ma, Shutao

    2013-10-01

    Bacterial infections are a constant and serious threat to human health. With the increase of multidrug resistance of clinically pathogenic bacteria, common antibiotic therapies have been less effective. Fatty acid synthesis type II (FASII) system enzymes are essential for bacterial membrane lipid biosynthesis and represent increasingly promising targets for the discovery of antibacterial agents with new mechanisms of action. This review highlights recent advances in inhibitors of bacterial FASII as potential antibacterial agents, paying special attention to the activities, mechanisms, and structure-activity relationships of those inhibitors that mainly target β-ketoacyl-ACP synthase, β-ketoacyl-ACP reductase, β-hydroxyacyl-ACP dehydratase, and enoyl-ACP reductase. Although inhibitors with low nanomolar and selective activity against various bacterial FASII have entered clinical trials, further research is needed to expand upon both available and yet unknown scaffolds to identify new FASII inhibitors that may have antibacterial potential, particularly against resistant bacterial strains.

  20. 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.

  1. 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.

  2. Overexpression of NAD kinase in recombinant Escherichia coli harboring the phbCAB operon improves poly(3-hydroxybutyrate) production.

    PubMed

    Li, Zheng-Jun; Cai, Lei; Wu, Qiong; Chen, Guo-Qiang

    2009-07-01

    NAD kinase was overexpressed to enhance the accumulation of poly(3-hydroxybutyrate) (PHB) in recombinant Escherichia coli harboring PHB synthesis pathway via an accelerated supply of NADPH, which is one of the most crucial factors influencing PHB production. A high copy number expression plasmid pE76 led to a stronger NAD kinase activity than that brought about by the low copy number plasmid pELRY. Overexpressing NAD kinase in recombinant E. coli was found not to have a negative effect on cell growth in the absence of PHB synthesis. Shake flask experiments demonstrated that excess NAD kinase in E. coli harboring the PHB synthesis operon could increase the accumulation of PHB to 16-35 wt.% compared with the controls; meanwhile, NADP concentration was enhanced threefold to sixfold. Although the two NAD kinase overexpression recombinants exhibited large disparity on NAD kinase activity, their influence on cell growth and PHB accumulation was not proportional. Under the same growth conditions without process optimization, the NAD kinase-overexpressing recombinant produced 14 g/L PHB compared with 7 g/L produced by the control in a 28-h fermentor study. In addition, substrate to PHB yield Y (PHB/glucose) showed an increase from 0.08 g PHB/g glucose for the control to 0.15 g PHB/g glucose for the NAD kinase-overexpressing strain, a 76% increase for the Y (PHB/glucose). These results clearly showed that the overexpression of NAD kinase could be used to enhance the PHB synthesis.

  3. Discovery of Novel Oral Protein Synthesis Inhibitors of Mycobacterium tuberculosis That Target Leucyl-tRNA Synthetase

    PubMed Central

    Palencia, Andrés; Li, Xianfeng; Bu, Wei; Choi, Wai; Ding, Charles Z.; Easom, Eric E.; Feng, Lisa; Hernandez, Vincent; Houston, Paul; Liu, Liang; Meewan, Maliwan; Mohan, Manisha; Rock, Fernando L.; Sexton, Holly; Zhang, Suoming; Zhou, Yasheen; Wan, Baojie; Wang, Yuehong; Franzblau, Scott G.; Woolhiser, Lisa; Gruppo, Veronica; Lenaerts, Anne J.; O'Malley, Theresa; Parish, Tanya; Cooper, Christopher B.; Waters, M. Gerard; Ma, Zhenkun; Ioerger, Thomas R.; Sacchettini, James C.; Rullas, Joaquín; Angulo-Barturen, Iñigo; Pérez-Herrán, Esther; Mendoza, Alfonso; Barros, David; Cusack, Stephen; Plattner, Jacob J.

    2016-01-01

    The recent development and spread of extensively drug-resistant and totally drug-resistant resistant (TDR) strains of Mycobacterium tuberculosis highlight the need for new antitubercular drugs. Protein synthesis inhibitors have played an important role in the treatment of tuberculosis (TB) starting with the inclusion of streptomycin in the first combination therapies. Although parenteral aminoglycosides are a key component of therapy for multidrug-resistant TB, the oxazolidinone linezolid is the only orally available protein synthesis inhibitor that is effective against TB. Here, we show that small-molecule inhibitors of aminoacyl-tRNA synthetases (AARSs), which are known to be excellent antibacterial protein synthesis targets, are orally bioavailable and effective against M. tuberculosis in TB mouse infection models. We applied the oxaborole tRNA-trapping (OBORT) mechanism, which was first developed to target fungal cytoplasmic leucyl-tRNA synthetase (LeuRS), to M. tuberculosis LeuRS. X-ray crystallography was used to guide the design of LeuRS inhibitors that have good biochemical potency and excellent whole-cell activity against M. tuberculosis. Importantly, their good oral bioavailability translates into in vivo efficacy in both the acute and chronic mouse models of TB with potency comparable to that of the frontline drug isoniazid. PMID:27503647

  4. Effects of protein synthesis inhibitors during reactivation of associative memory in the common snail induces reversible and irreversible amnesia.

    PubMed

    Solntseva, S V; Nikitin, V P; Kozyrev, S A; Shevelkin, A V; Lagutin, A V; Sherstnev, V V

    2007-11-01

    The effects of protein synthesis inhibitors on the reactivation of an associative skill consisting of refusing a particular food by common snails were studied. Animals were given single injections of a protein synthesis inhibitor (cycloheximide at 0.6 mg/snail or anisomycin at 0.4 mg) 24 h after three days of training, and were then presented with a "reminding" stimulus (the "conditioned reflex" food-banana) and tested for retention of the skill. Observations revealed an impairment of reproduction of the acquired skill 2.5 h after the "reminder," with spontaneous restoration at 4.5-5.5 h. Other snails were given single 1.8-mg doses of cycloheximide or three 0.6-mg doses with intervals of 2 h. "Reminders" were presented after each injection. In these conditions, impairment of reproduction of the conditioned reflex also appeared 2.5 h after the first "reminder," though amnesia lasted at least 30 days and repeat training of the animals produced only partial recovery of the skill. Thus, we have provided the first demonstration that recovery of a long-term memory "trace" on exposure to relatively low doses of protein synthesis inhibitors produces transient and short-lived amnesia, lasting 2-3 h, while long-term, irreversible amnesia occurs after longer-lasting or more profound suppression of protein synthesis. These results suggest that the "reminding" process induces reconsolidation of the " initial" memory, suppression of which by protein synthesis inhibitors leads to "erasure" of the memory "trace" and impairs consolidation on repeat training.

  5. Design, synthesis, functional and structural characterization of an inhibitor of N-acetylneuraminate-9-phosphate phosphatase: observation of extensive dynamics in an enzyme/inhibitor complex.

    PubMed

    Kim, Soong-Hoon; Constantine, Keith L; Duke, Gerald J; Goldfarb, Valentina; Hunt, John T; Johnson, Stephen; Kish, Kevin; Klei, Herbert E; McDonnell, Patricia A; Metzler, William J; Mueller, Luciano; Poss, Michael A; Fairchild, Craig R; Bhide, Rajeev S

    2013-07-15

    The design, synthesis and characterization of a phosphonate inhibitor of N-acetylneuraminate-9-phosphate phosphatase (HDHD4) is described. Compound 3, where the substrate C-9 oxygen was replaced with a nonlabile CH2 group, inhibits HDHD4 with a binding affinity (IC50 11μM) in the range of the native substrate Neu5Ac-9-P (compound 1, Km 47μM). Combined SAR, modeling and NMR studies are consistent with the phosphonate group in inhibitor 3 forming a stable complex with native Mg(2+). In addition to this key interaction, the C-1 carboxylate of the sugar interacts with a cluster of basic residues, K141, R104 and R72. Comparative NMR studies of compounds 3 and 1 with Ca(2+) and Mg(2+) are indicative of a highly dynamic process in the active site for the HDHD4/Mg(2+)/3 complex. Possible explanations for this observation are discussed.

  6. 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.

  7. Structure-Based Design, Synthesis, and Biological Evaluation of Dihydroquinazoline-Derived Potent β-Secretase Inhibitors

    PubMed Central

    Ghosh, Arun K.; Pandey, Satyendra; Gangarajula, Sudhakar; Kulkarni, Sarang; Xu, Xiaoming; Rao, Kalapala Venkateswara; Huang, Xiangping; Tang, Jordan

    2012-01-01

    Structure-based design, synthesis, and biological evaluation of a series of dihydroquinazoline-derived β-secretase inhibitors incorporating thiazole and pyrazole-derived P2-ligands are described. We have identified inhibitor 4f which has shown potent enzyme inhibitory (Ki = 13 nM) and cellular (IC50 = 21 nM in neuroblastoma cells) assays. A model of 4f was created based upon the X-ray structure of 3a-bound β-Secretase. The model revealed critical interactions in the active site. PMID:22863204

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

    PubMed Central

    2016-01-01

    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

  9. 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.

  10. Synthesis, Pharmacological Profile and Docking Studies of New Sulfonamides Designed as Phosphodiesterase-4 Inhibitors

    PubMed Central

    Cardozo, Suzana Vanessa S.; Carvalho, Vinicius de Frias; Romeiro, Nelilma Correia; Silva, Patrícia Machado Rodrigues e; Martins, Marco Aurélio; Barreiro, Eliezer J.; Lima, Lídia Moreira

    2016-01-01

    Prior investigations showed that increased levels of cyclic AMP down-regulate lung inflammatory changes, stimulating the interest in phosphodiesterase (PDE)4 as therapeutic target. Here, we described the synthesis, pharmacological profile and docking properties of a novel sulfonamide series (5 and 6a-k) designed as PDE4 inhibitors. Compounds were screened for their selectivity against the four isoforms of human PDE4 using an IMAP fluorescence polarized protocol. The effect on allergen- or LPS-induced lung inflammation and airway hyper-reactivity (AHR) was studied in A/J mice, while the xylazine/ketamine-induced anesthesia test was employed as a behavioral correlate of emesis in rodents. As compared to rolipram, the most promising screened compound, 6a (LASSBio-448) presented a better inhibitory index concerning PDE4D/PDE4A or PDE4D/PDE4B. Accordingly, docking analyses of the putative interactions of LASSBio-448 revealed similar poses in the active site of PDE4A and PDE4C, but slight unlike orientations in PDE4B and PDE4D. LASSBio-448 (100 mg/kg, oral), 1 h before provocation, inhibited allergen-induced eosinophil accumulation in BAL fluid and lung tissue samples. Under an interventional approach, LASSBio-448 reversed ongoing lung eosinophilic infiltration, mucus exacerbation, peribronchiolar fibrosis and AHR by allergen provocation, in a mechanism clearly associated with blockade of pro-inflammatory mediators such as IL-4, IL-5, IL-13 and eotaxin-2. LASSBio-448 (2.5 and 10 mg/kg) also prevented inflammation and AHR induced by LPS. Finally, the sulfonamide derivative was shown to be less pro-emetic than rolipram and cilomilast in the assay employed. These findings suggest that LASSBio-448 is a new PDE4 inhibitor with marked potential to prevent and reverse pivotal pathological features of diseases characterized by lung inflammation, such as asthma. PMID:27695125

  11. Synthesis of 2-arylindole derivatives and evaluation as nitric oxide synthase and NFκB inhibitors.

    PubMed

    Yu, Xufen; Park, Eun-Jung; Kondratyuk, Tamara P; Pezzuto, John M; Sun, Dianqing

    2012-11-28

    Development of small molecule drug-like inhibitors blocking both nitric oxide synthase and NFκB could offer a synergistic therapeutic approach in the prevention and treatment of inflammation and cancer. During the course of evaluating the biological potential of a commercial compound library, 2-phenylindole (1) displayed inhibitory activity against nitrite production and NFκB with IC(50) values of 38.1 ± 1.8 and 25.4 ± 2.1 μM, respectively. Based on this lead, synthesis and systematic optimization have been undertaken in an effort to find novel and more potent nitric oxide synthase and NFκB inhibitors with antiinflammatory and cancer preventive potential. First, chemical derivatizations of 1 and 2-phenylindole-3-carboxaldehyde (4) were performed to generate a panel of N-alkylated indoles and 3-oxime derivatives 2–7. Second, a series of diversified 2-arylindole derivatives (10) were synthesized from an array of substituted 2-iodoanilines (8) and terminal alkynes (9) by applying a one-pot palladium catalyzed Sonogashira-type alkynylation and base-assisted cycloaddition. Subsequent biological evaluations revealed 3-carboxaldehyde oxime and cyano substituted 2-phenylindoles 5 and 7 exhibited the strongest nitrite inhibitory activities (IC(50) = 4.4 ± 0.5 and 4.8 ± 0.4 μM, respectively); as well as NFκB inhibition (IC(50) = 6.9 ± 0.8 and 8.5 ± 2.0 μM, respectively). In addition, the 6′-MeO-naphthalen-2′-yl indole derivative 10at displayed excellent inhibitory activity against NFκB with an IC(50) value of 0.6 ± 0.2 μM.

  12. ANTITUMOR ACTIVITY OF HYALURONIC ACID SYNTHESIS INHIBITOR 4-METHYLUMBELLIFERONE IN PROSTATE CANCER CELLS

    PubMed Central

    Lokeshwar, Vinata B.; Lopez, Luis E.; Munoz, Daniel; Chi, Andrew; Shirodkar, Samir P.; Lokeshwar, Soum D.; Escudero, Diogo O.; Dhir, Neetika; Altman, Norman

    2010-01-01

    4-methylumbelliferone (4-MU) is a hyaluronic acid (HA) synthesis inhibitor with anticancer properties; the mechanism of its anticancer effects is unknown. We evaluated the effects of 4-MU on prostate cancer cells. 4-MU inhibited proliferation, motility and invasion of DU145, PC3-ML, LNCaP, C4-2B and/or LAPC-4 cells. At IC50 for HA synthesis (0.4 mM), 4-MU induced > 3-fold apoptosis in prostate cancer cells, which could be prevented by HA addition. 4-MU induced caspase-8, -9 and -3 activation, PARP cleavage, up-regulation of Fas-L, Fas, FADD and DR4 and down regulation of bcl-2, phospho-bad, bcl-XL, phospho-Akt, phospho-IKB, phospho-ErbB2 and phospho-EGFR. At IC50, 4-MU also caused > 90% inhibition of NFkB reporter activity which was prevented partially by HA addition. With the exception of caveolin-1, HA prevented the 4-MU induced down regulation of HA receptors (CD44, RHAMM), matrix-degrading enzymes (MMP-2, MMP-9), IL-8, and chemokine receptors (CXCR1, CXCR4, CXCR7) at protein and mRNA levels. Expression of myristoylated-Akt rescued 4-MU induced apoptosis and inhibition of cell growth and IL-8, RHAMM, HAS2, CD44 and MMP-9 expression. Oral administration of 4-MU significantly decreased PC3-ML tumor growth (> 3-fold), when treatment was started either on the day of tumor cell injection or after the tumors became palpable, without organ toxicity, changes in serum chemistry or body weight. Tumors from 4-MU treated animals showed reduced microvessel density (~ 3-fold) and HA expression but increased TUNEL positive cells and expression of apoptosis-related molecules. Therefore, anticancer effects of 4-MU, an orally bioavailable and relatively non-toxic agent, are primarily mediated by inhibition of HA signaling. PMID:20332231

  13. Carbimazole is an inhibitor of protein synthesis and protects from neuronal hypoxic damage in vitro.

    PubMed

    Lehane, Cornelius; Guelzow, Timo; Zenker, Simone; Erxleben, Anika; Schwer, Christian I; Heimrich, Bernd; Buerkle, Hartmut; Humar, Matjaz

    2013-12-01

    Oxygen deprivation during ischemic or hemorrhagic stroke results in ATP depletion, loss of ion homeostasis, membrane depolarization, and excitotoxicity. Pharmacologic restoration of cellular energy supply may offer a promising concept to reduce hypoxic cell injury. In this study, we investigated whether carbimazole, a thionamide used to treat hyperthyroidism, reduces neuronal cell damage in oxygen-deprived human SK-N-SH cells or primary cortical neurons. Our results revealed that carbimazole induces an inhibitory phosphorylation of eukaryotic elongation factor 2 (eEF2) that was associated with a marked inhibition of global protein synthesis. Translational inhibition resulted in significant bioenergetic savings, preserving intracellular ATP content in oxygen-deprived neuronal cells and diminishing hypoxic cellular damage. Phosphorylation of eEF2 was mediated by AMP-activated protein kinase and eEF2 kinase. Carbimazole also induced a moderate calcium influx and a transient cAMP increase. To test whether translational inhibition generally diminishes hypoxic cell damage when ATP availability is limiting, the translational repressors cycloheximide and anisomycin were used. Cycloheximide and anisomycin also preserved ATP content in hypoxic SK-N-SH cells and significantly reduced hypoxic neuronal cell damage. Taken together, these data support a causal relation between the pharmacologic inhibition of global protein synthesis and efficient protection of neurons from ischemic damage by preservation of high-energy metabolites in oxygen-deprived cells. Furthermore, our results indicate that carbimazole or other translational inhibitors may be interesting candidates for the development of new organ-protective compounds. Their chemical structure may be used for computer-assisted drug design or screening of compounds to find new agents with the potential to diminish neuronal damage under ATP-limited conditions.

  14. 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

  15. An integrated approach for discovery of highly potent and selective Mnk inhibitors: Screening, synthesis and SAR analysis.

    PubMed

    Teo, Theodosia; Yang, Yuchao; Yu, Mingfeng; Basnet, Sunita K C; Gillam, Todd; Hou, Jinqiang; Schmid, Raffaella M; Kumarasiri, Malika; Diab, Sarah; Albrecht, Hugo; Sykes, Matthew J; Wang, Shudong

    2015-10-20

    Deregulation of protein synthesis is a common event in cancer. As MAPK-interacting kinases (Mnks) play critical roles in regulation of protein synthesis, they have emerged as novel anti-cancer targets. Mnks phosphorylate eukaryotic initiation factor 4E (eIF4E) and promote eIF4E-mediated oncogenic activity. Given that the kinase activity of Mnks is essential for oncogenesis but is dispensable for normal development, the discovery of potent and selective pharmacological Mnk inhibitors provides pharmacological target validation and offers a new strategy for cancer treatment. Herein, comprehensive in silico screening approaches were deployed, and three thieno[2,3-d]pyrimidine and pyrazolo[3,4-d]pyrimidine derivatives were identified as hit compounds. Further chemical modification of thieno[2,3-d]pyrimidine derivative 3 has given rise to a series of highly potent Mnk2 inhibitors that could be potential leads for the treatment of acute myeloid leukemia.

  16. Synthesis of 7-oxo-dihydrospiro[indazole-5,4'-piperidine] acetyl-CoA carboxylase inhibitors.

    PubMed

    Bagley, Scott W; Southers, James A; Cabral, Shawn; Rose, Colin R; Bernhardson, David J; Edmonds, David J; Polivkova, Jana; Yang, Xiaojing; Kung, Daniel W; Griffith, David A; Bader, Scott J

    2012-02-03

    Synthesis of oxo-dihydrospiroindazole-based acetyl-CoA carboxylase (ACC) inhibitors is reported. The dihydrospiroindazoles were assembled in a regioselective manner in six steps from substituted hydrazines and protected 4-formylpiperidine. Enhanced regioselectivity in the condensation between a keto enamine and substituted hydrazines was observed when using toluene as the solvent, leading to selective formation of 1-substituted spiroindazoles. The 2-substituted spiroindazoles were formed selectively from alkyl hydrazones by ring closure with Vilsmeier reagent. The key step in the elaboration to the final products is the conversion of an intermediate olefin to the desired ketone through elimination of HBr from an O-methyl bromohydrin. This methodology enabled the synthesis of each desired regioisomer on 50-75 g scale with minimal purification. Acylation of the resultant spirocyclic amines provided potent ACC inhibitors.

  17. Selective inhibition of cholesterol synthesis in liver versus extrahepatic tissues by HMG-CoA reductase inhibitors.

    PubMed

    Parker, R A; Clark, R W; Sit, S Y; Lanier, T L; Grosso, R A; Wright, J J

    1990-07-01

    Hepatic specificity of inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase may be achieved by efficient first-pass liver extraction resulting in low circulating drug levels, as with lovastatin, or by lower cellular uptake in peripheral tissues, seen with pravastatin. BMY-21950 and its lactone form BMY-22089, new synthetic inhibitors of HMG-CoA reductase, were compared with the major reference agent lovastatin and with the synthetic inhibitor fluindostatin in several in vitro and in vivo models of potency and tissue selectivity. The kinetic mechanism and the potency of BMY-21950 as a competitive inhibitor of isolated HMG-CoA reductase were comparable to the reference agents. The inhibitory potency (cholesterol synthesis assayed by 3H2O or [14C]acetate incorporation) of BMY-21950 in rat hepatocytes (IC50 = 21 nM) and dog liver slices (IC50 = 23 nM) equalled or exceeded the potencies of the reference agents. Hepatic cholesterol synthesis in vivo in rats was effectively inhibited by BMY-21950 and its lactone form BMY-22089 (ED50 = 0.1 mg/kg p.o.), but oral doses (20 mg/kg) that suppressed liver synthesis by 83-95% inhibited sterol synthesis by only 17-24% in the ileum. In contrast, equivalent doses of lovastatin markedly inhibited cholesterol synthesis in both organs. In tissue slices from rat ileum, cell dispersions from testes, adrenal, and spleen, and in bovine ocular lens epithelial cells, BMY-21950 inhibited sterol synthesis weakly in vitro with IC50 values 76- and 188-times higher than in hepatocytes; similar effects were seen for BMY-22089. However, the IC50 ratios (tissue/hepatocyte) for lovastatin and fluindostatin were near unity in these models. Thus, BMY-21950 and BMY-22089 are the first potent synthetic HMG-CoA reductase inhibitors that possess a very high degree of liver selectivity based upon differential inhibition sensitivities in tissues. This cellular uptake-based property of hepatic specificity of BMY-21950 and BMY-22089, also

  18. 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

  19. Towards inhibitors of glycosyltransferases: A novel approach to the synthesis of 3-acetamido-3-deoxy-D-psicofuranose derivatives

    PubMed Central

    Koóš, Miroslav; Lin, Chun-Hung

    2015-01-01

    Summary A novel synthetic strategy leading to 3-acetamido-3-deoxy-D-psicofuranose 9 is presented. The latter compound, after some manipulations, was transformed into fully protected 3-acetamido-3-deoxy-D-psicofuranose 11 as a potential substrate for the synthesis of N-acetylglucosaminyltransferase inhibitors designed by computational methods. After the attempted thioglycosylation of 11 with EtSH in the presence of BF3·OEt2, 2-methyloxazoline derivatives 13 and 14 were isolated. PMID:26425214

  20. Design and synthesis of non-hydrolyzable homoisoprenoid α-monofluorophosphonate inhibitors of PPAPDC family integral membrane lipid phosphatases.

    PubMed

    Subramanian, Thangaiah; Ren, Hongmei; Subramanian, Karunai Leela; Sunkara, Manjula; Onono, Fredrick O; Morris, Andrew J; Spielmann, H Peter

    2014-09-15

    An efficient, diversity oriented synthesis of homoisoprenoid α-monofluorophosphonates utilizing electrophilic fluorination is presented along with their activity as inhibitors of PPAPDC2 family integral membrane lipid phosphatases. These novel phosphatase-resistant analogues of isoprenoid monophosphates are a platform for further structure-activity relationship studies and provide access to other isoprenoid family members where the phosphate ester oxygen is replaced by a α-monofluoromethylene moiety.

  1. Synthesis of N-diisopropyl phosphoryl benzyltetrahydroisoquinoline, a new class of mitochondrial complexes I and III inhibitors.

    PubMed

    Andreu, I; Cabedo, N; Tormo, J R; Bermejo, A; Mello, R; Cortes, D

    2000-07-03

    The synthesis of N-(O,O-diisopropylphosphoryl)-benzyltetrahydroisoquinoline (3) has been achieved in a 'one pot' procedure from imine (2) and diisopropyl-phosphorochloridate (1) generated in situ (POCl3 + iPrOH). Compound 3 is the first benzyltetrahydroisoquinoline derivative found to be a potent inhibitor of mitochondrial complexes I and III, and therefore it opens a new perspective with this series of compounds as they can be considered as new class of antitumor agents.

  2. Effects of inhibitors of DNA, RNA and protein synthesis on frequencies and types of premature chromosome condensation from X-ray induced micronuclei.

    PubMed

    Madle, S; Nowak, J; Obe, G

    1976-10-28

    Cells containing X-ray induced micronuclei were treated for a few hours before fixation with inhibitors of DNA synthesis (cytosine arabinoside; azathioprine; thymidine; trenimon), of RNA synthesis (actinomycin D; ethidium bromide), and of protein synthesis (puromycin). Only the inhibitors of DNA synthesis lead to a significant suppression of the frequencies of mitoses with micronucleus derived premature chromosome condensation (PCC). We tend to interprete the result as follows: Micronuclei that are in the G1 phase of their cell cycles are accumulated at the G1/S border or in the early S phase of their cell cycles under the influence of the inhibitors of the DNA synthesis. Micronuclei blocked in this way cannot be induced to undergo PCC and seem to disappear from the cells.

  3. 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.

  4. 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.

  5. Treatment with the hyaluronic Acid synthesis inhibitor 4-methylumbelliferone suppresses LPS-induced lung inflammation.

    PubMed

    McKallip, Robert J; Ban, Hao; Uchakina, Olga N

    2015-01-01

    Exposure to bacterial endotoxins, such as lipopolysaccharide (LPS), can lead to the induction of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). To date, there are no known effective treatments for LPS-induced inflammation. In the current study, we investigated the potential use of the hyaluronic acid (HA) synthesis inhibitor 4-methylumbelliferone (4-MU) on LPS-induced acute lung inflammation. Culturing LPS-activated immune cells with 4-MU led to reduced proliferation, reduced cytokine production, and an increase in apoptosis when compared to untreated cells. Treatment of mice with 4-MU led to protection from LPS-induced lung injury. Specifically, 4-MU treatment led to a reduction in LPS-induced hyaluronic acid synthase (HAS) messenger RNA (mRNA) levels, reduction in lung permeability, and reduction in proinflammatory cytokine production. Taken together, these results suggest that use of 4-MU to target HA production may be an effective treatment for the inflammatory response following exposure to LPS.

  6. QSAR studies, synthesis and antibacterial assessment of new inhibitors against multidrug-resistant Mycobacterium tuberculosis.

    PubMed

    Kovalishyn, Vasyl; Brovarets, Volodymyr; Blagodatnyi, Volodymyr; Kopernyk, Iryna; Hodyna, Diana; Chumachenko, Svitlana; Shablykin, Oleg; Kozachenko, Oleksandr; Vovk, Myhailo; Barus, Marianna; Bratenko, Myhailo; Metelytsia, Larysa

    2016-11-08

    This paper describes Quantitative Structure-Activity Relationships (QSAR) studies using Artificial Neural Networks (ANN), synthesis and in vitro antitubercular activity of several potent compounds against H37Rv and resistant Mycobacterium tuberculosis (Mtb) strains. Eight QSAR models were built using various types of descriptors with four publicly available structurally diverse datasets, including recent data from PubChem and ChEMBL. The predictive power of the obtained QSAR models was evaluated with a cross-validation procedure, giving a q2=0.74-0.78 for regression models and overall accuracy 78.9-94.4% for classification models. The external test sets were predicted with accuracies in the range of 84.1-95.0% (for the active/inactive classifications) and q2=0.80-0.83 for regressions. The 15 synthesized compounds showed inhibitory activity against H37Rv strain whereas the compounds 1-7 were also active against resistant Mtb strain (resistant to isoniazid and rifampicin). The results indicated that compounds 1-7 could serve as promising leads for further optimization as novel antibacterial inhibitors, in particular, for the treatment of drug resistance of Mtb forms.

  7. Design and Synthesis of Novel Small-molecule Inhibitors of the Hypoxia Inducible Factor Pathway

    PubMed Central

    Mooring, Suazette Reid; Jin, Hui; Devi, Narra S.; Jabbar, Adnan A.; Kaluz, Stefan; Liu, Yuan; Van Meir, Erwin G.; Wang, Binghe

    2012-01-01

    Hypoxia, a reduction in partial oxygen pressure, is a salient property of solid tumors. Hypoxia drives malignant progression and metastasis in tumors and participates in tumor resistance to radio- and chemotherapies. Hypoxia activates the hypoxia-inducible factor (HIF) family of transcription factors, which induce target genes that regulate adaptive biological processes such as anaerobic metabolism, cell motility and angiogenesis. Clinical evidence has demonstrated that expression of HIF-1 is strongly associated with poor patient prognosis and activation of HIF-1 contributes to malignant behavior and therapeutic resistance. Consequently, HIF-1 has become an important therapeutic target for inhibition by small molecules. Herein, we describe the design and synthesis of small molecules that inhibit the HIF-1 signaling pathway. Many of these compounds exhibit inhibitory activity in the nanomolar range. Separate mechanistic studies indicate that these inhibitors do not alter HIF-1 levels, but interfere with the HIF-1α/HIF-1β/p300/CBP complex formation by interacting with p300 and CBP. PMID:22032632

  8. Alpha-fluoromethylhistidine, a histamine synthesis inhibitor, inhibits orexin-induced wakefulness in rats.

    PubMed

    Yasuko, Seki; Atanda, Akanmu Moses; Masato, Matsuura; Kazuhiko, Yanai; Kazuki, Honda

    2010-02-11

    Orexins A and B are involved in the regulation of feeding and arousal state. Previously, we reported that third intracerebroventricular (icv) infusion of both orexins A and B induced a significant arousal effect in rats. We determined the effects of intraperitoneal (i.p.) injection of alpha-fluoromethylhistidine (alpha-FMH), a histamine synthesis inhibitor, on orexin-induced wakefulness in freely behaving rats. Male Sprague-Dawley rats were chronically implanted with cortical electroencephalogram (EEG) and neck electromyogram (EMG) electrodes, and a cannula for icv infusion. EEG and EMG were monitored for three consecutive days during continuous icv saline infusion at a rate of 10 microl/h. For a 5-h diurnal period, orexin-B (10 nmol/50 microl saline) replaced the icv infusion of saline. alpha-FMH (100mg/kg, i.p.) was administered 6h before icv infusion of orexin-B. Orexin-B at a dose of 10 nmol/h markedly increased the amount of wakefulness by 99.4% (p<0.05) over the baseline value, whereas alpha-FMH decreased orexin-B-induced wakefulness by 48.8%. Orexin-B-induced suppression of non-REM sleep was reversed by alpha-FMH treatment. Pretreatment with alpha-FMH, significantly inhibited orexin-B-induced wakefulness in rats. The findings of this study therefore suggest that arousal-state regulation by orexin neurons is possibly mediated via the histaminergic system in the tuberomammilary nucleus.

  9. 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

  10. Design, Synthesis, and Evaluation of Acrylamide Derivatives as Direct NLRP3 Inflammasome Inhibitors.

    PubMed

    Cocco, Mattia; Miglio, Gianluca; Giorgis, Marta; Garella, Davide; Marini, Elisabetta; Costale, Annalisa; Regazzoni, Luca; Vistoli, Giulio; Orioli, Marica; Massulaha-Ahmed, Raïhane; Détraz-Durieux, Isabelle; Groslambert, Marine; Py, Bénédicte F; Bertinaria, Massimo

    2016-08-19

    NLRP3 inflammasome plays a key role in the intracellular activation of caspase-1, processing of pro-inflammatory interleukin-1β (IL-1β), and pyroptotic cell death cascade. The overactivation of NLRP3 is implicated in the pathogenesis of autoinflammatory diseases, known as cryopyrin-associated periodic syndromes (CAPS), and in the progression of several diseases, such as atherosclerosis, type-2 diabetes, gout, and Alzheimer's disease. In this study, the synthesis of acrylamide derivatives and their pharmaco-toxicological evaluation as potential inhibitors of NLRP3-dependent events was undertaken. Five hits were identified and evaluated for their efficiency in inhibiting IL-1β release from different macrophage subtypes, including CAPS mutant macrophages. The most attractive hits were tested for their ability to inhibit NLRP3 ATPase activity on human recombinant NLRP3. This screening allowed the identification of 14, 2-(2-chlorobenzyl)-N-(4-sulfamoylphenethyl)acrylamide, which was able to concentration-dependently inhibit NLRP3 ATPase with an IC50 value of 74 μm. The putative binding pose of 14 in the ATPase domain of NLRP3 was also proposed. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. 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.

  12. Synthesis and biological evaluation of flexible and conformationally constrained LpxC inhibitors.

    PubMed

    Löppenberg, Marius; Müller, Hannes; Pulina, Carla; Oddo, Alberto; Teese, Mark; Jose, Joachim; Holl, Ralph

    2013-09-28

    Inhibitors of the UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine deacetylase (LpxC) represent promising candidates for the development of antibiotics possessing a so far unexploited mechanism of action. In a chiral pool synthesis, starting from the D-mannose derived mannonolactone 4, conformationally constrained C-glycosidic as well as open chained hydroxamic acids with a defined stereochemistry were prepared. Diversity was introduced by performing C–C coupling reactions like the Sonogashira and Suzuki cross-coupling reactions. The biological evaluation of the synthesized compounds revealed that in the case of the C-glycosides a long, linear and rigid hydrophobic side chain is required for antibiotic activity against E. coli. The open chain derivatives show higher biological activity than the conformationally constrained C-glycosides. The morpholinomethyl substituted open chain derivative 43, being the most potent compound presented in this paper, inhibits LpxC with a Ki value of 0.35 μM and represents a promising lead structure.

  13. Synthesis and Identification of Pregnenolone Derivatives as Inhibitors of Isozymes of 5α-Reductase.

    PubMed

    Chávez-Riveros, Alejandra; Bratoeff, Eugene; Heuze, Yvonne; Soriano, Juan; Moreno, Isabel; Sánchez-Márquez, Araceli; Cabeza, Marisa

    2015-09-17

    Hyperplasia of the prostate gland and prostate cancer have been associated with high levels of serum 5α-dihydrotestosterone. This steroid is formed from testosterone by the activity of the enzyme 5α-reductase (5α-R) present in the prostate. Thus, inhibition of this enzyme could be a goal for therapies to treat these diseases. This study reports the synthesis and effects of five different 21-esters of pregnenolone derivatives as inhibitors of 5α-R types 1 and 2. The activity of these steroidal compounds was determined using in vivo and in vitro experiments. The results indicate that of the five steroids studied, the 21(p-fluoro)benzoyloxypregna-4,16-diene-3,6,20-trione derivative, whose structure has not yet been reported, has the best molecular conformation to inhibit the in vitro activity of both types of 5α-R. In addition, this steroid also displayed activity in vivo. Apparently, its pharmacological effect was increased by the presence of a keto group at C-6, because this group decreased the possibility that the steroid would be metabolized by hepatic enzymes. In addition, the double bond present at C-4 of this compound also enhanced its inhibitory activity on 5α-R, and the C-21 ester moiety increased its liphophilicity. Therefore, its solubility in the cell membrane and its pharmacological activity were both increased.

  14. 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

  15. Synthesis of new pyrimidine-fused derivatives as potent and selective antidiabetic α-glucosidase inhibitors.

    PubMed

    Panahi, Farhad; Yousefi, Reza; Mehraban, Mohammad Hossein; Khalafi-Nezhad, Ali

    2013-10-18

    The synthesis of a set of pyrimidine-fused derivatives (L1-L8), resulting from the incorporation of different fragments on the pyrimidine-fused heterocycle (PFH) of the earlier reported α-glucosidase (α-Gls) inhibitor (C1-C5), allowed the discovery of new ligands with modest and selective inhibitory activity. The PFH core (substructure 2) was proved to play a significant role in their inhibitory properties. Additionally, the substituent on substructures 1 and 3 of the heterocyclic ring was demonstrated to be important in the enzyme inhibitory action of the pyrimidine-fused derivatives. Moreover, these ligands show selective inhibitory properties for α-Gls over porcine pancreatic α-amylase (α-Amy) which is important in terms of their reduced susceptibility for the possible development of intestinal disturbance side effects. Therefore, low to moderate α-Amy inhibition with effective α-Gls inhibitory action may offer a better therapeutic strategy. Overall, these compounds can potentially offer a new opportunity to develop novel antidiabetic drugs with selective inhibitory action against α-Gls. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. 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.

  17. 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.

  18. Molecular design and synthesis of novel peptides from amphibians skin acting as inhibitors of cholinesterase enzymes.

    PubMed

    Siano, Alvaro; Garibotto, Francisco F; Andujar, Sebastian A; Baldoni, Hector A; Tonarelli, Georgina G; Enriz, Ricardo D

    2017-03-01

    Cholinesterases are a family of enzymes that catalyze the hydrolysis of neurotransmitter acetylcholine. There are two types of cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), which differ in their distribution in the body. Currently, cholinesterase inhibitors (ChEI) represent the treatment of choice for Alzheimer's disease (AD). In this paper, we report the synthesis and inhibitory effect on both enzymes of four new peptides structurally related to P1-Hp-1971 (amphibian skin peptide found in our previous work. Sequence: TKPTLLGLPLGAGPAAGPGKR-NH2 ). The bioassay data and cytotoxicity test show that some of the compounds possess a significant AChE and BChE inhibition and no toxic effect. The present work demonstrates that diminution of the size of the original peptide could potentially result in new compounds with significant cholinesterase inhibition activity, although it appears that there is an optimal size for the sequence. We also conducted an exhaustive molecular modeling study to better understand the mechanism of action of these compounds by combining docking techniques with molecular dynamics simulations on BChE. This is the first report about amphibian peptides and the second one of natural peptides with ChE inhibitory activity. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

  19. 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

  20. 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

  1. The importance of NAD in multiple sclerosis.

    PubMed

    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 Wld(S) 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 Wld(S) 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

  2. Bisindolylmaleimide protein-kinase-C inhibitors delay the decline in DNA synthesis in mouse hair follicle organ cultures.

    PubMed

    Harmon, C S; Nevins, T D; Ducote, J; Lutz, D

    1997-01-01

    We have used a series of bisindolylmaleimide selective protein-kinase C (PKC) inhibitors to investigate the role of this enzyme in the regulation of cell proliferation in mouse hair follicle organ cultures. Mouse whisker follicles were isolated by microdissection, and rates of DNA synthesis during culture were determined from 3H-thymidine incorporation. The bisindolylmaleimides Ro 31-7549, Ro 31-8161, Ro 31-8425 and Ro 31-8830 inhibit isolated brain PKC with IC50 values of 8-80 nM, are > 60-fold less potent against protein kinase A, and inhibit PKC-mediated protein phosphorylation in platelets with IC50 values in the range 0.25-4.4 microM. These PKC inhibitors were found to increase levels of mouse hair follicle DNA synthesis, with EC50 values in the range 1-4 microM and maximal levels in the range 151-197% of control. Ro 31-7549 had an IC50 value 50-fold lower than that of minoxidil, while the maximal level of DNA synthesis for the PKC inhibitor was 86% higher. Incubation of mouse hair follicles with Ro 31-7549 resulted in a delay of approximately 24 h in the onset of decline in follicular DNA synthesis rates. Ro 31-6045 and Ro 31-7208, bisindolylmaleimides without activity in the platelet PKC assay, did not affect mouse hair follicle DNA synthesis rates. Taken together, these findings show that PKC mediates, at least in part, the rapid loss of proliferative activity that occurs in mouse whisker follicles in culture, and provide further evidence that PKC plays a role as a negative proliferative signal in hair follicles.

  3. 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

    2007-05-04

    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.

  4. Cloning, expression, purification, crystallization and preliminary X-ray diffraction studies of NAD synthetase from methicillin-resistant Staphylococcus aureus.

    PubMed

    Arbade, Gajanan Kashinathrao; Srivastava, Sandeep Kumar

    2015-06-01

    Staphylococcus aureus is an important human and animal pathogen that causes a wide range of infections. The prevalence of multidrug-resistant S. aureus strains in both hospital and community settings makes it imperative to characterize new drug targets to combat S. aureus infections. In this context, enzymes involved in NAD metabolism and synthesis are significant drug targets as NAD is a central player in several cellular processes. NAD synthetase catalyzes the last step in the biosynthesis of nicotinamide adenine dinucleotide, making it a crucial intermediate enzyme linked to the biosynthesis of several amino acids, purine and pyrimidine nucleotides, coenzymes and antibiotics.

  5. Design, synthesis and structure-activity relationships of substituted oxazole-benzamide antibacterial inhibitors of FtsZ.

    PubMed

    Stokes, Neil R; Baker, Nicola; Bennett, James M; Chauhan, Pramod K; Collins, Ian; Davies, David T; Gavade, Maruti; Kumar, Dushyant; Lancett, Paul; Macdonald, Rebecca; Macleod, Leanne; Mahajan, Anu; Mitchell, Jeffrey P; Nayal, Narendra; Nayal, Yashodanand Nandan; Pitt, Gary R W; Singh, Mahipal; Yadav, Anju; Srivastava, Anil; Czaplewski, Lloyd G; Haydon, David J

    2014-01-01

    The design, synthesis and structure-activity relationships of a series of oxazole-benzamide inhibitors of the essential bacterial cell division protein FtsZ are described. Compounds had potent anti-staphylococcal activity and inhibited the cytokinesis of the clinically-significant bacterial pathogen Staphylococcus aureus. Selected analogues possessing a 5-halo oxazole also inhibited a strain of S. aureus harbouring the glycine-to-alanine amino acid substitution at residue 196 of FtsZ which conferred resistance to previously reported inhibitors in the series. Substitutions to the pseudo-benzylic carbon of the scaffold improved the pharmacokinetic properties by increasing metabolic stability and provided a mechanism for creating pro-drugs. Combining multiple substitutions based on the findings reported in this study has provided small-molecule inhibitors of FtsZ with enhanced in vitro and in vivo antibacterial efficacy.

  6. Regioselective Synthesis of a Family of β‐Lactams Bearing a Triazole Moiety as Potential Apoptosis Inhibitors

    PubMed Central

    Garrido, Maria; Corredor, Miriam; Orzáez, Mar; Alfonso, Ignacio

    2016-01-01

    Abstract Apoptosis is a biological process important to several human diseases; it is strongly regulated through protein–protein interactions and complex formation. We previously reported the synthesis of apoptosis inhibitors bearing an exocyclic triazole amide isoster by using an Ugi four‐component coupling reaction (Ugi‐4CC), followed by a base‐promoted intramolecular cyclization. Depending on the substitution patterns and the reaction conditions, this cyclization forms the six‐ or four‐membered ring. Two compounds bearing the β‐lactam scaffold turned out to be the most potent inhibitors. This encouraged us to optimize the modulation of the cyclization, and prepare a library of 15 β‐lactams with total regioselectivity. Moreover, we aimed to improve the bioavailability of these compounds through the introduction of diversity at different substitution positions. The activity of these compounds as apoptosis inhibitors in cellular extracts has been evaluated, showing an increase in their potency. PMID:27777842

  7. Regioselective Synthesis of a Family of β-Lactams Bearing a Triazole Moiety as Potential Apoptosis Inhibitors.

    PubMed

    Garrido, Maria; Corredor, Miriam; Orzáez, Mar; Alfonso, Ignacio; Messeguer, Angel

    2016-10-01

    Apoptosis is a biological process important to several human diseases; it is strongly regulated through protein-protein interactions and complex formation. We previously reported the synthesis of apoptosis inhibitors bearing an exocyclic triazole amide isoster by using an Ugi four-component coupling reaction (Ugi-4CC), followed by a base-promoted intramolecular cyclization. Depending on the substitution patterns and the reaction conditions, this cyclization forms the six- or four-membered ring. Two compounds bearing the β-lactam scaffold turned out to be the most potent inhibitors. This encouraged us to optimize the modulation of the cyclization, and prepare a library of 15 β-lactams with total regioselectivity. Moreover, we aimed to improve the bioavailability of these compounds through the introduction of diversity at different substitution positions. The activity of these compounds as apoptosis inhibitors in cellular extracts has been evaluated, showing an increase in their potency.

  8. 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

  9. Depletion of the central metabolite NAD leads to oncosis-mediated cell death.

    PubMed

    Del Nagro, Christopher; Xiao, Yang; Rangell, Linda; Reichelt, Mike; O'Brien, Thomas

    2014-12-19

    Depletion of the central metabolite NAD in cells results in broad metabolic defects leading to cell death and is a proposed novel therapeutic strategy in oncology. There is, however, a limited understanding of the underlying mechanisms that connect disruption of this central metabolite with cell death. Here we utilize GNE-617, a small molecule inhibitor of NAMPT, a rate-limiting enzyme required for NAD generation, to probe the pathways leading to cell death following NAD depletion. In all cell lines examined, NAD was rapidly depleted (average t½ of 8.1 h) following NAMPT inhibition. Concurrent with NAD depletion, there was a decrease in both cell proliferation and motility, which we attribute to reduced activity of NAD-dependent deacetylases because cells fail to deacetylate α-tubulin-K40 and histone H3-K9. Following depletion of NAD by >95%, cells lose the ability to regenerate ATP. Cell lines with a slower rate of ATP depletion (average t½ of 45 h) activate caspase-3 and show evidence of apoptosis and autophagy, whereas cell lines with rapid depletion ATP (average t½ of 32 h) do not activate caspase-3 or show signs of apoptosis or autophagy. However, the predominant form of cell death in all lines is oncosis, which is driven by the loss of plasma membrane homeostasis once ATP levels are depleted by >20-fold. Thus, our work illustrates the sequence of events that occurs in cells following depletion of a key metabolite and reveals that cell death caused by a loss of NAD is primarily driven by the inability of cells to regenerate ATP.

  10. Targeting tumor-initiating cells: Eliminating anabolic cancer stem cells with inhibitors of protein synthesis or by mimicking caloric restriction

    PubMed Central

    Lamb, Rebecca; Harrison, Hannah; Smith, Duncan L.; Townsend, Paul A.; Jackson, Thomas; Ozsvari, Bela; Martinez-Outschoorn, Ubaldo E.; Pestell, Richard G.; Howell, Anthony; Lisanti, Michael P.; Sotgia, Federica

    2015-01-01

    We have used an unbiased proteomic profiling strategy to identify new potential therapeutic targets in tumor-initiating cells (TICs), a.k.a., cancer stem cells (CSCs). Towards this end, the proteomes of mammospheres from two breast cancer cell lines were directly compared to attached monolayer cells. This allowed us to identify proteins that were highly over-expressed in CSCs and/or progenitor cells. We focused on ribosomal proteins and protein folding chaperones, since they were markedly over-expressed in mammospheres. Overall, we identified >80 molecules specifically associated with protein synthesis that were commonly upregulated in mammospheres. Most of these proteins were also transcriptionally upregulated in human breast cancer cells in vivo, providing evidence for their potential clinical relevance. As such, increased mRNA translation could provide a novel mechanism for enhancing the proliferative clonal expansion of TICs. The proteomic findings were functionally validated using known inhibitors of protein synthesis, via three independent approaches. For example, puromycin (which mimics the structure of tRNAs and competitively inhibits protein synthesis) preferentially targeted CSCs in both mammospheres and monolayer cultures, and was ~10-fold more potent for eradicating TICs, than “bulk” cancer cells. In addition, rapamycin, which inhibits mTOR and hence protein synthesis, was very effective at reducing mammosphere formation, at nanomolar concentrations. Finally, mammosphere formation was also markedly inhibited by methionine restriction, which mimics the positive effects of caloric restriction in cultured cells. Remarkably, mammosphere formation was >18-fold more sensitive to methionine restriction and replacement, as directly compared to monolayer cell proliferation. Methionine is absolutely required for protein synthesis, since every protein sequence starts with a methionine residue. Thus, the proliferation and survival of CSCs is very sensitive to

  11. Regioselective synthesis of 5- and 6-methoxybenzimidazole-1,3,5-triazines as inhibitors of phosphoinositide 3-kinase.

    PubMed

    Miller, Michelle S; Pinson, Jo-Anne; Zheng, Zhaohua; Jennings, Ian G; Thompson, Philip E

    2013-02-01

    Phosphoinositide 3-kinases (PI3K) hold significant therapeutic potential as novel targets for the treatment of cancer. ZSTK474 (4a) is a potent, pan-PI3K inhibitor currently under clinical evaluation for the treatment of cancer. Structural studies have shown that derivatisation at the 5- or 6-position of the benzimidazole ring may influence potency and isoform selectivity. However, synthesis of these derivatives by the traditional route results in a mixture of the two regioisomers. We have developed a straightforward regioselective synthesis that gave convenient access to 5- and 6-methoxysubstituted benzimidazole derivatives of ZSTK474. While 5-methoxy substitution abolished activity at all isoforms, the 6-methoxy substitution is consistently 10-fold more potent. This synthesis will allow convenient access to further 6-position derivatives, thus allowing the full scope of the structure-activity relationships of ZSTK474 to be probed.

  12. Structure-based Design of Potent HIV-1 Protease Inhibitors with Modified P1 - Biphenyl Ligands: Synthesis, Biological Evaluation, and Enzyme-inhibitor X-ray Structural studies

    PubMed Central

    Ghosh, Arun K.; Yu, Xufen; Osswald, Heather L.; Agniswamy, Johnson; Wang, Yuan-Fang; Amano, Masayuki; Weber, Irene T.; Mitsuya, Hiroaki

    2016-01-01

    We report the design, synthesis, X-ray structural studies, and biological evaluation of a novel series of HIV-1 protease inhibitors. We designed a variety of functionalized biphenyl derivatives to make enhanced van der Waals interactions in the S1 subsite of HIV-1 protease. These biphenyl derivatives were conveniently synthesized using a Suzuki-Miyaura cross-coupling reaction as the key step. We examined the potential of these functionalized biphenyl-derived P1 ligands in combination with 3-(S)-tetrahydrofuranyl urethane and bis-tetrahydrofuranyl urethane as the P2 ligands. Inhibitor 21e, with a 2-methoxy-1, 1’-biphenyl derivative as P1 ligand and bis-THF as the P2 ligand, displayed the most potent enzyme inhibitory and antiviral activity. This inhibitor also exhibited potent activity against a panel of multidrug-resistant HIV-1 variants. A high resolution X-ray crystal structure of related Boc-derivative 17a-bound HIV-1 protease provided important molecular insight into the ligand-binding site interactions of the biphenyl core in the S1 subsite of HIV-1 protease. PMID:26107245

  13. Synthesis and optimization of thiadiazole derivatives as a novel class of substrate competitive c-Jun N-terminal kinase inhibitors

    PubMed Central

    De, Surya K.; Chen, Vida; Stebbins, John L.; Chen, Li-Hsing; Cellitti, Jason F.; Machleidt, Thomas; Barile, Elisa; Riel-Mehan, Megan; Dahl, Russell; Yang, Li; Emdadi, Aras; Murphy, Ria; Pellecchia, Maurizio

    2009-01-01

    A series of thiadiazole derivatives has been designed as potential allosteric, substrate competitive inhibitors of the protein kinase JNK. We report on the synthesis, characterization and evaluation of a series of compounds that resulted in the identification of potent and selective JNK inhibitors targeting its JIP-1 docking site. PMID:20045647

  14. Synthesis and SAR of new pyrazolo[4,3-h]quinazoline-3-carboxamide derivatives as potent and selective MPS1 kinase inhibitors.

    PubMed

    Caldarelli, Marina; Angiolini, Mauro; Disingrini, Teresa; Donati, Daniele; Guanci, Marco; Nuvoloni, Stefano; Posteri, Helena; Quartieri, Francesca; Silvagni, Marco; Colombo, Riccardo

    2011-08-01

    The synthesis and SAR of a series of novel pyrazolo-quinazolines as potent and selective MPS1 inhibitors are reported. We describe the optimization of the initial hit, identified by screening the internal library collection, into an orally available, potent and selective MPS1 inhibitor.

  15. NAD+ Modulates p53 DNA Binding Specificity and Function

    PubMed Central

    McLure, Kevin G.; Takagi, Masatoshi; Kastan, Michael B.

    2004-01-01

    DNA damage induces p53 DNA binding activity, which affects tumorigenesis, tumor responses to therapies, and the toxicities of cancer therapies (B. Vogelstein, D. Lane, and A. J. Levine, Nature 408:307-310, 2000; K. H. Vousden and X. Lu, Nat. Rev. Cancer 2:594-604, 2002). Both transcriptional and transcription-independent activities of p53 contribute to DNA damage-induced cell cycle arrest, apoptosis, and aneuploidy prevention (M. B. Kastan et al., Cell 71:587-597, 1992; K. H. Vousden and X. Lu, Nat. Rev. Cancer 2:594-604, 2002). Small-molecule manipulation of p53 DNA binding activity has been an elusive goal, but here we show that NAD+ binds to p53 tetramers, induces a conformational change, and modulates p53 DNA binding specificity in vitro. Niacinamide (vitamin B3) increases the rate of intracellular NAD+ synthesis, alters radiation-induced p53 DNA binding specificity, and modulates activation of a subset of p53 transcriptional targets. These effects are likely due to a direct effect of NAD+ on p53, as a molecule structurally related to part of NAD+, TDP, also inhibits p53 DNA binding, and the TDP precursor, thiamine (vitamin B1), inhibits intracellular p53 activity. Niacinamide and thiamine affect two p53-regulated cellular responses to ionizing radiation: rereplication and apoptosis. Thus, niacinamide and thiamine form a novel basis for the development of small molecules that affect p53 function in vivo, and these results suggest that changes in cellular energy metabolism may regulate p53. PMID:15509798

  16. Ultrasonic synthesis of tyramine derivatives as novel inhibitors of α-glucosidase in vitro.

    PubMed

    Siddiqui, Hina; Bashir, Muhammad Arslan; Javaid, Kulsoom; Nizamani, Arsalan; Bano, Huma; Yousuf, Sammer; Rahman, Atta-Ur; Choudhary, M Iqbal

    2016-12-01

    Tyramine derivatives 3-27 were synthesized by using conventional and environmental friendly ultrasonic techniques. These derivatives were then evaluated for the first time for their α-glucosidase (Sources: Saccharomyces cerevisiae and mammalian rat-intestinal acetone powder) inhibitory activity by using in vitro mechanism-based biochemical assays. Compounds 7, 14, 20, 21 and 26 were found to be more active (IC50 = 49.7 ± 0.4, 318.8 ± 3.7, 23.5 ± 0.9, 302.0 ± 7.3 and 230.7 ± 4.0 μM, respectively) than the standard drug, acarbose (IC50 = 840.0 ± 1.73 μM (observed) and 780 ± 0.028 μM (reported)) against α-glucosidase obtained from Saccharomyces cerevisiae. Kinetic studies were carried out on the most active members of the series in order to determine their mode of inhibition and dissociation constants. Compounds 7, 20 and 26 were found to be the competitive inhibitors of α-glucosidase. These compounds were also screened for their protein antiglycation, and dipeptidyl peptidase-IV (DPP-IV) inhibitory activities. Only compounds 20, 22 and 27 showed weak antiglycation activity with IC50 values 505.27 ± 5.95, 581.87 ± 5.50 and 440.58 ± 2.74 μM, respectively. All the compounds were found to be inactive against DDP-IV enzyme. Inhibition of α-glucosidase, DPP-IV enzymes and glycation of proteins are valid targets for the discovery of antidiabetic drugs. Cytotoxicity of compounds 3-27 was also evaluated by using mouse fibroblast 3T3 cell lines. All the compounds were found to be noncytotoxic. The current study describes the synthesis α-glucosidase inhibitory activity of derivatives, based on a natural product tyramine template. The compounds reported here may serve as the starting point for the design and development of novel α-glucosidase inhibitors as antidiabetic agents.

  17. [Synthesis and activity evaluation of PARP-1 inhibitors with azaindole skeleton].

    PubMed

    Zhou, Jie; Zhu, Zhi-Xiang; Chen, Xiao-Guang; Xu, Bai-Ling

    2013-12-01

    PARP [poly(ADP-ribose)polymerase] represents a novel potential target in cancer therapy. It is involved in a DNA repair process by catalyzing the transfer of ADP-ribose units from NAD to a number of its substrate proteins. In this work, a series of novel azaindole derivatives was designed and synthesized. Moreover, 16 target molecules were screened and 8 compounds displayed inhibitory activity against PARP-1. It has been demonstrated that these azaindoles bearing cycloamine substituents at 2-position were active to both PARP-1 and PARP-2.

  18. Discrimination of Potent Inhibitors of Toxoplasma gondii Enoyl-Acyl Carrier Protein Reductase by Thermal Shift Assay

    PubMed Central

    Afanador, Gustavo A.; Muench, Stephen P.; McPhillie, Martin; Fomovska, Alina; Schön, Arne; Zhou, Ying; Cheng, Gang; Stec, Jozef; Freundlich, Joel S.; Shieh, Hong-Ming; Anderson, John W.; Jacobus, David P.; Fidock, David A.; Kozikowski, Alan P.; Fishwick, Colin W.; Rice, David W.; Freire, Ernesto; McLeod, Rima; Prigge, Sean T.

    2014-01-01

    Many microbial pathogens rely on a type II fatty acid synthesis (FASII) pathway which is distinct from the type I pathway found in humans. Enoyl-Acyl Carrier Protein Reductase (ENR) is an essential FASII pathway enzyme and the target of a number of antimicrobial drug discovery efforts. The biocide triclosan is established as a potent inhibitor of ENR and has been the starting point for medicinal chemistry studies. We evaluated a series of triclosan analogs for their ability to inhibit the growth of Toxoplasma gondii, a pervasive human pathogen, and its ENR enzyme (TgENR). Several compounds were identified that inhibited TgENR at low nanomolar concentrations, but could not be further differentiated due to the limited dynamic range of the TgENR activity assay. Thus, we adapted a thermal shift assay (TSA) to directly measure the dissociation constant (Kd) of the most potent inhibitors identified in this study as well as inhibitors from previous studies. Furthermore, the TSA allowed us to determine the mode of action of these compounds in the presence of NADH or NAD+ cofactors. We found that all of the inhibitors bind to a TgENR/NAD+ complex, but that they differed in their dependence on NAD+ concentration. Ultimately, we were able to identify compounds which bind to the TgENR/NAD+ complex in the low femtomolar range. This shows how TSA data combined with enzyme inhibition, parasite growth inhibition data and ADMET predictions allow for better discrimination between potent ENR inhibitors for future medicine development. PMID:24295325

  19. Synthesis and evaluation of heteroarylalanine diacids as potent and selective neutral endopeptidase inhibitors.

    PubMed

    Glossop, Melanie S; Bazin, Richard J; Dack, Kevin N; Fox, David N A; MacDonald, Graeme A; Mills, Mark; Owen, Dafydd R; Phillips, Chris; Reeves, Keith A; Ringer, Tracy J; Strang, Ross S; Watson, Christine A L

    2011-06-01

    Heteroarylalanine derivatives 4 were designed as potential inhibitors of neutral endopeptidase (NEP EC 3.4.24.11). Selectivity over other zinc metalloproteinases was explored through occupation of the S2' subsite within NEP. Structural optimisation led to the identification of 5-phenyl oxazole 4f, a potent and selective NEP inhibitor. A crystal structure of the inhibitor bound complex is reported.

  20. 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

  1. Evidence synthesis and decision modelling to support complex decisions: stockpiling neuraminidase inhibitors for pandemic influenza usage.

    PubMed

    Watson, Samuel I; Chen, Yen-Fu; Nguyen-Van-Tam, Jonathan S; Myles, Puja R; Venkatesan, Sudhir; Zambon, Maria; Uthman, Olalekan; Chilton, Peter J; Lilford, Richard J

    2016-01-01

    Objectives: The stockpiling of neuraminidase inhibitor (NAI) antivirals as a defence against pandemic influenza is a significant public health policy decision that must be made despite a lack of conclusive evidence from randomised controlled trials regarding the effectiveness of NAIs on important clinical end points such as mortality. The objective of this study was to determine whether NAIs should be stockpiled for treatment of pandemic influenza on the basis of current evidence. Methods: A decision model for stockpiling was designed. Data on previous pandemic influenza epidemiology was combined with data on the effectiveness of NAIs in reducing mortality obtained from a recent individual participant meta-analysis using observational data. Evidence synthesis techniques and a bias modelling method for observational data were used to incorporate the evidence into the model. The stockpiling decision was modelled for adults (≥16 years old) and the United Kingdom was used as an example. The main outcome was the expected net benefits of stockpiling in monetary terms. Health benefits were estimated from deaths averted through stockpiling. Results: After adjusting for biases in the estimated effectiveness of NAIs, the expected net benefit of stockpiling in the baseline analysis was £444 million, assuming a willingness to pay of £20,000/QALY ($31,000/QALY). The decision would therefore be to stockpile NAIs. There was a greater probability that the stockpile would not be utilised than utilised. However, the rare but catastrophic losses from a severe pandemic justified the decision to stockpile. Conclusions: Taking into account the available epidemiological data and evidence of effectiveness of NAIs in reducing mortality, including potential biases, a decision maker should stockpile anti-influenza medication in keeping with the postulated decision rule.

  2. Evidence synthesis and decision modelling to support complex decisions: stockpiling neuraminidase inhibitors for pandemic influenza usage

    PubMed Central

    Watson, Samuel I.; Chen, Yen-Fu; Nguyen-Van-Tam, Jonathan S.; Myles, Puja R.; Venkatesan, Sudhir; Zambon, Maria; Uthman, Olalekan; Chilton, Peter J.; Lilford, Richard J.

    2017-01-01

    Objectives: The stockpiling of neuraminidase inhibitor (NAI) antivirals as a defence against pandemic influenza is a significant public health policy decision that must be made despite a lack of conclusive evidence from randomised controlled trials regarding the effectiveness of NAIs on important clinical end points such as mortality. The objective of this study was to determine whether NAIs should be stockpiled for treatment of pandemic influenza on the basis of current evidence. Methods: A decision model for stockpiling was designed. Data on previous pandemic influenza epidemiology was combined with data on the effectiveness of NAIs in reducing mortality obtained from a recent individual participant meta-analysis using observational data. Evidence synthesis techniques and a bias modelling method for observational data were used to incorporate the evidence into the model. The stockpiling decision was modelled for adults (≥16 years old) and the United Kingdom was used as an example. The main outcome was the expected net benefits of stockpiling in monetary terms. Health benefits were estimated from deaths averted through stockpiling. Results: After adjusting for biases in the estimated effectiveness of NAIs, the expected net benefit of stockpiling in the baseline analysis was £444 million, assuming a willingness to pay of £20,000/QALY ($31,000/QALY). The decision would therefore be to stockpile NAIs. There was a greater probability that the stockpile would not be utilised than utilised. However, the rare but catastrophic losses from a severe pandemic justified the decision to stockpile. Conclusions: Taking into account the available epidemiological data and evidence of effectiveness of NAIs in reducing mortality, including potential biases, a decision maker should stockpile anti-influenza medication in keeping with the postulated decision rule. PMID:28413608

  3. 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.

  4. The protein synthesis inhibitor anisomycin reduces sex behavior during a critical period after testosterone treatment in male Syrian hamsters.

    PubMed

    Piekarski, David J; Seto, Tiffany; Zucker, Irving

    2012-01-18

    Testosterone (T) is critical for maintaining male sexual behavior (MSB) in rodents, in part by altering protein synthesis in a well-defined neural circuit. The specific timing of protein synthesis essential for expression of MSB has never been investigated. We administered the protein synthesis inhibitor anisomycin (Ani) to castrated male Syrian hamsters treated sc with 100 μg T in an aqueous vehicle once weekly; this T regimen maintains MSB while elevating circulating T concentrations for only a few hours after each injection. Hamsters were injected s.c. with the vehicle or 12.5 mg Ani at one of several times relative to T administration; MSB was assessed once per week, 6 days after the previous T injection, for 5 weeks. Anisomycin administered 6-12 h after T injection significantly reduced the expression of sexual behavior, whereas Ani treatment between 3 h before and 3 h after T injection did not impair MSB. This experiment is the first to assess the specific timing of protein synthesis relative to a T pulse that is required for the expression of MSB. The demarcation of a critical interval for T-induced protein synthesis necessary for maintenance of MSB should facilitate specification of the genomic, proteomic, and biochemical cascades that subserve actions of T on male copulation.

  5. Regulation of leukotriene and 5oxoETE synthesis and the effect of 5-lipoxygenase inhibitors: a mathematical modeling approach

    PubMed Central

    2012-01-01

    Background 5-lipoxygenase (5-LO) is a key enzyme in the synthesis of leukotrienes and 5-Oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (oxoETE). These inflammatory signaling molecules play a role in the pathology of asthma and so 5-LO inhibition is a promising target for asthma therapy. The 5-LO redox inhibitor zileuton (Zyflo IR/CR®) is currently marketed for the treatment of asthma in adults and children, but widespread use of zileuton is limited by its efficacy/safety profile, potentially related to its redox characteristics. Thus, a quantitative, mechanistic description of its functioning may be useful for development of improved anti-inflammatory targeting this mechanism. Results A mathematical model describing the operation of 5-LO, phospholipase A2, glutathione peroxidase and 5-hydroxyeicosanoid dehydrogenase was developed. The catalytic cycles of the enzymes were reconstructed and kinetic parameters estimated on the basis of available experimental data. The final model describes each stage of cys-leukotriene biosynthesis and the reactions involved in oxoETE production. Regulation of these processes by substrates (phospholipid concentration) and intracellular redox state (concentrations of reduced glutathione, glutathione (GSH), and lipid peroxide) were taken into account. The model enabled us to reveal differences between redox and non-redox 5-LO inhibitors under conditions of oxidative stress. Despite both redox and non-redox inhibitors suppressing leukotriene A4 (LTA4) synthesis, redox inhibitors are predicted to increase oxoETE production, thus compromising efficacy. This phenomena can be explained in terms of the pseudo-peroxidase activity of 5-LO and the ability of lipid peroxides to transform 5-LO into its active form even in the presence of redox inhibitors. Conclusions The mathematical model developed described quantitatively different mechanisms of 5-LO inhibition and simulations revealed differences between the potential therapeutic outcomes for these

  6. 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

  7. 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

  8. Synthesis of chromone, quinolone, and benzoxazinone sulfonamide nucleosides as conformationally constrained inhibitors of adenylating enzymes required for siderophore biosynthesis.

    PubMed

    Engelhart, Curtis A; Aldrich, Courtney C

    2013-08-02

    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 on the basis of 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.

  9. OXPHOS-Mediated Induction of NAD+ Promotes Complete Oxidation of Fatty Acids and Interdicts Non-Alcoholic Fatty Liver Disease

    PubMed Central

    Nam, Minwoo; Lei, Shi; Cooper, Marcus P.

    2015-01-01

    OXPHOS is believed to play an important role in non-alcoholic fatty liver disease (NAFLD), however, precise mechanisms whereby OXPHOS influences lipid homeostasis are incompletely understood. We previously reported that ectopic expression of LRPPRC, a protein that increases cristae density and OXPHOS, promoted fatty acid oxidation in cultured primary hepatocytes. To determine the biological significance of that observation and define underlying mechanisms, we have ectopically expressed LRPPRC in mouse liver in the setting of NAFLD. Interestingly, ectopic expression of LRPPRC in mouse liver completely interdicted NAFLD, including inflammation. Consistent with mitigation of NAFLD, two markers of hepatic insulin resistance—ROS and PKCε activity—were both modestly reduced. As reported by others, improvement of NAFLD was associated with improved whole-body insulin sensitivity. Regarding hepatic lipid homeostasis, the ratio of NAD+ to NADH was dramatically increased in mouse liver replete with LRPPRC. Pharmacological activators and inhibitors of the cellular respiration respectively increased and decreased the [NAD+]/[NADH] ratio, indicating respiration-mediated control of the [NAD+]/[NADH] ratio. Supporting a prominent role for NAD+, increasing the concentration of NAD+ stimulated complete oxidation of fatty acids. Importantly, NAD+ rescued impaired fatty acid oxidation in hepatocytes deficient for either OXPHOS or SIRT3. These data are consistent with a model whereby augmented hepatic OXPHOS increases NAD+, which in turn promotes complete oxidation of fatty acids and protects against NAFLD. PMID:25933096

  10. Human heat shock protein (Hsp) 90 interferes with Neisseria meningitidis adhesin A (NadA)-mediated adhesion and invasion.

    PubMed

    Montanari, Paolo; Bozza, Giuseppe; Capecchi, Barbara; Caproni, Elena; Barrile, Riccardo; Norais, Nathalie; Capitani, Mirco; Sallese, Michele; Cecchini, Paola; Ciucchi, Laura; Gao, Zhenai; Rappuoli, Rino; Pizza, Mariagrazia; Aricò, Beatrice; Merola, Marcello

    2012-03-01

    NadA (N eisseria meningitidisadhesin A), a meningococcal surface protein, mediates adhesion to and invasion of human cells, an activity in which host membrane proteins have been implicated. While investigating these host factors in human epithelial cells by affinity chromatography, we discovered an unanticipated interaction of NadA with heat shock protein (Hsp) 90, a molecular chaperone. The specific in vitro interaction of recombinant soluble NadA and Hsp90 was confirmed by co-immunoprecipitations, dot and far-Western blot. Intriguingly, ADP, but not ATP, was required for this association, and the Hsp90 inhibitor 17-AAG promoted complex formation. Hsp90 binding to an Escherichia coli strain used as carrier to express surface exposed NadA confirmed these results in live bacteria. We also examined RNA interference, plasmid-driven overexpression, addition of exogenous rHsp90 and 17-AAG inhibition in human epithelial cells to further elucidate the involvement of Hsp90 in NadA-mediated adhesion and invasion. Together, these data suggest an inverse correlation between the amount of host Hsp90 and the NadA adhesive/invasive phenotype. Confocal microscopy also demonstrated that meningococci interact with cellular Hsp90, a completely novel finding. Altogether our results show that variation of host Hsp90 expression or activity interferes with adhesive and invasive events driven by NadA. © 2011 Blackwell Publishing Ltd.

  11. Novel assay for simultaneous measurement of pyridine mononucleotides synthesizing activities allows dissection of the NAD(+) biosynthetic machinery in mammalian cells.

    PubMed

    Zamporlini, Federica; Ruggieri, Silverio; Mazzola, Francesca; Amici, Adolfo; Orsomando, Giuseppe; Raffaelli, Nadia

    2014-11-01

    The redox coenzyme NAD(+) is also a rate-limiting co-substrate for several enzymes that consume the molecule, thus rendering its continuous re-synthesis indispensable. NAD(+) biosynthesis has emerged as a therapeutic target due to the relevance of NAD(+) -consuming reactions in complex intracellular signaling networks whose alteration leads to many neurologic and metabolic disorders. Distinct metabolic routes, starting from various precursors, are known to support NAD(+) biosynthesis with tissue/cell-specific efficiencies, probably reflecting differential expression of the corresponding rate-limiting enzymes, i.e. nicotinamide phosphoribosyltransferase, quinolinate phosphoribosyltransferase, nicotinate phosphoribosyltransferase and nicotinamide riboside kinase. Understanding the contribution of these enzymes to NAD(+) levels depending on the tissue/cell type and metabolic status is necessary for the rational design of therapeutic strategies aimed at modulating NAD(+) availability. Here we report a simple, fast and sensitive coupled fluorometric assay that enables simultaneous determination of the four activities in whole-cell extracts and biological fluids. Its application to extracts from various mouse tissues, human cell lines and plasma yielded for the first time an overall picture of the tissue/cell-specific distribution of the activities of the various enzymes. The screening enabled us to gather novel findings, including (a) the presence of quinolinate phosphoribosyltransferase and nicotinamide riboside kinase in all examined tissues/cell lines, indicating that quinolinate and nicotinamide riboside are relevant NAD(+) precursors, and (b) the unexpected occurrence of nicotinate phosphoribosyltransferase in human plasma.

  12. A mutation in At-nMat1a, which encodes a nuclear gene having high similarity to group II intron maturase, causes impaired splicing of mitochondrial NAD4 transcript and altered carbon metabolism in Arabidopsis thaliana.

    PubMed

    Nakagawa, Naoki; Sakurai, Naoki

    2006-06-01

    To elucidate the mechanism of cellulose synthesis, we isolated a mutant of Arabidopsis (changed sensitivity to cellulose synthesis inhibitors 1, css1) that showed changed sensitivity to cellulose biosynthesis inhibitor. The analysis of phenotypes indicated that the css1 mutation influenced various fundamental metabolic pathways including amino acid metabolism, triacylglycerol degradation and polysaccharide synthesis (cellulose and starch) during the early stage of plant growth. Unexpectedly, the map-based cloning of the gene responsible for the css1 mutation identified a protein (At-nMat1a) that was assumed to be a splicing factor of the mitochondrial group II intron. In accordance with this result, this mutant exhibited improper splicing of the mitochondrial NAD4 transcript. We noticed that the phenotypes of the css1 mutant are similar to the responses to anoxia that hinders mitochondrial aerobic respiration. It seems that the defect in the function of mitochondria influences various aspects of fundamental cellular metabolism including cellulose synthesis. Our results suggested that sucrose synthase (SuSy), an enzyme involved in the biosynthesis of cellulose, plays key roles in the connection between mitochondria and cellulose synthesis. The isolation of the css1 mutant also provides a useful resource in the study of post-transcriptional gene regulation in mitochondria.

  13. Synthesis of ST7612AA1, a Novel Oral HDAC Inhibitor, via Radical 
Thioacetic Acid Addition

    PubMed Central

    Battistuzzi, Gianfranco; Giannini, Giuseppe

    2016-01-01

    Abstract: Background In the expanding field of anticancer drugs, HDAC inhibitors are playing an increasingly important role. To date, four/five HDAC inhibitors have been approved by FDA. All these compounds fit the widely accepted HDAC inhibitors pharmacophore model characterized by a cap group, a linker chain and a zinc binding group (ZBG), able to bind the Zn2+ ion in a pocket of the HDAC active site. Romidepsin, a natural compound, is the only thiol derivative. We have selected a new class of synthetic HDAC inhibitors, the thio-ω(lactam-carboxamide) derivatives, with ST7612AA1 as drug candidate, pan-inhibitor active in the range of single- to two-digit nanomolar concentrations. Preliminary results of a synthetic optimization attempt towards a fast scale-up process are here proposed. Methods In the four steps of synthesis, from unsaturated amino acid intermediate to the final product, we explored different synthetic conditions in order to have a transferable process for a scale-up synthetic laboratory. Results In the first step, isobutyl chloroformate was used and, after a simple work up with 1M HCl, 2 (96% yield) was obtained as a white solid, which was used directly in the next step. For thioacetic acid addition to the double bond of intermediate 2, two different routes were possible, with addition reaction in the first (D’) or last step (D). Reactions of 2 to give 5 or of 4 to give ST7612AA1 were both performed in dioxane. Reactions were fast and did not need the usually advised radical quenching with cyclohexene. The corresponding products were obtained in good yields (step D’, 89%; step D, 81%) after a flash chromatography. Conclusion: ST7612AA1 , a thiol derivative prodrug of ST7464AA1, is the first of a new generation of HDAC inhibitors, very potent, orally administered, and well tolerated. Here, we have identified a synthetic route, competitive, versatile and easily transferable to industrial processes. PMID:27917100

  14. Reduced estradiol synthesis by letrozole, an aromatase inhibitor, is protective against development of pentylenetetrazole-induced kindling in mice.

    PubMed

    Rashid, Davood; Panda, B P; Vohora, Divya

    2015-11-01

    Neurosteroids, such as testosterone and their metabolites, are known to modulate neuronal excitability. The enzymes regulating the metabolism of these neurosteroids, thus, may be targeted as a noval strategy for the development of new antiepileptic drugs. The present work targeted two such enzymes i,e aromatase and 5α-reductase in order to explore the potential of letrozole (an aromatase inhibitor) on pentylenetetrazole (PTZ)-induced kindling in mice and the ability of finasteride (a 5α-reductase inhibitor) to modulate any such effects. PTZ (30 mg/kg, i.p.), when administered once every two days (for a total of 24 doses) induced kindling in Swiss albino mice. Letrozole (1 mg/kg, p.o.), administered prior to PTZ, significantly reduced the % incidence of kindling, delayed mean onset time of seizures and reduced seizure severity score. Letrozole reduced the levels of plasma 17β-estradiol after induction of kindling. The concurrent administration of finasteride and letrozole produced effects similar to letrozole on PTZ-kindling and on estradiol levels. This implies that the ability of letrozole to redirect the synthesis of dihydrotestosterone (DHT) and 5α-androstanediol from testosterone doesn't appear to play a significant role in the protective effects of letrozole against PTZ kindling. Letrozole, however, increased the levels of 5α-DHT in mice plasma. The aromatase inhibitors, thus, may be exploited for inhibiting the synthesis of proconvulsant (17β-estradiol) and/or redirecting the synthesis of anticonvulsant (DHT and 5α-androstanediol) neurosteroids.

  15. Inhibitors of HIV-protease from computational design. A history of theory and synthesis still to be fully appreciated.

    PubMed

    Berti, Federico; Frecer, Vladimir; Miertus, Stanislav

    2014-01-01

    Despite the fact that HIV-Protease is an over 20 years old target, computational approaches to rational design of its inhibitors still have a great potential to stimulate the synthesis of new compounds and the discovery of new, potent derivatives, ever capable to overcome the problem of drug resistance. This review deals with successful examples of inhibitors identified by computational approaches, rather than by knowledge-based design. Such methodologies include the development of energy and scoring functions, docking protocols, statistical models, virtual combinatorial chemistry. Computations addressing drug resistance, and the development of related models as the substrate envelope hypothesis are also reviewed. In some cases, the identified structures required the development of synthetic approaches in order to obtain the desired target molecules; several examples are reported.

  16. Synthesis and biological evaluation of phosphate prodrugs of 4-phospho-D-erythronohydroxamic acid, an inhibitor of 6-phosphogluconate dehydrogenase.

    PubMed

    Ruda, Gian Filippo; Alibu, Vincent P; Mitsos, Christos; Bidet, Olivier; Kaiser, Marcel; Brun, Reto; Barrett, Michael P; Gilbert, Ian H

    2007-08-01

    We have previously reported the discovery of potent and selective inhibitors of 6-phosphogluconate dehydrogenase, the third enzyme of the phosphate pentose pathway, from Trypanosoma brucei, the causative organism of human African trypanosomiasis. These inhibitors were charged phosphate derivatives with restricted capacity to enter cells. Herein, we report the synthesis of five different classes of prodrugs: phosphoramidate; bis-S-acyl thioethyl esters (bis-SATE); bis-pivaloxymethyl (bis-POM); CycloSaligenyl; and phenyl, S-acyl thioethyl mixed phosphate esters (mix-SATE). Prodrugs were studied for stability and activity against the intact parasites. Most prodrugs caused inhibition of the growth of the parasites. The activity of the prodrugs against the parasites appeared to be related to their stability in aqueous buffer.

  17. Potential New H1N1 Neuraminidase Inhibitors from Ferulic Acid and Vanillin: Molecular Modelling, Synthesis and in Vitro Assay

    PubMed Central

    Hariono, Maywan; Abdullah, Nurshariza; Damodaran, K.V.; Kamarulzaman, Ezatul E.; Mohamed, Nornisah; Hassan, Sharifah Syed; Shamsuddin, Shaharum; Wahab, Habibah A.

    2016-01-01

    We report the computational and experimental efforts in the design and synthesis of novel neuraminidase (NA) inhibitors from ferulic acid and vanillin. Two proposed ferulic acid analogues, MY7 and MY8 were predicted to inhibit H1N1 NA using molecular docking. From these two analogues, we designed, synthesised and evaluated the biological activities of a series of ferulic acid and vanillin derivatives. The enzymatic H1N1 NA inhibition assay showed MY21 (a vanillin derivative) has the lowest IC50 of 50 μM. In contrast, the virus inhibition assay showed MY15, a ferulic acid derivative has the best activity with the EC50 of ~0.95 μM. Modelling studies further suggest that these predicted activities might be due to the interactions with conserved and essential residues of NA with ΔGbind values comparable to those of oseltamivir and zanamivir, the two commercial NA inhibitors. PMID:27995961

  18. Synthesis and molecular modeling studies of derivatives of a highly potent peptidomimetic vinyl ester as falcipain-2 inhibitors.

    PubMed

    Ettari, Roberta; Micale, Nicola; Grazioso, Giovanni; Bova, Floriana; Schirmeister, Tanja; Grasso, Silvana; Zappalà, Maria

    2012-09-01

    Herein we report the synthesis of a set of constrained peptidomimetics endowed with an electrophilic vinyl ester warhead and structurally related to a previously identified lead compound, a potent and irreversible inhibitor of falcipain-2 (FP-2). FP-2 is the main hemoglobinase of the malaria parasite P. falciparum. The new compounds were evaluated for their inhibition against FP-2, and the results were rationalized on the basis of docking experiments. These studies underscore the pivotal role of both the ester function at the P1' site and the trifluoromethyl group of the P3 side chain in determining the correct orientation of the Michael acceptor warhead in the catalytic site, and as a consequence, the potency of the inhibitors as well as their reversible or irreversible mode of inhibition.

  19. Potential New H1N1 Neuraminidase Inhibitors from Ferulic Acid and Vanillin: Molecular Modelling, Synthesis and in Vitro Assay.

    PubMed

    Hariono, Maywan; Abdullah, Nurshariza; Damodaran, K V; Kamarulzaman, Ezatul E; Mohamed, Nornisah; Hassan, Sharifah Syed; Shamsuddin, Shaharum; Wahab, Habibah A

    2016-12-20

    We report the computational and experimental efforts in the design and synthesis of novel neuraminidase (NA) inhibitors from ferulic acid and vanillin. Two proposed ferulic acid analogues, MY7 and MY8 were predicted to inhibit H1N1 NA using molecular docking. From these two analogues, we designed, synthesised and evaluated the biological activities of a series of ferulic acid and vanillin derivatives. The enzymatic H1N1 NA inhibition assay showed MY21 (a vanillin derivative) has the lowest IC50 of 50 μM. In contrast, the virus inhibition assay showed MY15, a ferulic acid derivative has the best activity with the EC50 of ~0.95 μM. Modelling studies further suggest that these predicted activities might be due to the interactions with conserved and essential residues of NA with ΔGbind values comparable to those of oseltamivir and zanamivir, the two commercial NA inhibitors.

  20. Potential New H1N1 Neuraminidase Inhibitors from Ferulic Acid and Vanillin: Molecular Modelling, Synthesis and in Vitro Assay

    NASA Astrophysics Data System (ADS)

    Hariono, Maywan; Abdullah, Nurshariza; Damodaran, K. V.; Kamarulzaman, Ezatul E.; Mohamed, Nornisah; Hassan, Sharifah Syed; Shamsuddin, Shaharum; Wahab, Habibah A.

    2016-12-01

    We report the computational and experimental efforts in the design and synthesis of novel neuraminidase (NA) inhibitors from ferulic acid and vanillin. Two proposed ferulic acid analogues, MY7 and MY8 were predicted to inhibit H1N1 NA using molecular docking. From these two analogues, we designed, synthesised and evaluated the biological activities of a series of ferulic acid and vanillin derivatives. The enzymatic H1N1 NA inhibition assay showed MY21 (a vanillin derivative) has the lowest IC50 of 50 μM. In contrast, the virus inhibition assay showed MY15, a ferulic acid derivative has the best activity with the EC50 of ~0.95 μM. Modelling studies further suggest that these predicted activities might be due to the interactions with conserved and essential residues of NA with ΔGbind values comparable to those of oseltamivir and zanamivir, the two commercial NA inhibitors.

  1. Oxazin-5-Ones as a Novel Class of Penicillin Binding Protein Inhibitors: Design, Synthesis and Structure Activity Relationship.

    PubMed

    Onoabedje, Efeturi Abraham; Ibezim, Akachukwu; Okafor, Sunday Nwankwor; Onoabedje, Ufuoma Shalom; Okoro, Uchechukwu Chris

    2016-01-01

    Penicillin binding proteins (PBPs) are normal constituents of bacterial which are absent in mammalian cells. The theoretical binding modes of known oxazin-5-ones toward the protein were used as a guide to synthesis new inhibitors. Structural studies of protein-ligand complexes revealed that conformational discrepancies of the derivatives in the protein's binding site gave rise to the variation in their inhibition constant which ranged from 68.58 μM to 2.04 mM. Biological assay results further confirmed the antibiotic potencies of the studied compounds. Although the outcome of biological screening does not parallel computational predictions, the results obtained from both methods suggest that the oxazin-5-one derivatives are potential PBP inhibitors, hence interesting antibiotic lead agents.

  2. Oxazin-5-Ones as a Novel Class of Penicillin Binding Protein Inhibitors: Design, Synthesis and Structure Activity Relationship

    PubMed Central

    Onoabedje, Efeturi Abraham; Ibezim, Akachukwu; Okafor, Sunday Nwankwor; Onoabedje, Ufuoma Shalom; Okoro, Uchechukwu Chris

    2016-01-01

    Penicillin binding proteins (PBPs) are normal constituents of bacterial which are absent in mammalian cells. The theoretical binding modes of known oxazin-5-ones toward the protein were used as a guide to synthesis new inhibitors. Structural studies of protein-ligand complexes revealed that conformational discrepancies of the derivatives in the protein’s binding site gave rise to the variation in their inhibition constant which ranged from 68.58 μM to 2.04 mM. Biological assay results further confirmed the antibiotic potencies of the studied compounds. Although the outcome of biological screening does not parallel computational predictions, the results obtained from both methods suggest that the oxazin-5-one derivatives are potential PBP inhibitors, hence interesting antibiotic lead agents. PMID:27749913

  3. Synthesis and Evaluation of Macrocyclic Peptide Aldehydes as Potent and Selective Inhibitors of the 20S Proteasome.

    PubMed

    Wilson, David L; Meininger, Isabel; Strater, Zack; Steiner, Stephanie; Tomlin, Frederick; Wu, Julia; Jamali, Haya; Krappmann, Daniel; Götz, Marion G

    2016-03-10

    This research explores the first design and synthesis of macrocyclic peptide aldehydes as potent inhibitors of the 20S proteasome. Two novel macrocyclic peptide aldehydes based on the ring-size of the macrocyclic natural product TMC-95 were prepared and evaluated as inhibitors of the 20S proteasome. Both compounds inhibited in the low nanomolar range and proved to be selective for the proteasome over other serine and cysteine proteases, particularly when compared to linear analogues with similar amino acid sequences. In HeLa cells, both macrocycles efficiently inhibited activation of nuclear factor-κB (NF-κB) transcription factor by blocking proteasomal degradation of the inhibitor protein IκBα after cytokine stimulation. Due to their covalent mechanism of binding these compounds represent a 1000-fold increase in inhibitory potency over previously reported noncovalently binding TMC-95 analogues. Molecular modeling of the macrocyclic peptides confirms the preference of the large S3 pocket for large, hydrophobic residues and the ability to exploit this to improve selectivity of proteasome inhibitors.

  4. Synthesis and biological evaluation of non-glucose glycoconjugated N-hydroyxindole class LDH inhibitors as anticancer agents

    PubMed Central

    Di Bussolo, Valeria; Calvaresi, Emilia C.; Granchi, Carlotta; Del Bino, Linda; Frau, Ileana; Lang, Maria Chiara Dasso; Tuccinardi, Tiziano; Macchia, Marco; Martinelli, Adriano

    2015-01-01

    Inhibitors of human lactate dehydrogenase A (LDH-A) are promising therapeutic agents against cancer. The development of LDH-A inhibitors that possess cellular activities has so far proved to be particularly challenging, since the enzyme’s active site is narrow and highly polar. In the recent past, we were able to develop a glucose-conjugated N-hydroxyindole-based LDH-A inhibitor designed to exploit the sugar avidity expressed by cancer cells (the Warburg effect). Herein we describe a structural modulation of the sugar moiety of this class of inhibitors, with the insertion of α-D-mannose, β-D-gulose, or β-N-acetyl-D-glucosamine portions in their structures. Their stereospecific chemical synthesis, which involves a substrate-dependent stereospecific glycosylation step, and their biological activity in reducing lactate production and proliferation in cancer cells are reported. Interestingly, the α-D-mannose conjugate displayed the best properties in the cellular assays, demonstrating an efficient antiglycolytic and antiproliferative activity in cancer cells. PMID:26167277

  5. Synthesis and Evaluation of Macrocyclic Peptide Aldehydes as Potent and Selective Inhibitors of the 20S Proteasome

    PubMed Central

    2016-01-01

    This research explores the first design and synthesis of macrocyclic peptide aldehydes as potent inhibitors of the 20S proteasome. Two novel macrocyclic peptide aldehydes based on the ring-size of the macrocyclic natural product TMC-95 were prepared and evaluated as inhibitors of the 20S proteasome. Both compounds inhibited in the low nanomolar range and proved to be selective for the proteasome over other serine and cysteine proteases, particularly when compared to linear analogues with similar amino acid sequences. In HeLa cells, both macrocycles efficiently inhibited activation of nuclear factor-κB (NF-κB) transcription factor by blocking proteasomal degradation of the inhibitor protein IκBα after cytokine stimulation. Due to their covalent mechanism of binding these compounds represent a 1000-fold increase in inhibitory potency over previously reported noncovalently binding TMC-95 analogues. Molecular modeling of the macrocyclic peptides confirms the preference of the large S3 pocket for large, hydrophobic residues and the ability to exploit this to improve selectivity of proteasome inhibitors. PMID:26985310

  6. Design and Synthesis of Hydroxyethylene-Based BACE-1 Inhibitors Incorporating Extended P1 Substituents

    PubMed Central

    Sandgren, Veronica; Bäck, Marcus; Kvarnström, Ingemar; Dahlgren, Anders

    2013-01-01

    Novel BACE-1 inhibitors with a hydroxyethylene central core have been developed. Modified P1´ and extended P1 substituents were incorporated with the aim to explore potential interactions with the S1´ and the S1-S3 pocket, respectively, of BACE-1. Inhibitors were identified displaying IC50 values in the nanomolar range, i.e. 69 nM for the most potent compound. Possible inhibitor interactions with the enzyme are also discussed. PMID:23585822

  7. Generating a Generation of Proteasome Inhibitors: From Microbial Fermentation to Total Synthesis of Salinosporamide A (Marizomib) and Other Salinosporamides

    PubMed Central

    Potts, Barbara C.; Lam, Kin S.

    2010-01-01

    The salinosporamides are potent proteasome inhibitors among which the parent marine-derived natural product salinosporamide A (marizomib; NPI-0052; 1) is currently in clinical trials for the treatment of various cancers. Methods to generate this class of compounds include fermentation and natural products chemistry, precursor-directed biosynthesis, mutasynthesis, semi-synthesis, and total synthesis. The end products range from biochemical tools for probing mechanism of action to clinical trials materials; in turn, the considerable efforts to produce the target molecules have expanded the technologies used to generate them. Here, the full complement of methods is reviewed, reflecting remarkable contributions from scientists of various disciplines over a period of 7 years since the first publication of the structure of 1. PMID:20479958

  8. Synthesis of Highly Selective Submicromolar Microcystin‐Based Inhibitors of Protein Phosphatase (PP)2A over PP1

    PubMed Central

    Fontanillo, Miriam; Zemskov, Ivan; Häfner, Maximilian; Uhrig, Ulrike; Salvi, Francesca; Simon, Bernd; Wittmann, Valentin

    2016-01-01

    Abstract Research and therapeutic targeting of the phosphoserine/threonine phosphatases PP1 and PP2A is hindered by the lack of selective inhibitors. The microcystin (MC) natural toxins target both phosphatases with equal potency, and their complex synthesis has complicated structure–activity relationship studies in the past. We report herein the synthesis and biochemical evaluation of 11 MC analogues, which was accomplished through an efficient strategy combining solid‐ and solution‐phase approaches. Our approach led to the first MC analogue with submicromolar inhibitory potency that is strongly selective for PP2A over PP1 and does not require the complex lipophilic Adda group. Through mutational and structural analyses, we identified a new key element for binding, as well as reasons for the selectivity. This work gives unprecedented insight into how selectivity between these phosphatases can be achieved with MC analogues. PMID:27723199

  9. The design and synthesis of novel SGLT2 inhibitors: C-glycosides with benzyltriazolopyridinone and phenylhydantoin as the aglycone moieties.

    PubMed

    Guo, Cheng; Hu, Min; DeOrazio, Russell J; Usyatinsky, Alexander; Fitzpatrick, Kevin; Zhang, Zhenjun; Maeng, Jun-Ho; Kitchen, Douglas B; Tom, Susan; Luche, Michele; Khmelnitsky, Yuri; Mhyre, Andrew J; Guzzo, Peter R; Liu, Shuang

    2014-07-01

    The sodium glucose co-transporter 2 (SGLT2) has received considerable attention in recent years as a target for the treatment of type 2 diabetes mellitus. This report describes the design, synthesis and structure-activity relationship (SAR) of C-glycosides with benzyltriazolopyridinone and phenylhydantoin as the aglycone moieties as novel SGLT2 inhibitors. Compounds 5p and 33b demonstrated high potency in inhibiting SGLT2 and high selectivity against SGLT1. The in vitro ADMET properties of these compounds will also be discussed.

  10. Synthesis and Biological Evaluation of Macrocyclized Betulin Derivatives as a Novel Class of Anti-HIV-1 Maturation Inhibitors.

    PubMed

    Tang, Jun; Jones, Stacey A; Jeffery, Jerry L; Miranda, Sonia R; Galardi, Cristin M; Irlbeck, David M; Brown, Kevin W; McDanal, Charlene B; Han, Nianhe; Gao, Daxin; Wu, Yongyong; Shen, Bin; Liu, Chunyu; Xi, Caiming; Yang, Heping; Li, Rui; Yu, Yajun; Sun, Yufei; Jin, Zhimin; Wang, Erjuan; Johns, Brian A

    2014-01-01

    A macrocycle provides diverse functionality and stereochemical complexity in a conformationally preorganized ring structure, and it occupies a unique chemical space in drug discovery. However, the synthetic challenge to access this structural class is high and hinders the exploration of macrocycles. In this study, efficient synthetic routes to macrocyclized betulin derivatives have been established. The macrocycle containing compounds showed equal potency compared to bevirimat in multiple HIV-1 antiviral assays. The synthesis and biological evaluation of this novel series of HIV-1 maturation inhibitors will be discussed.

  11. Total synthesis and structural revision of TMG-chitotriomycin, a specific inhibitor of insect and fungal beta-N-acetylglucosaminidases.

    PubMed

    Yang, You; Li, Yao; Yu, Biao

    2009-09-02

    TMG-chitotriomycin, a potent and selective inhibitor of the beta-N-acetylglucosaminidases that possesses an unique N,N,N-trimethyl-d-glucosamine (TMG) residue, is revised to be the TMG-beta-(1-->4)-chitotriose instead of the originally proposed alpha-anomer via its total synthesis, for which a highly convergent approach was developed in which the sterically demanding (1-->4)-glycosidic linkages are efficiently constructed by the Au(I)-catalyzed glycosylation protocol with glycosyl o-hexynylbenzoates as donors.

  12. Nitric oxide degradation by potato tuber mitochondria: evidence for the involvement of external NAD(P)H dehydrogenases.

    PubMed

    de Oliveira, Halley Caixeta; Wulff, Alfredo; Saviani, Elzira Elisabeth; Salgado, Ione

    2008-05-01

    The mechanisms of nitric oxide (NO) synthesis in plants have been extensively investigated. NO degradation can be just as important as its synthesis in controlling steady-state levels of NO. Here, we examined NO degradation in mitochondria isolated from potato tubers and the contribution of the respiratory chain to this process. NO degradation was faster in mitochondria energized with NAD(P)H than with succinate or malate. Oxygen consumption and the inner membrane potential were transiently inhibited by NO in NAD(P)H-energized mitochondria, in contrast to the persistent inhibition seen with succinate. NO degradation was abolished by anoxia and superoxide dismutase, which suggested that NO was consumed by its reaction with superoxide anion (O2(-)). Antimycin-A stimulated and myxothiazol prevented NO consumption in succinate- and malate-energized mitochondria. Although favored by antimycin-A, NAD(P)H-mediated NO consumption was not abolished by myxothiazol, indicating that an additional site of O2(-) generation, besides complex III, stimulated NO degradation. Larger amounts of O2(-) were generated in NAD(P)H- compared to succinate- or malate-energized mitochondria. NAD(P)H-mediated NO degradation and O2(-) production were stimulated by free Ca2+ concentration. Together, these results indicate that Ca2+-dependent external NAD(P)H dehydrogenases, in addition to complex III, contribute to O2(-) production that favors NO degradation in potato tuber mitochondria.

  13. Evidence for two NAD kinases in Salmonella typhimurium.

    PubMed Central

    Cheng, W.; Roth, J. R.

    1994-01-01

    The electron-carrying cofactor NADP is formed by phosphorylation of NAD. A strategy for the isolation of NAD kinase mutants revealed two classes of temperature-sensitive mutations, nadF and nadG, mapping at min 13 and 72 of the Salmonella chromosome. Both mutant types grew on nutrient broth at both 30 and 42 degrees C but on minimal medium showed a temperature-sensitive growth defect which was not corrected by any of the single nutritional supplements tested. A nadF deletion mutant grew on nutrient broth but not on minimal medium. A double mutant with the nadF deletion and a nadG(Ts) mutation showed temperature-sensitive growth on all media. We propose that Salmonella typhimurium has two NAD kinases, one encoded by the nadF and one by the nadG gene. This is supported by the fact that temperature-sensitive mutants of both genes produce kinase activity with altered heat stability. Results suggest that either one of two NAD kinases is sufficient for growth on rich medium, but that both are needed for growth on minimal media. Enzyme assays show that the nadF gene is responsible for about 70% of total NAD kinase activity, and that the nadG gene dictates the remaining 30%. While testing nutritional phenotypes of nadF and nadG mutants, we found that the biosynthetic intermediate, quinolinic acid (QA) inhibited growth of nadF mutants on nutrient broth. This suggested that the NadG enzyme might be inhibited by QA. Enzyme assays demonstrated that QA inhibits the NadG but not the NadF enzyme. This suggests the existence of a regulatory mechanism which controls NADP levels. PMID:8021211

  14. Rapid discovery of a novel series of Abl kinase inhibitors by application of an integrated microfluidic synthesis and screening platform.

    PubMed

    Desai, Bimbisar; Dixon, Karen; Farrant, Elizabeth; Feng, Qixing; Gibson, Karl R; van Hoorn, Willem P; Mills, James; Morgan, Trevor; Parry, David M; Ramjee, Manoj K; Selway, Christopher N; Tarver, Gary J; Whitlock, Gavin; Wright, Adrian G

    2013-04-11

    Drug discovery faces economic and scientific imperatives to deliver lead molecules rapidly and efficiently. Using traditional paradigms the molecular design, synthesis, and screening loops enforce a significant time delay leading to inefficient use of data in the iterative molecular design process. Here, we report the application of a flow technology platform integrating the key elements of structure-activity relationship (SAR) generation to the discovery of novel Abl kinase inhibitors. The platform utilizes flow chemistry for rapid in-line synthesis, automated purification, and analysis coupled with bioassay. The combination of activity prediction using Random-Forest regression with chemical space sampling algorithms allows the construction of an activity model that refines itself after every iteration of synthesis and biological result. Within just 21 compounds, the automated process identified a novel template and hinge binding motif with pIC50 > 8 against Abl kinase--both wild type and clinically relevant mutants. Integrated microfluidic synthesis and screening coupled with machine learning design have the potential to greatly reduce the time and cost of drug discovery within the hit-to-lead and lead optimization phases.

  15. Aflatoxin-Exposure of Vibrio gazogenes as a Novel System for the Generation of Aflatoxin Synthesis Inhibitors.

    PubMed

    Gummadidala, Phani M; Chen, Yung Pin; Beauchesne, Kevin R; Miller, Kristen P; Mitra, Chandrani; Banaszek, Nora; Velez-Martinez, Michelle; Moeller, Peter D R; Ferry, John L; Decho, Alan W; Chanda, Anindya

    2016-01-01

    Aflatoxin is a mycotoxin and a secondary metabolite, and the most potent known liver carcinogen that contaminates several important crops, and represents a significant threat to public health and the economy. Available approaches reported thus far have been insufficient to eliminate this threat, and therefore provide the rational to explore novel methods for preventing aflatoxin accumulation in the environment. Many terrestrial plants and microbes that share ecological niches and encounter the aflatoxin producers have the ability to synthesize compounds that inhibit aflatoxin synthesis. However, reports of natural aflatoxin inhibitors from marine ecosystem components that do not share ecological niches with the aflatoxin producers are rare. Here, we show that a non-pathogenic marine bacterium, Vibrio gazogenes, when exposed to low non-toxic doses of aflatoxin B1, demonstrates a shift in its metabolic output and synthesizes a metabolite fraction that inhibits aflatoxin synthesis without affecting hyphal growth in the model aflatoxin producer, Aspergillus parasiticus. The molecular mass of the predominant metabolite in this fraction was also different from the known prodigiosins, which are the known antifungal secondary metabolites synthesized by this Vibrio. Gene expression analyses using RT-PCR demonstrate that this metabolite fraction inhibits aflatoxin synthesis by down-regulating the expression of early-, middle-, and late- growth stage aflatoxin genes, the aflatoxin pathway regulator, aflR and one global regulator of secondary metabolism, laeA. Our study establishes a novel system for generation of aflatoxin synthesis inhibitors, and emphasizes the potential of the under-explored Vibrio's silent genome for generating new modulators of fungal secondary metabolism.

  16. Aflatoxin-Exposure of Vibrio gazogenes as a Novel System for the Generation of Aflatoxin Synthesis Inhibitors

    PubMed Central

    Gummadidala, Phani M.; Chen, Yung Pin; Beauchesne, Kevin R.; Miller, Kristen P.; Mitra, Chandrani; Banaszek, Nora; Velez-Martinez, Michelle; Moeller, Peter D. R.; Ferry, John L.; Decho, Alan W.; Chanda, Anindya

    2016-01-01

    Aflatoxin is a mycotoxin and a secondary metabolite, and the most potent known liver carcinogen that contaminates several important crops, and represents a significant threat to public health and the economy. Available approaches reported thus far have been insufficient to eliminate this threat, and therefore provide the rational to explore novel methods for preventing aflatoxin accumulation in the environment. Many terrestrial plants and microbes that share ecological niches and encounter the aflatoxin producers have the ability to synthesize compounds that inhibit aflatoxin synthesis. However, reports of natural aflatoxin inhibitors from marine ecosystem components that do not share ecological niches with the aflatoxin producers are rare. Here, we show that a non-pathogenic marine bacterium, Vibrio gazogenes, when exposed to low non-toxic doses of aflatoxin B1, demonstrates a shift in its metabolic output and synthesizes a metabolite fraction that inhibits aflatoxin synthesis without affecting hyphal growth in the model aflatoxin producer, Aspergillus parasiticus. The molecular mass of the predominant metabolite in this fraction was also different from the known prodigiosins, which are the known antifungal secondary metabolites synthesized by this Vibrio. Gene expression analyses using RT-PCR demonstrate that this metabolite fraction inhibits aflatoxin synthesis by down-regulating the expression of early-, middle-, and late- growth stage aflatoxin genes, the aflatoxin pathway regulator, aflR and one global regulator of secondary metabolism, laeA. Our study establishes a novel system for generation of aflatoxin synthesis inhibitors, and emphasizes the potential of the under-explored Vibrio’s silent genome for generating new modulators of fungal secondary metabolism. PMID:27375561

  17. Synthesis and PET studies of [11C-cyano]letrozole (Femara), an aromatase inhibitor drug

    SciTech Connect

    kil K. E.; Biegon A.; Kil, K.-E.; Biegon, A.; Ding, Y.-S.; Fischer, A.; Ferrieri, R.A.; Kim, S.-W.; Pareto, D.; Schueller, M.J.; Fowler, J.S.

    2008-11-10

    Aromatase, a member of the cytochrome P450 family, converts androgens such as androstenedione and testosterone to estrone and estradiol respectively. Letrozole (1-[bis-(4-cyanophenyl)methyl]-1H-1,2,4-triazole, Femara{reg_sign}) is a high affinity aromatase inhibitor (K{sub i}=11.5 nM) which has FDA approval for breast cancer treatment. Here we report the synthesis of carbon-11 labeled letrozole and its assessment as a radiotracer for brain aromatase in the baboon. Letrozole and its precursor (4-[(4-bromophenyl)-1H-1,2,4-triazol-1-ylmethyl]benzonitrile, 3) were prepared in two-step syntheses from 4-cyanobenzyl bromide and 4-bromobenzyl bromide, respectively. The [{sup 11}C]cyano group was introduced via the tetrakis(triphenylphosphine)palladium(0) catalyzed coupling of [{sup 11}C]cyanide with the bromo-precursor (3). PET studies in the baboon brain were carried out to assess regional distribution and kinetics, reproducibility of repeated measures and saturability. The free fraction of letrozole in the plasma, log D, and the [{sup 11}C-cyano]letrozole fraction in the arterial plasma were also measured. [{sup 11}C-cyano]Letrozole was synthesized in 60 min with a radiochemical yield of 79-80%, with a radiochemical purity greater than 98% and a specific activity of 4.16 {+-} 2.21 Ci/{micro}mol at the end of bombardment (n=4). PET studies in the baboon revealed initial rapid and high uptake and initial rapid clearance followed by slow clearance of carbon-11 from the brain with no difference between brain regions. The brain kinetics was not affected by co-injection of unlabeled letrozole (0.1 mg/kg). The free fraction of letrozole in plasma was 48.9% and log D was 1.84. [{sup 11}C-cyano]Letrozole is readily synthesized via a palladium catalyzed coupling reaction with [{sup 11}C]cyanide. Although it is unsuitable as a PET radiotracer for brain aromatase as revealed by the absence of regional specificity and saturability in brain regions, such as amygdala, which are known

  18. Hepatic and nonhepatic sterol synthesis and tissue distribution following administration of a liver selective HMG-CoA reductase inhibitor, CI-981: comparison with selected HMG-CoA reductase inhibitors.

    PubMed

    Bocan, T M; Ferguson, E; McNally, W; Uhlendorf, P D; Bak Mueller, S; Dehart, P; Sliskovic, D R; Roth, B D; Krause, B R; Newton, R S

    1992-01-24

    Since cholesterol biosynthesis is an integral part of cellular metabolism, several HMG-CoA reductase inhibitors were systematically analyzed in in vitro, ex vivo and in vivo sterol synthesis assays using [14C]acetate incorporation into digitonin precipitable sterols as a marker of cholesterol synthesis. Tissue distribution of radiolabeled CI-981 and lovastatin was also performed. In vitro, CI-981 and PD134967-15 were equipotent in liver, spleen, testis and adrenal, lovastatin was more potent in extrahepatic tissues than liver and BMY21950, pravastatin and PD135023-15 were more potent in liver than peripheral tissues. In ex vivo assays, all inhibitors except lovastatin preferentially inhibited liver sterol synthesis; however, pravastatin and BMY22089 were strikingly less potent in the liver. CI-981 inhibited sterol synthesis in vivo in the liver, spleen and adrenal while not affecting the testis, kidney, muscle and brain. Lovastatin inhibited sterol synthesis to a greater extent than CI-981 in the spleen, adrenal and kidney while pravastatin and BMY22089 primarily affected liver and kidney. The tissue distribution of radiolabeled CI-981 and lovastatin support the changes observed in tissue sterol synthesis. Thus, we conclude that a spectrum of liver selective HMG-CoA reductase inhibitors exist and that categorizing agents as liver selective is highly dependent upon method of analysis.

  19. Mycofactocin-associated mycobacterial dehydrogenases with non-exchangeable NAD cofactors

    PubMed Central

    Haft, Daniel H.; Pierce, Phillip G.; Mayclin, Stephen J.; Sullivan, Amy; Gardberg, Anna S.; Abendroth, Jan; Begley, Darren W.; Phan, Isabelle Q.; Staker, Bart L.; Myler, Peter J.; Marathias, Vasilios M.; Lorimer, Donald D.; Edwards, Thomas E.

    2017-01-01

    During human infection, Mycobacterium tuberculosis (Mtb) survives the normally bacteriocidal phagosome of macrophages. Mtb and related species may be able to combat this harsh acidic environment which contains reactive oxygen species due to the mycobacterial genomes encoding a large number of dehydrogenases. Typically, dehydrogenase cofactor binding sites are open to solvent, which allows NAD/NADH exchange to support multiple turnover. Interestingly, mycobacterial short chain dehydrogenases/reductases (SDRs) within family TIGR03971 contain an insertion at the NAD binding site. Here we present crystal structures of 9 mycobacterial SDRs in which the insertion buries the NAD cofactor except for a small portion of the nicotinamide ring. Line broadening and STD-NMR experiments did not show NAD or NADH exchange on the NMR timescale. STD-NMR demonstrated binding of the potential substrate carveol, the potential product carvone, the inhibitor tricyclazol, and an external redox partner 2,6-dichloroindophenol (DCIP). Therefore, these SDRs appear to contain a non-exchangeable NAD cofactor and may rely on an external redox partner, rather than cofactor exchange, for multiple turnover. Incidentally, these genes always appear in conjunction with the mftA gene, which encodes the short peptide MftA, and with other genes proposed to convert MftA into the external redox partner mycofactocin. PMID:28120876

  20. The Sensitivity of Memory Consolidation and Reconsolidation to Inhibitors of Protein Synthesis and Kinases: Computational Analysis

    ERIC Educational Resources Information Center

    Zhang, Yili; Smolen, Paul; Baxter, Douglas A.; Byrne, John H.

    2010-01-01

    Memory consolidation and reconsolidation require kinase activation and protein synthesis. Blocking either process during or shortly after training or recall disrupts memory stabilization, which suggests the existence of a critical time window during which these processes are necessary. Using a computational model of kinase synthesis and…

  1. The Sensitivity of Memory Consolidation and Reconsolidation to Inhibitors of Protein Synthesis and Kinases: Computational Analysis

    ERIC Educational Resources Information Center

    Zhang, Yili; Smolen, Paul; Baxter, Douglas A.; Byrne, John H.

    2010-01-01

    Memory consolidation and reconsolidation require kinase activation and protein synthesis. Blocking either process during or shortly after training or recall disrupts memory stabilization, which suggests the existence of a critical time window during which these processes are necessary. Using a computational model of kinase synthesis and…

  2. Inhibition of thyrotropin-stimulated DNA synthesis by microinjection of inhibitors of cellular Ras and cyclic AMP-dependent protein kinase.

    PubMed Central

    Kupperman, E; Wen, W; Meinkoth, J L

    1993-01-01

    Microinjection of a dominant interfering mutant of Ras (N17 Ras) caused a significant reduction in thyrotropin (thyroid-stimulating hormone [TSH])-stimulated DNA synthesis in rat thyroid cells. A similar reduction was observed following injection of the heat-stable protein kinase inhibitor of the cyclic AMP-dependent protein kinase. Coinjection of both inhibitors almost completely abolished TSH-induced DNA synthesis. In contrast to TSH, overexpression of cellular Ras protein did not stimulate the expression of a cyclic AMP response element-regulated reporter gene. Similarly, injection of N17 Ras had no effect on TSH-stimulated reporter gene expression. Moreover, overexpression of cellular Ras protein stimulated similar levels of DNA synthesis in the presence or absence of the heat-stable protein kinase inhibitor. Together, these results suggest that in Wistar rat thyroid cells, a full mitogenic response to TSH requires both Ras and cyclic APK-dependent protein kinase. Images PMID:8336696

  3. Inhibition of thyrotropin-stimulated DNA synthesis by microinjection of inhibitors of cellular Ras and cyclic AMP-dependent protein kinase.

    PubMed

    Kupperman, E; Wen, W; Meinkoth, J L

    1993-08-01

    Microinjection of a dominant interfering mutant of Ras (N17 Ras) caused a significant reduction in thyrotropin (thyroid-stimulating hormone [TSH])-stimulated DNA synthesis in rat thyroid cells. A similar reduction was observed following injection of the heat-stable protein kinase inhibitor of the cyclic AMP-dependent protein kinase. Coinjection of both inhibitors almost completely abolished TSH-induced DNA synthesis. In contrast to TSH, overexpression of cellular Ras protein did not stimulate the expression of a cyclic AMP response element-regulated reporter gene. Similarly, injection of N17 Ras had no effect on TSH-stimulated reporter gene expression. Moreover, overexpression of cellular Ras protein stimulated similar levels of DNA synthesis in the presence or absence of the heat-stable protein kinase inhibitor. Together, these results suggest that in Wistar rat thyroid cells, a full mitogenic response to TSH requires both Ras and cyclic APK-dependent protein kinase.

  4. In vivo NAD assay reveals the intracellular NAD contents and redox state in healthy human brain and their age dependences.

    PubMed

    Zhu, Xiao-Hong; Lu, Ming; Lee, Byeong-Yeul; Ugurbil, Kamil; Chen, Wei

    2015-03-03

    NAD is an essential metabolite that exists in NAD(+) or NADH form in all living cells. Despite its critical roles in regulating mitochondrial energy production through the NAD(+)/NADH redox state and modulating cellular signaling processes through the activity of the NAD(+)-dependent enzymes, the method for quantifying intracellular NAD contents and redox state is limited to a few in vitro or ex vivo assays, which are not suitable for studying a living brain or organ. Here, we present a magnetic resonance (MR) -based in vivo NAD assay that uses the high-field MR scanner and is capable of noninvasively assessing NAD(+) and NADH contents and the NAD(+)/NADH redox state in intact human brain. The results of this study provide the first insight, to our knowledge, into the cellular NAD concentrations and redox state in the brains of healthy volunteers. Furthermore, an age-dependent increase of intracellular NADH and age-dependent reductions in NAD(+), total NAD contents, and NAD(+)/NADH redox potential of the healthy human brain were revealed in this study. The overall findings not only provide direct evidence of declined mitochondrial functions and altered NAD homeostasis that accompany the normal aging process but also, elucidate the merits and potentials of this new NAD assay for noninvasively studying the intracellular NAD metabolism and redox state in normal and diseased human brain or other organs in situ.

  5. In vivo NAD assay reveals the intracellular NAD contents and redox state in healthy human brain and their age dependences

    PubMed Central

    Zhu, Xiao-Hong; Lu, Ming; Lee, Byeong-Yeul; Ugurbil, Kamil; Chen, Wei

    2015-01-01

    NAD is an essential metabolite that exists in NAD+ or NADH form in all living cells. Despite its critical roles in regulating mitochondrial energy production through the NAD+/NADH redox state and modulating cellular signaling processes through the activity of the NAD+-dependent enzymes, the method for quantifying intracellular NAD contents and redox state is limited to a few in vitro or ex vivo assays, which are not suitable for studying a living brain or organ. Here, we present a magnetic resonance (MR) -based in vivo NAD assay that uses the high-field MR scanner and is capable of noninvasively assessing NAD+ and NADH contents and the NAD+/NADH redox state in intact human brain. The results of this study provide the first insight, to our knowledge, into the cellular NAD concentrations and redox state in the brains of healthy volunteers. Furthermore, an age-dependent increase of intracellular NADH and age-dependent reductions in NAD+, total NAD contents, and NAD+/NADH redox potential of the healthy human brain were revealed in this study. The overall findings not only provide direct evidence of declined mitochondrial functions and altered NAD homeostasis that accompany the normal aging process but also, elucidate the merits and potentials of this new NAD assay for noninvasively studying the intracellular NAD metabolism and redox state in normal and diseased human brain or other organs in situ. PMID:25730862

  6. Design and synthesis of thiazolo[5,4-f]quinazolines as DYRK1A inhibitors, part II.

    PubMed

    Foucourt, Alicia; Hédou, Damien; Dubouilh-Benard, Carole; Girard, Angélique; Taverne, Thierry; Casagrande, Anne-Sophie; Désiré, Laurent; Leblond, Bertrand; Besson, Thierry

    2014-09-26

    The convenient synthesis of a focused library (forty molecules) of novel 6,6,5-tricyclic thiazolo[5,4-f]quinazolines was realized mainly under microwave irradiation. A novel 6-aminobenzo[d]thiazole-2,7-dicarbonitrile (1) was used as a versatile molecular platform for the synthesis of various derivatives. Kinase inhibition, of the obtained final compounds, was evaluated on a panel of two kinases (DYRK1A/1B) together with some known reference DYRK1A and DYRK1B inhibitors (harmine, TG003, NCGC-00189310 and leucettine L41). Compound IC50 values were obtained and compared. Five of the novel thiazolo[5,4-f]quinazoline derivatives prepared, EHT 5372 (8c), EHT 6840 (8h), EHT 1610 (8i), EHT 9851 (8k) and EHT 3356 (9b) displayed single-digit nanomolar or subnanomolar IC50 values and are among the most potent DYRK1A/1B inhibitors disclosed to date. DYRK1A/1B kinases are known to be involved in the regulation of various molecular pathways associated with oncology, neurodegenerative diseases (such as Alzheimer disease, AD, or other tauopathies), genetic diseases (such as Down Syndrome, DS), as well as diseases involved in abnormal pre-mRNA splicing. The compounds described in this communication constitute a highly potent set of novel molecular probes to evaluate the biology/pharmacology of DYR1A/1B in such diseases.

  7. Programmed cell death in plants: effect of protein synthesis inhibitors and structural changes in pea guard cells.

    PubMed

    Dzyubinskaya, E V; Kiselevsky, D B; Bakeeva, L E; Samuilov, V D

    2006-04-01

    Pea leaf epidermis incubated with cyanide displayed ultrastructural changes in guard cells that are typical of apoptosis. Cycloheximide, an inhibitor of cytoplasmic protein synthesis, and lincomycin, an inhibitor of protein synthesis in chloroplasts and mitochondria, produced different effects on the dynamics of programmed death of guard cells. According to light microscopy data, cycloheximide reinforced and lincomycin suppressed the CN(-)-induced destruction of cell nuclei. Lincomycin lowered the effect of cycloheximide in the light and prevented it in the dark. According to electron microscopy data, the most pronounced effects of cycloheximide in the presence of cyanide were autophagy and a lack of apoptotic condensation of nuclear chromatin, the prevention of chloroplast envelope rupturing and its invagination inside the stroma, and the appearance of particular compartments with granular inclusions in mitochondria. Lincomycin inhibited the CN(-)-induced ultrastructural changes in guard cell nuclei. The data show that programmed death of guard cells may have a combined scenario involving both apoptosis and autophagy and may depend on the action of both cytoplasm synthesized and chloroplast and mitochondrion synthesized proteins.

  8. Discovery of potent KIFC1 inhibitors using a method of integrated high-throughput synthesis and screening.

    PubMed

    Yang, Bin; Lamb, Michelle L; Zhang, Tao; Hennessy, Edward J; Grewal, Gurmit; Sha, Li; Zambrowski, Mark; Block, Michael H; Dowling, James E; Su, Nancy; Wu, Jiaquan; Deegan, Tracy; Mikule, Keith; Wang, Wenxian; Kaspera, Rüdiger; Chuaqui, Claudio; Chen, Huawei

    2014-12-11

    KIFC1 (HSET), a member of the kinesin-14 family of motor proteins, plays an essential role in centrosomal bundling in cancer cells, but its function is not required for normal diploid cell division. To explore the potential of KIFC1 as a therapeutic target for human cancers, a series of potent KIFC1 inhibitors featuring a phenylalanine scaffold was developed from hits identified through high-throughput screening (HTS). Optimization of the initial hits combined both design-synthesis-test cycles and an integrated high-throughput synthesis and biochemical screening method. An important aspect of this integrated method was the utilization of DMSO stock solutions of compounds registered in the corporate compound collection as synthetic reactants. Using this method, over 1500 compounds selected for structural diversity were quickly assembled in assay-ready 384-well plates and were directly tested after the necessary dilutions. Our efforts led to the discovery of a potent KIFC1 inhibitor, AZ82, which demonstrated the desired centrosome declustering mode of action in cell studies.

  9. Design, Synthesis and Evaluation of 2,5-Diketopiperazines as Inhibitors of the MDM2-p53 Interaction.

    PubMed

    Pettersson, Mariell; Quant, Maria; Min, Jaeki; Iconaru, Luigi; Kriwacki, Richard W; Waddell, M Brett; Guy, R Kiplin; Luthman, Kristina; Grøtli, Morten

    2015-01-01

    The transcription factor p53 is the main tumour suppressor in cells and many cancer types have p53 mutations resulting in a loss of its function. In tumours that retain wild-type p53 function, p53 activity is down-regulated by MDM2 (human murine double minute 2) via a direct protein-protein interaction. We have designed and synthesised two series of 2,5-diketopiperazines as inhibitors of the MDM2-p53 interaction. The first set was designed to directly mimic the α-helical region of the p53 peptide, containing key residues in the i, i+4 and i+7 positions of a natural α-helix. Conformational analysis indicated that 1,3,6-trisubstituted 2,5-diketopiperazines were able to place substituents in the same spatial orientation as an α-helix template. The key step of the synthesis involved the cyclisation of substituted dipeptides. The other set of tetrasubstituted 2,5-diketopiperazines were designed based on structure-based docking studies and the Ugi multicomponent reaction was used for the synthesis. This latter set comprised the most potent inhibitors which displayed micromolar IC50-values in a biochemical fluorescence polarisation assay.

  10. I. Development of Metal-Mediated SPOT-Synthesis Methods for the Efficient Construction of Small-Molecule Macroarrays. II. Design and Synthesis of Novel Bacterial Biofilm Inhibitors

    NASA Astrophysics Data System (ADS)

    Frei, Reto

    I. The use of small molecule probes to explore biological phenomena has become a valuable tool in chemical biology. As a result, methods that permit the rapid synthesis and biological evaluation of such compounds are highly sought-after. The small molecule macroarray represents one such approach for the synthesis and identification of novel bioactive agents. Macroarrays are readily constructed via the SPOT-synthesis technique on planar cellulose membranes, yielding spatially addressed libraries of ˜10-1000 unique compounds. We sought to expand the arsenal of chemical reactions compatible with this solid-phase platform, and developed highly efficient SPOT-synthesis protocols for the Mizoroki-Heck, Suzuki-Miyaura, and copper-catalyzed azide-alkyne cycloaddition reaction. We demonstrated that these metal-mediated reactions can be implemented, either individually or sequentially, for the efficient construction of small molecules in high purity on rapid time scales. Utilizing these powerful C-C and C-N bond forming coupling reactions, we constructed a series of macroarrays based on novel stilbene, phenyl-naphthalene, and triazole scaliblds. Subsequent biological testing of the stilbene and phenyl-naphthalene libraries revealed several potent antagonists and agonists, respectively, of the quorum sensing (QS) receptor LuxR in Vibrio fischeri. II. Bacteria living within biofilms are notorious for their resistance to known antibiotic agents, and constitute a major human health threat. Methods to attenuate biofilm growth would have a significant impact on the management of bacterial infections. Despite intense research efforts, small molecules capable of either inhibiting or dispersing biolilms remain scarce. We utilized natural products with purported anti-biofilm or QS inhibitory activity as sources of structural insight to guide the synthesis of novel biofilm modulators with improved activities. These studies revealed 2-aminobenzimidazole derivatives as highly potent

  11. Synthesis and optimization of N-heterocyclic pyridinones as catechol-O-methyltransferase (COMT) inhibitors.

    PubMed

    Zhao, Zhijian; Harrison, Scott T; Schubert, Jeffrey W; Sanders, John M; Polsky-Fisher, Stacey; Zhang, Nanyan Rena; McLoughlin, Debra; Gibson, Christopher R; Robinson, Ronald G; Sachs, Nancy A; Kandebo, Monika; Yao, Lihang; Smith, Sean M; Hutson, Pete H; Wolkenberg, Scott E; Barrow, James C

    2016-06-15

    A series of N-heterocyclic pyridinone catechol-O-methyltransferase (COMT) inhibitors were synthesized. Physicochemical properties, including ligand lipophilic efficiency (LLE) and clogP, were used to guide compound design and attempt to improve inhibitor pharmacokinetics. Incorporation of heterocyclic central rings provided improvements in physicochemical parameters but did not significantly reduce in vitro or in vivo clearance. Nevertheless, compound 11 was identified as a potent inhibitor with sufficient in vivo exposure to significantly affect the dopamine metabolites homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC), and indicate central COMT inhibition.

  12. Synthesis and evaluation of potential inhibitors of human and Escherichia coli histidine triad nucleotide binding proteins.

    PubMed

    Bardaweel, Sanaa K; Ghosh, Brahma; Wagner, Carston R

    2012-01-01

    Based on recent substrate specificity studies, a series of ribonucleotide based esters and carbamates were synthesized and screened as inhibitors of the phosphoramidases and acyl-AMP hydrolases, Escherichia coli Histidine Triad Nucleotide Binding Protein (ecHinT) and human Histidine Triad Nucleotide Binding Protein 1 (hHint1). Using our established phosphoramidase assay, K(i) values were determined. All compounds exhibited non-competitive inhibition profiles. The carbamate based inhibitors were shown to successfully suppress the Hint1-associated phenotype in E. coli, suggesting that they are permeable intracellular inhibitors of ecHinT.

  13. Genetic structure of the nadA and nadB antivirulence loci in Shigella spp.

    PubMed

    Prunier, Anne-Laure; Schuch, Raymond; Fernández, Reinaldo E; Maurelli, Anthony T

    2007-09-01

    Comparison of nadA and nadB in 14 Shigella strains and enteroinvasive Escherichia coli versus E. coli showed that at least one locus is altered in all strains. These observations explain the characteristic nicotinic acid auxotrophy of Shigella organisms and are consistent with the previously identified antivirulence nature of these genes for these pathogens.

  14. Salmonella typhimurium nit Is nadE: Defective Nitrogen Utilization and Ammonia-Dependent NAD Synthetase

    PubMed Central

    Schneider, Barbara L.; Reitzer, Lawrence J.

    1998-01-01

    S. typhimurium nit mutants are defective in nitrogen assimilation, despite having normal levels of assimilatory enzymes. Complementation, enzyme assays, and genetic mapping show that nit is nadE. We present evidence that ammonia, not glutamine, is the physiological substrate for eubacterial NAD synthetases and that low activity completely accounts for the mutant phenotype. PMID:9721319

  15. Toward analogues of MraY natural inhibitors: synthesis of 5'-triazole-substituted-aminoribosyl uridines through a Cu-catalyzed azide-alkyne cycloaddition.

    PubMed

    Fer, Mickaël J; Olatunji, Samir; Bouhss, Ahmed; Calvet-Vitale, Sandrine; Gravier-Pelletier, Christine

    2013-10-18

    A straightforward strategy for the synthesis of triazole-containing MraY inhibitors has been developed. It involves the sequential introduction of a terminal alkyne at the 5' position of an uridine derivative and O-glycosylation with a protected aminoribose leading to an elaborated alkyne scaffold. An efficient Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) allowed the introduction of chemical diversity toward a small library of inhibitors.

  16. Synthesis, biological evaluation, and molecular docking of Ugi products containing a zinc-chelating moiety as novel inhibitors of histone deacetylases.

    PubMed

    Grolla, Ambra A; Podestà, Valeria; Chini, Maria Giovanna; Di Micco, Simone; Vallario, Antonella; Genazzani, Armando A; Canonico, Pier Luigi; Bifulco, Giuseppe; Tron, Gian Cesare; Sorba, Giovanni; Pirali, Tracey

    2009-05-14

    HDAC inhibitors show great promise for the treatment of cancer. As part of a broader effort to explore the SAR of HDAC inhibitors, synthesis, biological evaluation, and molecular docking of novel Ugi products containing a zinc-chelating moiety are presented. One compound shows improved inhibitory potencies compared to SAHA, demonstrating that hindered lipophilic residues grafted on the peptide scaffold of the alpha-aminoacylamides can be favorable in the interaction with the enzyme.

  17. Identification of Novel Triazole-Based Nicotinamide Phosphoribosyltransferase (NAMPT) Inhibitors Endowed with Antiproliferative and Antiinflammatory Activity.

    PubMed

    Travelli, Cristina; Aprile, Silvio; Rahimian, Reza; Grolla, Ambra A; Rogati, Federica; Bertolotti, Mattia; Malagnino, Floriana; di Paola, Rosanna; Impellizzeri, Daniela; Fusco, Roberta; Mercalli, Valentina; Massarotti, Alberto; Stortini, Giorgio; Terrazzino, Salvatore; Del Grosso, Erika; Fakhfouri, Gohar; Troiani, Maria Pia; Alisi, Maria Alessandra; Grosa, Giorgio; Sorba, Giovanni; Canonico, Pier Luigi; Orsomando, Giuseppe; Cuzzocrea, Salvatore; Genazzani, Armando A; Galli, Ubaldina; Tron, Gian Cesare

    2017-03-09

    Nicotinamide phosphoribosyltransferase (NAMPT) is a key enzyme involved in the recycling of nicotinamide to maintain adequate NAD levels inside the cells. It has been postulated to be a pharmacological target, as it is overexpressed in cancer cells as well as in inflammatory diseases. We describe the synthesis and characterization of a novel class of one-digit nanomolar NAMPT inhibitors based on in vitro characterization. The most active compound tested, 30c, displayed activity in xenograft and allograft models, strengthening the potential of NAMPT inhibitors as antitumoral drugs. Furthermore, in the present contribution we describe the ability of 30c to significantly improve the outcome of colitis in mice. Given that this is the first report of an effect of NAMPT inhibitors in colitis, this result paves the way for novel applications for this class of compounds.

  18. Synthesis and multiparametric evaluation of thiadiazoles and oxadiazoles as diacylglycerol acyltransferase type 1 inhibitors.

    PubMed

    Mougenot, Patrick; Namane, Claudie; Fett, Eykmar; Goumy, Florence; Dadji-Faïhun, Rommel; Langot, Gwladys; Monseau, Catherine; Onofri, Bénédicte; Pacquet, François; Pascal, Cécile; Crespin, Olivier; Ben-Hassine, Majdi; Ragot, Jean-Luc; Van-Pham, Thao; Philippo, Christophe; Chatelain-Egger, Florence; Péron, Philippe; Le Bail, Jean-Christophe; Guillot, Etienne; Chamiot-Clerc, Philippe; Chabanaud, Marie-Aude; Pruniaux, Marie-Pierre; Ménegotto, Jérôme; Schmidt, Friedemann; Venier, Olivier; Viviani, Fabrice; Nicolai, Eric

    2016-01-01

    Chemical modulation of a formerly disclosed DGAT-1 inhibitor resulted in the identification of a compound with a suitable profile for preclinical development. Optimisation of solubility is discussed and a PK/PD study is presented.

  19. Seeking for Non-Zinc-Binding MMP-2 Inhibitors: Synthesis, Biological Evaluation and Molecular Modelling Studies

    PubMed Central

    Ammazzalorso, Alessandra; De Filippis, Barbara; Campestre, Cristina; Laghezza, Antonio; Marrone, Alessandro; Amoroso, Rosa; Tortorella, Paolo; Agamennone, Mariangela

    2016-01-01

    Matrix metalloproteinases (MMPs) are an important family of zinc-containing enzymes with a central role in many physiological and pathological processes. Although several MMP inhibitors have been synthesized over the years, none reached the market because of off-target effects, due to the presence of a zinc binding group in the inhibitor structure. To overcome this problem non-zinc-binding inhibitors (NZIs) have been recently designed. In a previous article, a virtual screening campaign identified some hydroxynaphtyridine and hydroxyquinoline as MMP-2 non-zinc-binding inhibitors. In the present work, simplified analogues of previously-identified hits have been synthesized and tested in enzyme inhibition assays. Docking and molecular dynamics studies were carried out to rationalize the activity data. PMID:27782083

  20. Synthesis of hydroxypyrone- and hydroxythiopyrone-based matrix metalloproteinase inhibitors: Developing a structure–activity relationship

    PubMed Central

    Yan, Yi-Long; Miller, Melissa T.; Cao, Yuchen; Cohen, Seth M.

    2010-01-01

    The zinc(II)-dependent matrix metalloproteinases (MMPs) are associated with a variety of diseases. Development of inhibitors to modulate MMP activity has been an active area of investigation for therapeutic development. Hydroxypyrones and hydroxythiopyrones are alternative zinc-binding groups (ZBGs) that, when combined with peptidomimetic backbones, comprise a novel class of MMP inhibitors (MMPi). In this report, a series of hydroxypyrone- and hydroxythiopyrone-based MMPi with aryl backbones at the 2-, 5-, and 6-positions of the hydroxypyrone ring have been synthesized. Synthetic routes for developing inhibitors with substituents at two of these positions (so-called double-handed inhibitors) are also explored. The MMP inhibition profiles and structure–activity relationship of synthesized hydroxypyrones and hydroxythiopyrones have been analyzed. The results here show that the ZBG, the position of the backbone on the ZBG, and the nature of the linker between the ZBG and backbone are critical for MMPi activities. PMID:19261472

  1. Plaque REgression with Cholesterol absorption Inhibitor or Synthesis inhibitor Evaluated by IntraVascular UltraSound (PRECISE-IVUS Trial): Study protocol for a randomized controlled trial.

    PubMed

    Tsujita, Kenichi; Sugiyama, Seigo; Sumida, Hitoshi; Shimomura, Hideki; Yamashita, Takuro; Yamanaga, Kenshi; Komura, Naohiro; Sakamoto, Kenji; Ono, Takamichi; Oka, Hideki; Nakao, Koichi; Nakamura, Sunao; Ishihara, Masaharu; Matsui, Kunihiko; Sakaino, Naritsugu; Nakamura, Natsuki; Yamamoto, Nobuyasu; Koide, Shunichi; Matsumura, Toshiyuki; Fujimoto, Kazuteru; Tsunoda, Ryusuke; Morikami, Yasuhiro; Matsuyama, Koushi; Oshima, Shuichi; Kaikita, Koichi; Hokimoto, Seiji; Ogawa, Hisao

    2015-10-01

    Although the positive association between achieved low-density lipoprotein cholesterol (LDL-C) level and the risk of coronary artery disease (CAD) has been confirmed by randomized studies with statins, many patients remain at high residual risk of events suggesting the necessity of novel pharmacologic strategies. The combination of ezetimibe/statin produces greater reductions in LDL-C compared to statin monotherapy. The Plaque REgression with Cholesterol absorption Inhibitor or Synthesis inhibitor Evaluated by IntraVascular UltraSound (PRECISE-IVUS) trial was aimed at evaluating the effects of ezetimibe addition to atorvastatin, compared with atorvastatin monotherapy, on coronary plaque regression and change in lipid profile in patients with CAD. The study is a prospective, randomized, controlled, multicenter study. The eligible patients undergoing IVUS-guided percutaneous coronary intervention will be randomly assigned to receive either atorvastatin alone or atorvastatin plus ezetimibe (10 mg) daily using a web-based randomization software. The dosage of atorvastatin will be increased by titration within the usual dose range with a treatment goal of lowering LDL-C below 70 mg/dL based on consecutive measures of LDL-C at follow-up visits. IVUS will be performed at baseline and 9-12 months follow-up time point at participating cardiovascular centers. The primary endpoint will be the nominal change in percent coronary atheroma volume measured by volumetric IVUS analysis. PRECISE-IVUS will assess whether the efficacy of combination of ezetimibe/atorvastatin is noninferior to atorvastatin monotherapy for coronary plaque reduction, and will translate into increased clinical benefit of dual lipid-lowering strategy in a Japanese population. Copyright © 2015 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.

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

    PubMed Central

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

    2015-01-01

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

  3. Small-molecule inhibitors of 25-hydroxyvitamin D-24-hydroxylase (CYP24A1): synthesis and biological evaluation.

    PubMed

    Ferla, Salvatore; Aboraia, Ahmed S; Brancale, Andrea; Pepper, Christopher J; Zhu, Jinge; Ochalek, Justin T; DeLuca, Hector F; Simons, Claire

    2014-09-25

    The synthesis of imidazole styrylbenzamide, tert-butyl styrylimidazole, and tert-butyl styrylsulfonate derivatives is described. Evaluation of binding affinity and inhibitory activity against CYP24A1 identified the imidazole styrylbenzamides as potent inhibitors of CYP24A1, having selectivity with respect to CYP27B1 comparable with or greater than that of the standard ketoconazole. Further evaluation of the 3,5-dimethoxy and 3,4,5-trimethoxy derivatives in chronic lymphocytic leukemia cells revealed that co-treatment of 1α,25-dihydroxyvitamin D3 plus inhibitor coordinately upregulated GADD45α and CDKN1A. Docking experiments on the inhibitors in the CYP24A1 enzyme active site suggest the compounds reach the active site through the vitamin D access tunnel and are exposed to multiple hydrophobic residues. The imidazole styrylbenzamides are optimally positioned to allow interaction of the imidazole with the heme, and, in the case of the methoxy derivatives, a hydrogen bond between the 3-methoxy group and Gln82 stabilizes the molecule in a favorable active conformation.

  4. 3-Heterocycle-phenyl N-alkylcarbamates as FAAH inhibitors: design, synthesis and 3D-QSAR studies.

    PubMed

    Käsnänen, Heikki; Myllymäki, Mikko J; Minkkilä, Anna; Kataja, Antti O; Saario, Susanna M; Nevalainen, Tapio; Koskinen, Ari M P; Poso, Antti

    2010-02-01

    Carbamates are a well-established class of fatty acid amide hydrolase (FAAH) inhibitors. Here we describe the synthesis of meta-substituted phenolic N-alkyl/aryl carbamates and their in vitro FAAH inhibitory activities. The most potent compound, 3-(oxazol-2yl)phenyl cyclohexylcarbamate (2 a), inhibited FAAH with a sub-nanomolar IC(50) value (IC(50)=0.74 nM). Additionally, we developed and validated three-dimensional quantitative structure-activity relationships (QSAR) models of FAAH inhibition combining the newly disclosed carbamates with our previously published inhibitors to give a total set of 99 compounds. Prior to 3D-QSAR modeling, the degree of correlation between FAAH inhibition and in silico reactivity was also established. Both 3D-QSAR methods used, CoMSIA and GRID/GOLPE, produced statistically significant models with coefficient of correlation for external prediction (R(2) (PRED)) values of 0.732 and 0.760, respectively. These models could be of high value in further FAAH inhibitor design.

  5. Design, synthesis and biological evaluation of 2-(substituted phenyl)thiazolidine-4-carboxylic acid derivatives as novel tyrosinase inhibitors.

    PubMed

    Ha, Young Mi; Park, Yun Jung; Lee, Ji Yeon; Park, Daeui; Choi, Yeon Ja; Lee, Eun Kyeong; Kim, Ji Min; Kim, Jin-Ah; Park, Ji Young; Lee, Hye Jin; Moon, Hyung Ryong; Chung, Hae Young

    2012-02-01

    Herein we describe the design, synthesis and biological activities of 2-(substituted phenyl)thiazolidine-4-carboxylic acid derivatives as novel tyrosinase inhibitors. The target compounds 2a-2j were designed and synthesized from the structural characteristics of N-phenylthiourea, tyrosinase inhibitor and tyrosine, and l-DOPA, the natural substrates of tyrosinase. Among them, (2R/S,4R)-2-(2,4-dimethoxyphenyl)thiazolidine-4-carboxylic acid (2g) caused the greatest inhibition 66.47% at 20 μM of l-DOPA oxidase activity of mushroom tyrosinase. Kinetic analysis of tyrosinase inhibition revealed that 2g is a competitive inhibitor. We predicted the tertiary structure of tyrosinase, and simulated the docking of mushroom tyrosinase with 2g. These results suggest that the binding affinity of 2g with tyrosinase is high. Also, 2g effectively inhibited tyrosinase activity and reduced melanin levels in B16 cells treated with α-MSH. These data strongly suggest that 2g can suppress the production of melanin via the inhibition of tyrosinase activity.

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

    SciTech Connect

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

    2009-01-01

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

  7. Design and synthesis of lactam-thiophene carboxylic acids as potent hepatitis C virus polymerase inhibitors.

    PubMed

    Barnes-Seeman, David; Boiselle, Carri; Capacci-Daniel, Christina; Chopra, Rajiv; Hoffmaster, Keith; Jones, Christopher T; Kato, Mitsunori; Lin, Kai; Ma, Sue; Pan, Guoyu; Shu, Lei; Wang, Jianling; Whiteman, Leah; Xu, Mei; Zheng, Rui; Fu, Jiping

    2014-08-15

    Herein we report the successful incorporation of a lactam as an amide replacement in the design of hepatitis C virus NS5B Site II thiophene carboxylic acid inhibitors. Optimizing potency in a replicon assay and minimizing potential risk for CYP3A4 induction led to the discovery of inhibitor 22a. This lead compound has a favorable pharmacokinetic profile in rats and dogs.

  8. Synthesis and application of polyaminoamide as new paraffin inhibitor from vegetable oil

    PubMed Central

    2011-01-01

    In this work, a series of novel paraffin inhibitor, polyaminoamide (PAA), was designed and prepared by aminolysis and poly-condensation using soybean oil and canola oil as the raw material. The property of the PAAs as paraffin inhibitor was investigated, the results show several PAA samples are potent in paraffin inhibition, and PPC-2 is the most effective one. Besides, the paraffin crystal morphology analysis was carried out to provide the mechanism of paraffin inhibition. PMID:22152091

  9. Proline-Based Macrocyclic Inhibitors of the Hepatitis C Virus: Stereoselective Synthesis and Biological Activity

    SciTech Connect

    Chen, Kevin X.; Njoroge, F. George; Vibulbhan, Bancha; Prongay, Andrew; Pichardo, John; Madison, Vincent; Buevich, Alexei; Chan, Tze-Ming

    2008-06-30

    Macrocyclization through a Mitsunobu reaction was used to synthesize a 17-membered macrocycle. The bicyclic acetal core was prepared completely diastereoselectively. The macrocyclic peptidomimetic surrogate of the P2-P3 dipeptide moiety was designed to function as a hepatitis C virus (HCV) NS3 serine protease inhibitor, and the pentapeptide {alpha}-ketoamides derived from the macrocycle were shown to be potent HCV inhibitors.

  10. Design and synthesis of a new type of non steroidal human aromatase inhibitors.

    PubMed

    Sonnet, P; Guillon, J; Enguehard, C; Dallemagne, P; Bureau, R; Rault S Auvray, P; Moslemi, S; Sourdiane, P; Galopin, S; Séralini, G E

    1998-05-05

    The structure-activity relationship study of one of recently described aromatase inhibitors, compound 1 (MR20814), allowed us to design some related derivatives as potential new inhibitors. Among those we synthesized, chlorophenylpyridylmethylenetetrahydroindolizinone 5 (MR20492) exhibited in vitro a ten-fold higher inhibition of the enzyme (IC50 = 0.2 +/- 0.0 microM and Ki = 10.3 +/- 3.3 nM).

  11. [Design, synthesis and activity evaluation of novel matrix metalloproteinases inhibitors based on the structure of enzyme].

    PubMed

    Jia, Hong; Guo, Yan-shen; Ge, Yi-yu; Wen, Hui; Yang, Jing; Yang, Xiu-ying; Du, Guan-hua; Yang, Guang-zhong

    2007-12-01

    A novel inhibitor series for matrix metalloproteinases (MMPs) were designed and synthesized. Using succinate and malonate as zinc binding groups and long hydrophobic substituents to bind with S1' pockets, the compounds showed micromolar inhibition and selectivity for MMP-2 over others. And we found a better activity compound. It is a chance to find a better precursor of MMP-2 inhibitors with activity and bioavailability by further optimization of compounds.

  12. Design, synthesis and evaluation of polar head group containing 2-keto-oxazole inhibitors of FAAH.

    PubMed

    Rusch, Marion; Zahov, Stefan; Vetter, Ingrid R; Lehr, Matthias; Hedberg, Christian

    2012-01-15

    2-α-Keto oxazoles containing polar head groups in their C5-side chains were designed as fatty acid amide hydrolase (FAAH) inhibitors. Variation in the spacer length resulted in submicromolar α-keto-oxazole FAAH inhibitor (IC(50)=436 nM) presenting electrostatic stabilizing interactions between its polar head group contained in the C5-side chain and the hydrophilic pocket of the enzyme.

  13. Natural and synthetic geiparvarins are strong and selective MAO-B inhibitors. Synthesis and SAR studies.

    PubMed

    Carotti, Angelo; Carrieri, Antonio; Chimichi, Stefano; Boccalini, Marco; Cosimelli, Barbara; Gnerre, Carmela; Carotti, Andrea; Carrupt, Pierre Alain; Testa, Bernard

    2002-12-16

    Natural geiparvarin 1 and a number of its analogues were prepared and tested as inhibitors of both monoamine oxidase isoforms, MAO-B and MAO-A. The desmethyl congener 6 of geiparvarin, proved potent and selective MAO-B inhibitor (pIC(50)=7.55 vs 4.62). X-ray crystallography and molecular modelling studies helped the understanding of the observed structure-activity relationships.

  14. Design, synthesis and structure-activity relationships of novel biarylamine-based Met kinase inhibitors

    SciTech Connect

    Williams, David K; Chen, Xiao-Tao; Tarby, Christine; Kaltenbach, Robert; Cai, Zhen-Wei; Tokarski, John S; An, Yongmi; Sack, John S; Wautlet, Barri; Gullo-Brown, Johnni; Henley, Benjamin J; Jeyaseelan, Robert; Kellar, Kristen; Manne, Veeraswamy; Trainor, George L; Lombardo, Louis J; Fargnoli, Joseph; Borzilleri, Robert M

    2010-09-03

    Biarylamine-based inhibitors of Met kinase have been identified. Lead compounds demonstrate nanomolar potency in Met kinase biochemical assays and significant activity in the Met-driven GTL-16 human gastric carcinoma cell line. X-ray crystallography revealed that these compounds adopt a bioactive conformation, in the kinase domain, consistent with that previously seen with 2-pyridone-based Met kinase inhibitors. Compound 9b demonstrated potent in vivo antitumor activity in the GTL-16 human tumor xenograft model.

  15. Synthesis, Structural Analysis, and Biological Evaluation of Thioxoquinazoline Derivatives as Phosphodiesterase 7 Inhibitors

    SciTech Connect

    Castano, T.; Wang, H; Campillo, N; Ballester, S; Gonzalez-Garcia, C; Hernandez, J; Perez, C; Cuenca, J; Perez-Castillo, A; et. al.

    2009-01-01

    PDE7 inhibitors regulate pro-inflammatory and immune T-cell functions, and are a potentially novel class of drugs particularly useful for treatment of a wide variety of immune and inflammatory disorders. Structural optimization of thioxoquinazoline derivatives led to new compounds with very interesting profiles as PDE7 or PDE7/PDE4 dual inhibitors, which may be further developed as new drugs for inflammatory and neurological diseases.

  16. Design, synthesis and structure-activity relationships of novel biarylamine-based Met kinase inhibitors.

    PubMed

    Williams, David K; Chen, Xiao-Tao; Tarby, Christine; Kaltenbach, Robert; Cai, Zhen-Wei; Tokarski, John S; An, Yongmi; Sack, John S; Wautlet, Barri; Gullo-Brown, Johnni; Henley, Benjamin J; Jeyaseelan, Robert; Kellar, Kristen; Manne, Veeraswamy; Trainor, George L; Lombardo, Louis J; Fargnoli, Joseph; Borzilleri, Robert M

    2010-05-01

    Biarylamine-based inhibitors of Met kinase have been identified. Lead compounds demonstrate nanomolar potency in Met kinase biochemical assays and significant activity in the Met-driven GTL-16 human gastric carcinoma cell line. X-ray crystallography revealed that these compounds adopt a bioactive conformation, in the kinase domain, consistent with that previously seen with 2-pyridone-based Met kinase inhibitors. Compound 9b demonstrated potent in vivo antitumor activity in the GTL-16 human tumor xenograft model.

  17. Assimilation of NAD(+) precursors in Candida glabrata.

    PubMed

    Ma, Biao; Pan, Shih-Jung; Zupancic, Margaret L; Cormack, Brendan P

    2007-10-01

    The yeast pathogen Candida glabrata is a nicotinamide adenine dinucleotide (NAD(+)) auxotroph and its growth depends on the environmental supply of vitamin precursors of NAD(+). C. glabrata salvage pathways defined in this article allow NAD(+) to be synthesized from three compounds - nicotinic acid (NA), nicotinamide (NAM) and nicotinamide riboside (NR). NA is salvaged through a functional Preiss-Handler pathway. NAM is first converted to NA by nicotinamidase and then salvaged by the Preiss-Handler pathway. Salvage of NR in C. glabrata occurs via two routes. The first, in which NR is phosphorylated by the NR kinase Nrk1, is independent of the Preiss-Handler pathway. The second is a novel pathway in which NR is degraded by the nucleosidases Pnp1 and Urh1, with a minor role for Meu1, and ultimately converted to NAD(+) via the nicotinamidase Pnc1 and the Preiss-Handler pathway. Using C. glabrata mutants whose growth depends exclusively on the external NA or NR supply, we also show that C. glabrata utilizes NR and to a lesser extent NA as NAD(+) sources during disseminated infection.

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

    PubMed Central

    2014-01-01

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

  19. Discovery of benzimidazole-diamide finger loop (Thumb Pocket I) allosteric inhibitors of HCV NS5B polymerase: Implementing parallel synthesis for rapid linker optimization.

    PubMed

    Goulet, Sylvie; Poupart, Marc-André; Gillard, James; Poirier, Martin; Kukolj, George; Beaulieu, Pierre L

    2010-01-01

    Previously described SAR of benzimidazole-based non-nucleoside finger loop (Thumb Pocket I) inhibitors of HCV NS5B polymerase was expanded. Prospecting studies using parallel synthesis techniques allowed the rapid identification of novel cinnamic acid right-hand sides that provide renewed opportunities for further optimization of these inhibitors. Novel diamide derivatives such as 44 exhibited comparable potency (enzymatic and cell-based HCV replicon) as previously described tryptophan-based inhibitors but physicochemical properties (e.g., aqueous solubility and lipophilicity) have been improved, resulting in molecules with reduced off-target liabilities (CYP inhibition) and increased metabolic stability.

  20. Gentamicin differentially alters cellular metabolism of cochlear hair cells as revealed by NAD(P)H fluorescence lifetime imaging

    NASA Astrophysics Data System (ADS)

    Zholudeva, Lyandysha V.; Ward, Kristina G.; Nichols, Michael G.; Smith, Heather Jensen

    2015-05-01

    Aminoglycoside antibiotics are implicated as culprits of hearing loss in more than 120,000 individuals annually. Research has shown that the sensory cells, but not supporting cells, of the cochlea are readily damaged and/or lost after use of such antibiotics. High-frequency outer hair cells (OHCs) show a greater sensitivity to antibiotics than high- and low-frequency inner hair cells (IHCs). We hypothesize that variations in mitochondrial metabolism account for differences in susceptibility. Fluorescence lifetime microscopy was used to quantify changes in NAD(P)H in sensory and supporting cells from explanted murine cochleae exposed to mitochondrial uncouplers, inhibitors, and an ototoxic antibiotic, gentamicin (GM). Changes in metabolic state resulted in a redistribution of NAD(P)H between subcellular fluorescence lifetime pools. Supporting cells had a significantly longer lifetime than sensory cells. Pretreatment with GM increased NAD(P)H intensity in high-frequency sensory cells, as well as the NAD(P)H lifetime within IHCs. GM specifically increased NAD(P)H concentration in high-frequency OHCs, but not in IHCs or pillar cells. Variations in NAD(P)H intensity in response to mitochondrial toxins and GM were greatest in high-frequency OHCs. These results demonstrate that GM rapidly alters mitochondrial metabolism, differentially modulates cell metabolism, and provides evidence that GM-induced changes in metabolism are significant and greatest in high-frequency OHCs.

  1. Regulation of brain mitochondrial H2O2 production by membrane potential and NAD(P)H redox state.

    PubMed

    Starkov, Anatoly A; Fiskum, Gary

    2003-09-01

    Mitochondrial production of reactive oxygen species (ROS) at Complex I of the electron transport chain is implicated in the etiology of neural cell death in acute and chronic neurodegenerative disorders. However, little is known regarding the regulation of mitochondrial ROS production by NADH-linked respiratory substrates under physiologically realistic conditions in the absence of respiratory chain inhibitors. This study used Amplex Red fluorescence measurements of H2O2 to test the hypothesis that ROS production by isolated brain mitochondria is regulated by membrane potential (DeltaPsi) and NAD(P)H redox state. DeltaPsi was monitored by following the medium concentration of the lipophilic cation tetraphenylphosphonium with a selective electrode. NAD(P)H autofluorescence was used to monitor NAD(P)H redox state. While the rate of H2O2 production was closely related to DeltaPsi and the level of NAD(P)H reduction at high values of DeltaPsi, 30% of the maximal rate of H2O2 formation was still observed in the presence of uncoupler (p-trifluoromethoxycarbonylcyanide phenylhydrazone) concentrations that provided for maximum depolarization of DeltaPsi and oxidation of NAD(P)H. Our findings indicate that ROS production by mitochondria oxidizing physiological NADH-dependent substrates is regulated by DeltaPsi and by the NAD(P)H redox state over ranges consistent with those that exist at different levels of cellular energy demand.

  2. CD73 protein as a source of extracellular precursors for sustained NAD+ biosynthesis in FK866-treated tumor cells.

    PubMed

    Grozio, Alessia; Sociali, Giovanna; Sturla, Laura; Caffa, Irene; Soncini, Debora; Salis, Annalisa; Raffaelli, Nadia; De Flora, Antonio; Nencioni, Alessio; Bruzzone, Santina

    2013-09-06

    NAD(+) is mainly synthesized in human cells via the "salvage" pathways starting from nicotinamide, nicotinic acid, or nicotinamide riboside (NR). The inhibition with FK866 of the enzyme nicotinamide phosphoribosyltransferase (NAMPT), catalyzing the first reaction in the "salvage" pathway from nicotinamide, showed potent antitumor activity in several preclinical models of solid and hematologic cancers. In the clinical studies performed with FK866, however, no tumor remission was observed. Here we demonstrate that low micromolar concentrations of extracellular NAD(+) or NAD(+) precursors, nicotinamide mononucleotide (NMN) and NR, can reverse the FK866-induced cell death, this representing a plausible explanation for the failure of NAMPT inhibition as an anti-cancer therapy. NMN is a substrate of both ectoenzymes CD38 and CD73, with generation of NAM and NR, respectively. In this study, we investigated the roles of CD38 and CD73 in providing ectocellular NAD(+) precursors for NAD(+) biosynthesis and in modulating cell susceptibility to FK866. By specifically silencing or overexpressing CD38 and CD73, we demonstrated that endogenous CD73 enables, whereas CD38 impairs, the conversion of extracellular NMN to NR as a precursor for intracellular NAD(+) biosynthesis in human cells. Moreover, cell viability in FK866-treated cells supplemented with extracellular NMN was strongly reduced in tumor cells, upon pharmacological inhibition or specific down-regulation of CD73. Thus, our study suggests that genetic or pharmacologic interventions interfering with CD73 activity may prove useful to increase cancer cell sensitivity to NAMPT inhibitors.

  3. Molecular Features of the YAP Inhibitor Verteporfin: Synthesis of Hexasubstituted Dipyrrins as Potential Inhibitors of YAP/TAZ, the Downstream Effectors of the Hippo Pathway.

    PubMed

    Gibault, Floriane; Bailly, Fabrice; Corvaisier, Matthieu; Coevoet, Mathilde; Huet, Guillemette; Melnyk, Patricia; Cotelle, Philippe

    2017-06-21

    Porphyrin derivatives, in particular verteporfin (VP), a photosensitizer initially designed for cancer therapy, have been identified as inhibitors of the YAP-TEAD interaction and transcriptional activity. Herein we report the efficient convergent synthesis of the dipyrrin half of protoporphyrin IX dimethyl ester (PPIX-DME), in which the sensitive vinyl group was created at the final stage by a dehydroiodination reaction. Two other dipyrrin derivatives were synthesized, including dipyrrin 19 [(Z)-2-((3,5-dimethyl-4-vinyl-2H-pyrrol-2-ylidene)methyl)-3,5-dimethyl-4-vinyl-1H-pyrrole], containing two vinyl groups. We found that VP and dipyrrin 19 showed significant inhibitory effects on TEAD transcriptional activity in MDA-MB-231 human breast cancer cells, whereas other compounds did not show significant changes. In addition, we observed a marked decrease in both YAP and TAZ levels following VP treatment, whereas dipyrrin 19 treatment primarily decreased the levels of YAP and receptor kinase AXL, a downstream target of YAP. Together, our data suggest that, due to their chemical structures, porphyrin- and dipyrrin-related derivatives can directly target YAP and/or TAZ proteins and inhibit TEAD transcriptional activity. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Metformin induces suppression of NAD(P)H oxidase activity in podocytes.

    PubMed

    Piwkowska, Agnieszka; Rogacka, Dorota; Jankowski, Maciej; Dominiczak, Marek Henryk; Stepiński, Jan Kazimierz; Angielski, Stefan

    2010-03-05

    Hyperglycemia increases the production of reactive oxygen species (ROS). NAD(P)H oxidase, producing superoxide anion, is the main source of ROS in diabetic podocytes and their production contributes to the development of diabetic nephropathy. We have investigated the effect of an antidiabetic drug, metformin on the production of superoxide anion in cultured podocytes and attempted to elucidate underlying mechanisms. The experiments were performed in normal (NG, 5.6mM) and high (HG, 30mM) glucose concentration. Overall ROS production was measured by fluorescence of a DCF probe. Activity of NAD(P)H oxidase was measured by chemiluminescence method. The AMP-dependent kinase (AMPK) activity was determined by immunobloting, measuring the ratio of phosphorylated AMPK to total AMPK. Glucose accumulation was measured using 2-deoxy-[1,2-(3)H]-glucose. ROS production increased by about 27% (187+/-8 vs. 238+/-9 arbitrary units AU, P<0.01) in HG. Metformin (2mM, 2h) markedly reduced ROS production by 45% in NG and 60% in HG. Metformin decreased NAD(P)H oxidase activity in NG (36%) and HG (86%). AMPK activity was increased by metformin in NG and HG (from 0.58+/-0.07 to. 0.99+/-0.06, and from 0.53+/-0.03 to 0.64+/-0.03; P<0.05). The effects of metformin on the activities of NAD(P)H oxidase and AMPK were abolished in the presence of AMPK inhibitor, compound C. We have shown that metformin decreases production of ROS through reduction of NAD(P)H oxidase activity. We also have demonstrated relationship between activity of NAD(P)H oxidase and AMPK. 2010 Elsevier Inc. All rights reserved.

  5. Synthesis, modelling and kinetic assays of potent inhibitors of purple acid phosphatase.

    PubMed

    Mohd-Pahmi, Siti Hajar; Hussein, Waleed M; Schenk, Gerhard; McGeary, Ross P

    2011-05-15

    Purple acid phosphatases (PAPs) are binuclear metallohydrolases that have been isolated from various mammals, plants, fungi and bacteria. In mammals PAP activity is associated with bone resorption and can lead to bone metabolic disorders such as osteoporosis; thus human PAP is an attractive target to develop anti-osteoporotic drugs. Based on a previous lead compound and rational drug design, acyl derivatives of α-aminonaphthylmethylphosphonic acid were synthesised and tested as PAP inhibitors. Kinetic analysis showed that they are good PAP inhibitors whose potencies improve with increasing acyl chain length. Maximum potency is reached when the number of carbons in the acyl chain is between 12 and 14. The most potent inhibitor of red kidney bean PAP is the dodecyl-derivative with K(ic)=5 μM, while the most potent pig PAP inhibitor is the tetradecyl-derivative with K(ic)=8 μM, the most potent inhibitor of a mammalian PAP yet reported. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

  6. Design of Gem-difluoro-bis-Tetrahydrofuran as P2-Ligand for HIV-1 Protease Inhibitors to Improve Brain Penetration: Synthesis, X-ray Studies, and Biological Evaluation

    PubMed Central

    Yashchuk, Sofiya; Mizuno, Akira; Chakraborty, Nilanjana; Agniswamy, Johnson; Wang, Yuan-Fang; Aoki, Manabu; Gomez, Pedro Miguel Salcedo; Amano, Masayuki; Weber, Irene T.; Mitsuya, Hiroaki

    2015-01-01

    Structure-based design, synthesis, biological evaluation and X-ray structural studies of fluorine containing HIV-1 protease inhibitors are described. The synthesis of both enantiomers of the gem-difluoro-bis-THF ligands was carried out in a stereoselective manner using a Reformatskii-Claisen reaction as the key step. Optically active ligands HIV-1LAI were converted to protease inhibitors. Two of these inhibitors (3 and 4) exhibited HIV-1 protease inhibitory Ki’s in picomolar range. Both inhibitors showed very potent antiviral activity with EC50 values of 0.8 nM and 3.1 nM respectively against the laboratory strain HIV-1LAI. Both inhibitors exhibited improved lipophilicity profiles compared to darunavir. Also, both inhibitors showed much improved blood-brain-barrier permeability in an in vitro model. A high resolution X-ray structure of inhibitor 4-bound HIV-1 protease was determined. The X-ray structure revealed that fluoro ligand makes extensive interactions with the HIV-1 protease S2 subsite, including hydrogen-bonding interactions with the protease backbone atoms. Also, both fluorine atoms on the bis-THF ligand formed strong interactions with the flap Gly48 carbonyl oxygen. PMID:25336073

  7. Improved loading and cleavage methods for solid-phase synthesis using chlorotrityl resins: synthesis and testing of a library of 144 discrete chemicals as potential farnesyltransferase inhibitors.

    PubMed

    Park, Jewn Giew; Langenwalter, Kevin J; Weinbaum, Carolyn A; Casey, Patrick J; Pang, Yuan-Ping

    2004-01-01

    The use of chlorotrityl resins for the immobilization of amines is sometimes deterred by the lengthy process of loading the reactants on the resins and product decomposition caused by the reactive chlorotrityl group in the presence of 1% TFA as a cleavage agent. Here, we report improved methods developed for selective and efficient loading of aminobenzoic acid derivatives on chlorotrityl resins and for cleavage of aniline-containing products from the resins without decomposition. These methods led to the synthesis of a library of 144 discrete chemicals as potential farnesyltransferase inhibitors (FTIs) using IRORI's radio-frequency-encoded sorting technique and to the study of the applicability of the bivalence approach to the development of FTIs.

  8. Free [NADH]/[NAD(+)] regulates sirtuin expression.

    PubMed

    Gambini, Juan; Gomez-Cabrera, Mari Carmen; Borras, Consuelo; Valles, Soraya L; Lopez-Grueso, Raul; Martinez-Bello, Vladimir E; Herranz, Daniel; Pallardo, Federico V; Tresguerres, Jesus A F; Serrano, Manuel; Viña, Jose

    2011-08-01

    Sirtuins are deacetylases involved in metabolic regulation and longevity. Our aim was to test the hypothesis that they are subjected to redox regulation by the [NADH]/[NAD(+)] ratio. We used NIH3T3 fibroblasts in culture, Drosophila fed with or without ethanol and exercising rats. In all three models an increase in [NADH]/[NAD(+)] came up with an increased expression of sirtuin mRNA and protein. PGC-1α (a substrate of sirtuins) protein level was significantly increased in fibroblasts incubated with lactate and pyruvate but this effect was lost in fibroblasts obtained from sirtuin-deficient mice. We conclude that the expression of sirtuins is subject to tight redox regulation by the [NADH]/[NAD(+)] ratio, which is a major sensor for metabolite availability conserved from invertebrates to vertebrates. Copyright © 2011. Published by Elsevier Inc.

  9. Synthesis and biological evaluation of thiazole derivatives as novel USP7 inhibitors.

    PubMed

    Chen, Chao; Song, Jiemei; Wang, Jinzheng; Xu, Chang; Chen, Caiping; Gu, Wei; Sun, Hongbin; Wen, Xiaoan

    2017-02-15

    Herpesvirus-associated Ubiquitin-Specific Protease (HAUSP, also called USP7) interacts with and stabilizes Mdm2, and represents one of the first examples that deubiquitinases oncogenic proteins. USP7 has been regarded as a potential drug target for cancer therapy. Inhibitors of USP7 have been recently shown to suppress tumor cell growth in vitro and in vivo. Based on leading USP7 inhibitors P5091 and P22077, we designed and synthesized a series of thiazole derivatives. The results of in vitro assays showed that the thiazole compounds exhibited low micromolar inhibition activity against both USP7 enzyme and cancer cell lines. The compounds induced cell death in a p53-dependent and p53-independent manner. Taken together, this study may provide thiazole compounds as a new class of USP7 inhibitors. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Synthesis, biological evaluation and docking of novel bisamidinohydrazones as non-peptide inhibitors of furin.

    PubMed

    Kibirev, V K; Osadchuk, T V; Kozachenko, O P; Kholodovych, V; Fedoryak, D; Brovarets, V S

    2015-01-01

    A series of novel non-peptidicfurin inhibitors with values of inhibitory constants (Ki) in the range of 0.74-1.54 μM was obtained by interactions of aminoguanidine hydrocarbonate with three diaryldicarbalde- hydes. Correspondingly p-hydroquinone, piperazine and adipic acid were used as linkers between their ben- zene moieties. Docking studies of these new inhibitors into recently published 3D-structure of human furin (PDB code 4OMC) showed that they were able to interact with subsites S1 and S4 of the enzyme. The overall arrangement of bisamidinohydrazones into furin active site was similar to the position of the ligand co- crystallized with a protease. Observations obtained with molecular modeling allowed further guidance into chemical modifications of the synthesized inhibitors which improve their inhibitory activity.

  11. Design, synthesis and biological evaluation of novel non-peptide boronic acid derivatives as proteasome inhibitors.

    PubMed

    Ge, Ying; Li, Aibo; Wu, Jianwei; Feng, Haiwei; Wang, Letian; Liu, Hongwu; Xu, Yungen; Xu, Qingxiang; Zhao, Li; Li, Yuyan

    2017-03-10

    A novel series of non-peptide proteasome inhibitors bearing the 1, 4-naphthoquinone scaffold and boronic acid warhead was developed. In the biological evaluation on the chymotrypsin-like activity of human 20S proteasome, five compounds showed IC50 values in the nanomolar range. Docking experiments into the yeast 20S proteasome rationalized their biological activities and allowed further optimization of this interesting class of inhibitors. Within the cellular proliferation inhibition assay and western blot analysis, compound 3e demonstrated excellent anti-proliferative activity against solid tumor cells and clear accumulation of ubiquitinated cellular proteins. Furthermore, in the microsomal stability assay compound 3e demonstrated much improved metabolic stability compared to bortezomib, emerging as a promising lead compound for further design of non-peptide proteasome inhibitors.

  12. Molecular Design, Synthesis and Trypanocidal Activity of Dipeptidyl Nitriles as Cruzain Inhibitors

    PubMed Central

    Avelar, Leandro A. A.; Camilo, Cristian D.; de Albuquerque, Sérgio; Fernandes, William B.; Gonçalez, Cristiana; Kenny, Peter W.; Leitão, Andrei; McKerrow, James H.; Montanari, Carlos A.; Orozco, Erika V. Meñaca; Ribeiro, Jean F. R.; Rocha, Josmar R.; Rosini, Fabiana; Saidel, Marta E.

    2015-01-01

    A series of compounds based on the dipeptidyl nitrile scaffold were synthesized and assayed for their inhibitory activity against the T. cruzi cysteine protease cruzain. Structure activity relationships (SARs) were established using three, eleven and twelve variations respectively at the P1, P2 and P3 positions. A Ki value of 16 nM was observed for the most potent of these inhibitors which reflects a degree of non-additivity in the SAR. An X-ray crystal structure was determined for the ligand-protein complex for the structural prototype for the series. Twenty three inhibitors were also evaluated for their anti-trypanosomal effects and an EC50 value of 28 μM was observed for the most potent of these. Although there remains scope for further optimization, the knowledge gained from this study is also transferable to the design of cruzain inhibitors based on warheads other than nitrile as well as alternative scaffolds. PMID:26173110

  13. Molecular Design, Synthesis and Trypanocidal Activity of Dipeptidyl Nitriles as Cruzain Inhibitors.

    PubMed

    Avelar, Leandro A A; Camilo, Cristian D; de Albuquerque, Sérgio; Fernandes, William B; Gonçalez, Cristiana; Kenny, Peter W; Leitão, Andrei; McKerrow, James H; Montanari, Carlos A; Orozco, Erika V Meñaca; Ribeiro, Jean F R; Rocha, Josmar R; Rosini, Fabiana; Saidel, Marta E

    2015-01-01

    A series of compounds based on the dipeptidyl nitrile scaffold were synthesized and assayed for their inhibitory activity against the T. cruzi cysteine protease cruzain. Structure activity relationships (SARs) were established using three, eleven and twelve variations respectively at the P1, P2 and P3 positions. A Ki value of 16 nM was observed for the most potent of these inhibitors which reflects a degree of non-additivity in the SAR. An X-ray crystal structure was determined for the ligand-protein complex for the structural prototype for the series. Twenty three inhibitors were also evaluated for their anti-trypanosomal effects and an EC50 value of 28 μM was observed for the most potent of these. Although there remains scope for further optimization, the knowledge gained from this study is also transferable to the design of cruzain inhibitors based on warheads other than nitrile as well as alternative scaffolds.

  14. Synthesis and biological evaluation of 6-substituted indolizinoquinolinediones as catalytic DNA topoisomerase I inhibitors.

    PubMed

    Yu, Le-Mao; Zhang, Xiao-Ru; Li, Xiao-Bing; Yang, Yuan; Wei, Hong-Yu; He, Xi-Xin; Gu, Lian-Quan; Huang, Zhi-Shu; Pommier, Yves; An, Lin-Kun

    2015-08-28

    In our previous work, indolizinoquinolinedione derivative 1 was identified as a Top1 catalytic inhibitor. Herein, a series of 6-substituted indolizinoquinolinedione derivatives were synthesized through modification of the parent compound 1. Top1 cleavage and relaxation assays indicate that none of these novel compounds act as classical Top1 poison, and that the compounds with alkylamino terminus at C-6 side chain, including 8, 11-16, 18-21, 25, 26 and 28-30, are the most potent Top1 catalytic inhibitors. Top1-mediated unwinding assay demonstrated that 14, 22 and 26 were Top1 catalytic inhibitors without Top1-mediated unwinding effect. Moreover, MTT results showed that compounds 26, 28-30 exhibit significant cytotoxicity against human leukemia HL-60 cells, and that compound 26 exerts potent cytotoxicity against A549 lung cancer cells at nanomolar range.

  15. Synthesis and biological evaluation of 6-substituted indolizinoquinolinediones as catalytic DNA topoisomerase I inhibitors

    PubMed Central

    Yu, Le-Mao; Zhang, Xiao-Ru; Li, Xiao-Bing; Yang, Yuan; Wei, Hong-Yu; He, Xi-Xin; Gu, Lian-Quan; Huang, Zhi-Shu; Pommier, Yves; An, Lin-Kun

    2015-01-01

    In our previous work, indolizinoquinolinedione derivative 1 was identified as a Top1 catalytic inhibitor. Herein, a series of 6-substituted indolizinoquinolinedione derivatives were synthesized through modification of the parent compound 1. Top1 cleavage and relaxation assays indicate that none of these novel compounds act as classical Top1 poison, and that the compounds with alkylamino terminus at C-6 side chain, including 8, 11–16, 18–21, 25, 26 and 28–30, are the most potent Top1 catalytic inhibitors. Top1-mediated unwinding assay demonstrated that 14, 22 and 26 were Top1 catalytic inhibitors without Top1-mediated unwinding effect. Moreover, MTT results showed that compounds 26, 28–30 exhibit significant cytotoxicity against human leukemia HL-60 cells, and that compound 26 exerts potent cytotoxicity against A549 lung cancer cells at nanomolar range. PMID:26188908

  16. BET Bromodomain Inhibitors with One-Step Synthesis Discovered from Virtual Screen.

    PubMed

    Ayoub, Alex M; Hawk, Laura M L; Herzig, Ryan J; Jiang, Jiewei; Wisniewski, Andrea J; Gee, Clifford T; Zhao, Peiliang; Zhu, Jin-Yi; Berndt, Norbert; Offei-Addo, Nana K; Scott, Thomas G; Qi, Jun; Bradner, James E; Ward, Timothy R; Schönbrunn, Ernst; Georg, Gunda I; Pomerantz, William C K

    2017-06-22

    Chemical inhibition of epigenetic regulatory proteins BrdT and Brd4 is emerging as a promising therapeutic strategy in contraception, cancer, and heart disease. We report an easily synthesized dihydropyridopyrimidine pan-BET inhibitor scaffold, which was uncovered via a virtual screen followed by testing in a fluorescence anisotropy assay. Dihydropyridopyimidine 3 was subjected to further characterization and is highly selective for the BET family of bromodomains. Structure-activity relationship data and ligand deconstruction highlight the importance of the substitution of the uracil moiety for potency and selectivity. Compound 3 was also cocrystallized with Brd4 for determining the ligand binding pose and rationalizing subsequent structure-activity data. An additional series of dihydropyridopyrimidines was synthesized to exploit the proximity of a channel near the ZA loop of Brd4, leading to compounds with submicromolar affinity and cellular target engagement. Given these findings, novel and easily synthesized inhibitors are being introduced to the growing field of bromodomain inhibitor development.

  17. Design and Synthesis of Phenylpyrrolidine Phenylglycinamides As Highly Potent and Selective TF-FVIIa Inhibitors

    PubMed Central

    2013-01-01

    Inhibitors of the Tissue Factor/Factor VIIa (TF-FVIIa) complex are promising novel anticoagulants that show excellent efficacy and minimal bleeding in preclinical models. On the basis of a zwitterionic phenylglycine acylsulfonamide 1, a phenylglycine benzylamide 2 was shown to possess improved permeability and oral bioavailability. Optimization of the benzylamide, guided by X-ray crystallography, led to a potent TF-FVIIa inhibitor 18i with promising oral bioavailability, but promiscuous activity in an in vitro safety panel of receptors and enzymes. Introducing an acid on the pyrrolidine ring, guided by molecular modeling, resulted in highly potent, selective, and efficacious TF-FVIIa inhibitors with clean in vitro safety profile. The pyrrolidine acid 20 showed a moderate clearance, low volume of distribution, and a short t1/2 in dog PK studies. PMID:24900796

  18. Synthesis of bicyclic N-arylmethyl-substituted iminoribitol derivatives as selective nucleoside hydrolase inhibitors.

    PubMed

    Berg, Maya; Bal, Gunther; Goeminne, Annelies; Van der Veken, Pieter; Versées, Wim; Steyaert, Jan; Haemers, Achiel; Augustyns, Koen

    2009-02-01

    The purine metabolism of Trypanosoma and Leishmania spp. provides a good target in the search for new selective drugs. Bicyclic N-arylmethyl-substituted iminoribitols were developed as inhibitors of T. vivax nucleoside hydrolase, a key enzyme of the purine salvage pathway. The obtained results and structure-activity data confirmed our model for inhibitor binding with a hydrogen bond between a nitrogen atom of the nucleobase mimetic and the protonated Asp40 from the enzyme. This interaction depends on an optimal pK(a) value, which can be influenced by the electronic properties of the substituents. These compounds are potent, selective inhibitors of nucleoside hydrolase and are inactive toward human nucleoside phosphorylase.

  19. Synthesis, Structure, and SAR of Tetrahydropyran-Based LpxC Inhibitors

    PubMed Central

    2014-01-01

    In the search for novel Gram-negative agents, we performed a comprehensive search of the AstraZeneca collection and identified a tetrahydropyran-based matrix metalloprotease (MMP) inhibitor that demonstrated nanomolar inhibition of UDP-3-O-(acyl)-N-acetylglucosamine deacetylase (LpxC). Crystallographic studies in Aquifex aeolicus LpxC indicated the tetrahydropyran engaged in the same hydrogen bonds and van der Waals interactions as other known inhibitors. Systematic optimization of three locales on the scaffold provided compounds with improved Gram-negative activity. However, the optimization of LpxC activity was not accompanied by reduced inhibition of MMPs. Comparison of the crystal structure of the native product, UDP-3-O-(acyl)-glucosamine, in Aquifex aeolicus to the structure of a tetrahydropyran-based inhibitor indicates pathways for future optimization. PMID:25408833

  20. Structural basis for the design and synthesis of selective HDAC inhibitors.

    PubMed

    Di Micco, Simone; Chini, Maria Giovanna; Terracciano, Stefania; Bruno, Ines; Riccio, Raffaele; Bifulco, Giuseppe

    2013-07-01

    Histone Deacetylases are considered promising targets for cancer epigenetic therapy, and small molecules able to modulate their biological function have recently gained an increasing interest as potential anticancer agents. In spite of their potential application in cancer therapy, most HDAC inhibitors unselectively bind the several HDAC isoforms, giving rise to different side-effects. In this context, we have traced out the structural elements responsible of selective binding for the therapeutically relevant different HDAC isoforms. The structural analysis has been carried out by molecular modeling, docking in the binding pockets of HDAC1-4 and HDAC6-8, 36 inhibitors presenting a well defined selectivity for the different isoforms. As quick proof of evidence, we have designed, synthesized and experimentally tested three selective ligands. The experimental data suggest that the obtained structural guidelines can be useful tools for the rational design of new potent inhibitors against selected HDAC isoforms. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. Synthesis of selective inhibitors against V. cholerae sialidase and human cytosolic sialidase NEU2.

    PubMed

    Khedri, Zahra; Li, Yanhong; Cao, Hongzhi; Qu, Jingyao; Yu, Hai; Muthana, Musleh M; Chen, Xi

    2012-08-14

    Sialidases or neuraminidases catalyze the hydrolysis of terminal sialic acid residues from sialyl oligosaccharides and glycoconjugates. Despite successes in developing potent inhibitors specifically against influenza virus neuraminidases, the progress in designing and synthesizing selective inhibitors against bacterial and human sialidases has been slow. Guided by sialidase substrate specificity studies and sialidase crystal structural analysis, a number of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA or Neu5Ac2en) analogues with modifications at C9 or at both C5 and C9 were synthesized. Inhibition studies of various bacterial sialidases and human cytosolic sialidase NEU2 revealed that Neu5Gc9N(3)2en and Neu5AcN(3)9N(3)2en are selective inhibitors against V. cholerae sialidase and human NEU2, respectively.

  2. Synthesis and Evaluation of Substituted Chroman-4-one and Chromone Derivatives as Sirtuin 2-Selective Inhibitors

    PubMed Central

    2012-01-01

    A series of substituted chromone/chroman-4-one derivatives has been synthesized and evaluated as novel inhibitors of SIRT2, an enzyme involved in aging-related diseases, e.g., neurodegenerative disorders. The analogues were efficiently synthesized in a one-step procedure including a base-mediated aldol condensation using microwave irradiation. The most potent compounds, with inhibitory concentrations in the low micromolar range, were substituted in the 2-, 6-, and 8-positions. Larger, electron-withdrawing substituents in the 6- and 8-positions were favorable. The most potent inhibitor of SIRT2 was 6,8-dibromo-2-pentylchroman-4-one with an IC50 of 1.5 μM. The synthesized compounds show high selectivity toward SIRT2 over SIRT1 and SIRT3 and represent an important starting point for the development of novel SIRT2 inhibitors. PMID:22746324

  3. Recent Methodologies toward the Synthesis of Valdecoxib: A Potential 3,4-diarylisoxazolyl COX-2 Inhibitor

    PubMed Central

    Dadiboyena, Sureshbabu; Nefzi, Adel

    2011-01-01

    Non-steroidal anti-inflammatory drugs are widely used therapeutic agents in the treatment of inflammation, pain and fever. Cyclooxygenase catalyzes the initial step of biotransformation of arachidonic acid to prostanoids, and exist as three distinct isozymes; COX-I, COX-II and COX-III. Selective COX-II inhibitors are a class of potential anti-inflammatory, analgesic, and antipyretic drugs with reduced gastrointestinal (GI) side effects compared to nonselective inhibitors. 3,4-diarylisoxazole scaffold is recurrently found in a wide variety of NSAIDs, protein kinase inhibitors, hypertensive agents, and estrogen receptor (ER) modulators. In the present review, we document on the recent synthetic strategies of 3,4-diarylisoxazolyl scaffolds of valdecoxib and its relevant structural analogues. PMID:20724040

  4. Synthesis and biological evaluation of urea derivatives as highly potent and selective rho kinase inhibitors.

    PubMed

    Yin, Yan; Lin, Li; Ruiz, Claudia; Khan, Susan; Cameron, Michael D; Grant, Wayne; Pocas, Jennifer; Eid, Nibal; Park, HaJeung; Schröter, Thomas; Lograsso, Philip V; Feng, Yangbo

    2013-05-09

    RhoA and its downstream effector ROCK mediate stress fiber formation and cell contraction through their effects on the phosphorylation of myosin light chain (MLC). Inhibition of the RhoA/ROCK pathway has proven to be a promising strategy for several indications such as cardiovascular disease, glaucoma, and inflammatory disease. In 2010, our group reported urea-based ROCK inhibitors as potential antiglaucoma agents. These compounds showed potent IC50 values in enzymatic and cell-based assays and significant intraocular pressure (IOP)-lowering effects in rats (∼7 mmHg). (22) To develop more advanced ROCK inhibitors targeting various potential applications (such as myocardial infarction, erectile dysfunction, multiple sclerosis, etc.) in addition to glaucoma, a thorough SAR for this urea-based scaffold was studied. The detailed optimization process, counter-screening, and in vitro and in vivo DMPK studies are discussed. Potent and selective ROCK inhibitors with various in vivo pharmacokinetic properties were discovered.

  5. Design and synthesis of 1,4-dihydropyridine derivatives as BACE-1 inhibitors.

    PubMed

    Choi, Soo-Jeong; Cho, Joong-Heui; Im, Isak; Lee, So-Deok; Jang, Ji-Yeon; Oh, Yu-Min; Jung, Yong-Keun; Jeon, Eun-Seok; Kim, Yong-Chul

    2010-06-01

    BACE-1 has been shown to be an attractive therapeutic target in Alzheimer's disease (AD). Using a 1,4-dihydropyridine (DHP) scaffold, we synthesized new inhibitors of BACE-1 by modifying the known BACE inhibitor 2 containing a hydroxyethylamine (HEA) motif. Using structure-based drug design based on computer-aided molecular docking, the isophthalamide ring of 2 was replaced with a 1,4-dihydropyridine ring as a brain-targeting strategy. Several of the new dihydropyridine derivatives were synthesized and their BACE-1-inhibitory activities were evaluated using a cell-based, reporter gene assay system that measures the cleavage of alkaline phosphatase (AP)-APP fusion protein by BACE-1. Most of the 1,4-DHP analogs showed BACE-1-inhibitory activities with IC50 values in the range 8-30 microM, suggesting that the 1,4-DHP skeleton may be utilized to develop brain-targeting BACE-1 inhibitors.

  6. Design, synthesis, and biological activity of diaryl ether inhibitors of Toxoplasma gondii enoyl reductase.

    PubMed

    Cheng, Gang; Muench, Stephen P; Zhou, Ying; Afanador, Gustavo A; Mui, Ernest J; Fomovska, Alina; Lai, Bo Shiun; Prigge, Sean T; Woods, Stuart; Roberts, Craig W; Hickman, Mark R; Lee, Patty J; Leed, Susan E; Auschwitz, Jennifer M; Rice, David W; McLeod, Rima

    2013-04-01

    Triclosan is a potent inhibitor of Toxoplasma gondii enoyl reductase (TgENR), which is an essential enzyme for parasite survival. In view of triclosan's poor druggability, which limits its therapeutic use, a new set of B-ring modified analogs were designed to optimize its physico-chemical properties. These derivatives were synthesized and evaluated by in vitro assay and TgENR enzyme assay. Some analogs display improved solubility, permeability and a comparable MIC50 value to that of triclosan. Modeling of these inhibitors revealed the same overall binding mode with the enzyme as triclosan, but the B-ring modifications have additional interactions with the strongly conserved Asn130.

  7. Design and synthesis of disubstituted thiophene and thiazole based inhibitors of JNK

    SciTech Connect

    Hom, Roy K.; Bowers, Simeon; Sealy, Jennifer M.; Truong, Anh P.; Probst, Gary D.; Neitzel, Martin L.; Neitz, R. Jeffrey; Fang, Larry; Brogley, Louis; Wu, Jing; Konradi, Andrei W.; Sham, Hing L.; Tóth, Gergely; Pan, Hu; Yao, Nanhua; Artis, Dean R.; Quinn, Kevin; Sauer, John-Michael; Powell, Kyle; Ren, Zhao; Bard, Frédérique; Yednock, Ted A.; Griswold-Prenner, Irene

    2012-02-28

    From high throughput screening, we discovered compound 1, the prototype for a series of disubstituted thiophene inhibitors of JNK which is selective towards closely related MAP kinases p38 and Erk2. Herein we describe the evolution of these compounds to a novel class of thiophene and thiazole JNK inhibitors that retain favorable solubility, permeability, and P-gp properties for development as CNS agents for treatment of neurodegeneration. Compound 61 demonstrated JNK3 IC{sub 50} = 77 nM and retained the excellent broad kinase selectivity observed for the series.

  8. Design, synthesis and biological evaluation of bambuterol analogues as novel inhibitors of butyrylcholinesterase.

    PubMed

    Wu, Jie; Tian, Yiguang; Wang, Shanping; Pistolozzi, Marco; Jin, Ya; Zhou, Ting; Roy, Gaurab; Xu, Ling; Tan, Wen

    2017-01-27

    An increase activity of butyrylcholinesterase is believed to contribute to Alzheimer's disease. Bambuterol is a known potent inhibitor of butyrylcholinesterase, but it has undesired cardiac effects and less lipophilicity. Thirteen bambuterol analogues were synthesized using 1-(3, 5-dihydroxyphenyl) ethanone as a starting material. In-vitro cholinesterase assay established that the majority of the compounds are specific butyrylcholinesterase inhibitors. Out of the 13 compounds, two bambuterol derivatives, BD-6 and BD-11 exhibited similar efficacies in inhibiting butyrylcholinesterase with fewer effects on heart and enhanced possibilities of permeating through the blood-brain barrier as compared to bambuterol. These bambuterol analogues may provide better alternatives for treatments of Alzheimer's disease.

  9. Design, Synthesis and Biological Evaluation of Biphenylamide Derivatives as Hsp90 C-terminal Inhibitors

    PubMed Central

    Zhao, Huiping; Garg, Gaurav; Zhao, Jinbo; Moroni, Elisabetta; Girgis, Antwan; Franco, Lucas S.; Singh, Swapnil; Colombo, Giorgio; Blagg, Brian S. J.

    2015-01-01

    Modulation of Hsp90 C-terminal function represents a promising therapeutic approach for the treatment of cancer and neurodegenerative diseases. Current drug discovery efforts toward Hsp90 C-terminal inhibition focus on novobiocin, an antibiotic that was transformed into an Hsp90 inhibitor. Based on structural information obtained during the development of novobiocin derivatives and molecular docking studies, scaffolds containing a biphenyl moiety in lieu of the coumarin ring present in novobiocin were identified as new Hsp90 C-terminal inhibitors. Structure-activity relationship studies produced new derivatives that inhibit the proliferation of breast cancer cell lines at nanomolar concentrations, which corresponded directly with Hsp90 inhibition. PMID:25462258

  10. New 7,8-benzoflavanones as potent aromatase inhibitors: synthesis and biological evaluation.

    PubMed

    Yahiaoui, Samir; Fagnere, Catherine; Pouget, Christelle; Buxeraud, Jacques; Chulia, Albert-José

    2008-02-01

    Some natural compounds such as flavonoids are known to possess a moderate inhibitory activity against aromatase, this enzyme being an interesting target for hormone-dependent breast cancer treatment. It has been demonstrated that the modulation of flavonoid skeleton could increase anti-aromatase effect. Therefore, new 7,8-benzoflavanones were synthesized and tested for their activity toward aromatase inhibition. It was observed that the introduction of a benzo ring at position C-7 and C-8 on flavanone skeleton led to new potent aromatase inhibitors, the resulting 7,8-benzoflavanones being until nine times more potent than aminogluthetimide (the first aromatase inhibitor used clinically).

  11. Design and synthesis of 3,3'-biscoumarin-based c-Met inhibitors.

    PubMed

    Xu, Jimin; Ai, Jing; Liu, Sheng; Peng, Xia; Yu, Linqian; Geng, Meiyu; Nan, Fajun

    2014-06-14

    A library of biscoumarin-based c-Met inhibitors was synthesized, based on optimization of 3,3'-biscoumarin hit 3, which was identified as a non-ATP competitive inhibitor of c-Met from a diverse library of coumarin derivatives. Among these compounds, 38 and 40 not only showed potent enzyme activities with IC50 values of 107 nM and 30 nM, respectively, but also inhibited c-Met phosphorylation in BaF3/TPR-Met and EBC-1 cells.

  12. Asymmetric synthesis and evaluation of epoxy-α-acyloxycarboxamides as selective inhibitors of cathepsin L.

    PubMed

    Dos Santos, Deborah A; Deobald, Anna Maria; Cornelio, Vivian E; Ávila, Roberta M D; Cornea, Renata C; Bernasconi, Gilberto C R; Paixão, Marcio W; Vieira, Paulo C; Corrêa, Arlene G

    2017-09-01

    Cathepsin L plays important roles in physiological processes as well as in the development of many pathologies. Recently the attentions were turned to its association with tumor progress what makes essential the development of more potent and selective inhibitors. In this work, epoxipeptidomimetics were investigated as new cathepsin inhibitors. This class of compounds is straightforward obtained by using a green one-pot asymmetric epoxidation/Passerini 3-MCR. A small library of 17 compounds was evaluated against cathepsin L, and among them LSPN423 showed to be the most potent. Investigations of the mechanism suggested a tight binding uncompetitive inhibition. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Synthesis of N-glyoxylyl peptides and their in vitro evaluation as HIV-1 protease inhibitors.

    PubMed

    Qasmi, D; de Rosny, E; René, L; Badet, B; Vergely, I; Boggetto, N; Reboud-Ravaux, M

    1997-04-01

    A series of novel synthetic peptides containing an N-terminal glyoxylyl function (CHOCO-) have been tested as inhibitors of HIV-1 protease. The N-glyoxylyl peptide CHOCO-Pro-Ile-Val-NH2, which fulfills the specificity requirements of the MA/CA protease cleavage site together with the criteria of transition state analogue of the catalyzed reaction, was found to be a moderate competitive inhibitor although favorable interactions were visualized between its hydrated form and the catalytic aspartates using molecular modeling. Increasing the length of the peptide sequence led to compounds acting only as substrates.

  14. Synthesis and biological evaluation of nojirimycin- and pyrrolidine-based trehalase inhibitors

    PubMed Central

    Bini, Davide; Cardona, Francesca; Forcella, Matilde; Parmeggiani, Camilla; Parenti, Paolo; Nicotra, Francesco

    2012-01-01

    Summary A small set of nojirimycin- and pyrrolidine-based iminosugar derivatives has been synthesized and evaluated as potential inhibitors of porcine and insect trehalases. Compounds 12, 13 and 20 proved to be active against both insect and porcine trehalases with selectivity towards the insect glycosidase, while compounds 10, 14 and 16 behaved as inhibitors only of insect trehalase. Despite the fact that the activity was found in the micromolar range, these findings may help in elucidating the structural features of this class of enzymes of different origin, which are still scarcely characterised. PMID:22509223

  15. Design, synthesis and insight into the structure-activity relationship of 1,3-disubstituted indazoles as novel HIF-1 inhibitors.

    PubMed

    An, Hongchan; Kim, Nam-Jung; Jung, Jong-Wha; Jang, Hannah; Park, Jong-Wan; Suh, Young-Ger

    2011-11-01

    Design, synthesis and insight into the structure-activity relationship (SAR) of 1,3-disubstituted indazoles as novel HIF-1 inhibitors are described. In particular, the substituted furan moiety on indazole skeleton as well as its substitution pattern turns out crucial for the high HIF-1 inhibition.

  16. Synthesis of a novel UDP-carbasugar as UDP-galactopyranose mutase inhibitor.

    PubMed

    El Bkassiny, Sandy; N'Go, Inès; Sevrain, Charlotte M; Tikad, Abdellatif; Vincent, Stéphane P

    2014-05-02

    The multistep synthesis of a novel UDP-C-cyclohexene, designed as a high energy intermediate analogue of the UDP-galactopyranose mutase (UGM) catalyzed isomerization reaction, is reported. The synthesis of the central carbasugar involved the preparation of a galactitol derivative bearing two olefins necessary for the construction of the cyclohexene ring by a ring-closing metathesis as a key step. Further successive phosphonylation, deprotection, and UMP coupling provided the target molecule. The final molecule was assayed against UGM and compared with UDP-C-Galf, the C-glycosidic UGM substrate analogue.

  17. Synthesis and biological evaluation of alkenyldiarylmethane HIV-1 non-nucleoside reverse transcriptase inhibitors that possess increased hydrolytic stability.

    PubMed

    Cullen, Matthew D; Deng, Bo-Liang; Hartman, Tracy L; Watson, Karen M; Buckheit, Robert W; Pannecouque, Christophe; Clercq, Erik De; Cushman, Mark

    2007-10-04

    Non-nucleoside inhibitors of HIV reverse transcriptase (NNRTIs), albeit not the mainstays of HIV/AIDS treatment, have become increasingly important in highly active antiretroviral therapy (HAART) due to their unique mechanism of action. Several years ago our group identified the alkenyldiarylmethanes (ADAMs) as a potent and novel class of NNRTIs; however, the most active compounds were found to be metabolically unstable. Subsequent work has led to the synthesis of 33 analogues, with improved metabolic profiles, through the replacement of labile esters with various heterocycles, nitriles, and thioesters. As a result, a number of hydrolytically stable NNRTIs were identified with anti-HIV activity in the nanomolar concentration range. Furthermore, an improved pharmacophore model has been developed based on the new ADAM series, in which a salicylic acid-derived aryl ring is oriented cis to the side chain and the aryl ring that is trans to the side chain contains a hydrogen bond acceptor site within the plane of the ring.

  18. Synthesis of chiral ND-322, ND-364 and ND-364 derivatives as selective inhibitors of human gelatinase.

    PubMed

    Yan, Yugang; Chen, Xueying; Yang, Xinying; Zhang, Jian; Xu, Wenfang; Zhang, Yingjie

    2015-10-15

    Compounds 10 (ND-322) and 15 (ND-364) are potent selective inhibitors for gelatinases, matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9). However, both of them are racemates. Herein we report facile synthesis of optically active (R)- and (S)-enantiomers of compounds 10 and 15. And the sulfonyl of 15 was transformed to sulfinyl to obtain four epimeric mixtures. All synthesized thiirane-based compounds were evaluated in MMP2 and MMP9 inhibitory assays. Our results indicated that the configuration of thiirane moiety had little effects on gelatinase inhibition, but the substitution of sulfinyl for sulfonyl was detrimental to gelatinase inhibition. Besides, all target compounds exhibited no inhibition against other two Zn(2+) dependant metalloproteases, aminopeptidase N (APN) and histone deacetylases (HDACs), which confirmed the unique Zn(2+) chelation mechanism of thiirane moiety against gelatinases. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Novel 2-arylazoimidazole derivatives as inhibitors of Trypanosoma cruzi proliferation: Synthesis and evaluation of their biological activity.

    PubMed

    Salerno, Alejandra; Celentano, Ana M; López, Julieta; Lara, Virginia; Gaozza, Carlos; Balcazar, Darío E; Carrillo, Carolina; Frank, Fernanda M; Blanco, María M

    2017-01-05

    In this work, the synthesis of a series of 2-arylazoimidazole derivatives 6-20 has been achieved through the reaction of imidazole with aryldiazonium salts, followed by ultrasound-assisted alkylation. This approach has important advantages including higher yield, shorter reaction times and milder reaction conditions. The structures of the compounds obtained were determined by MS, IR; and (1)H and (13)C NMR. The anti-Trypanosoma cruzi activity of the 15 compounds obtained was evaluated. Two compounds with piperidino substituents in the carboxamide moiety proved to be effective inhibitors of epimastigote proliferation, obtaining inhibition values comparable to those achieved with the reference drug Benznidazole. Besides, these compounds displayed low cytotoxicity on mammalian cells. In vivo, both compounds protected mice against a challenge with a lethal Trypanosoma cruzi strain. These results allow us to propose 2-arylazoimidazoles as lead compounds for the design of novel drugs to treat Chagas' disease.

  20. Design, synthesis and evaluation of semi-synthetic triazole-containing caffeic acid analogues as 5-lipoxygenase inhibitors.

    PubMed

    De Lucia, Daniela; Lucio, Oscar Méndez; Musio, Biagia; Bender, Andreas; Listing, Monika; Dennhardt, Sophie; Koeberle, Andreas; Garscha, Ulrike; Rizzo, Roberta; Manfredini, Stefano; Werz, Oliver; Ley, Steven V

    2015-08-28

    In this work the synthesis, structure-activity relationship (SAR) and biological evaluation of a novel series of triazole-containing 5-lipoxygenase (5-LO) inhibitors are described. The use of structure-guided drug design techniques provided compounds that demonstrated excellent 5-LO inhibition with IC50 of 0.2 and 3.2 μm in cell-based and cell-free assays, respectively. Optimization of binding and functional potencies resulted in the identification of compound 13d, which showed an enhanced activity compared to the parent bioactive compound caffeic acid 5 and the clinically approved zileuton 3. Compounds 15 and 16 were identified as lead compounds in inhibiting 5-LO products formation in neutrophils. Their interference with other targets on the arachidonic acid pathway was also assessed. Cytotoxicity tests were performed to exclude a relationship between cytotoxicity and the increased activity observed after structure optimization.

  1. Locomotion Inhibition of Cimex lectularius L. Following Topical, Sublethal Dose Application of the Chitin Synthesis Inhibitor Lufenuron

    PubMed Central

    Campbell, Brittany; Baldwin, Rebecca; Koehler, Philip

    2017-01-01

    To date, few studies have evaluated chitin synthesis inhibitors against bed bugs, although they would provide an alternative mode of action to circumvent insecticide resistance. Acute and sublethal effects of lufenuron were evaluated against two strains of the common bed bug. Combined acute and sublethal effects were used to calculate effective doses. The dose that was effective against 50% of Harlan strain bed bugs was 0.0081% (w/v), and was much higher against Bradenton strain bed bugs (1.11% w/v). Sublethal doses were chosen to determine the effect that leg abnormalities had on pulling force. Both Harlan and Bradenton strain bed bugs had significantly lower locomotion ability (p < 0.0001) following topical application of lufenuron. The observed sublethal effects that limit locomotion could prevent bed bugs from moving within a domicile and taking a blood meal, subsequently reducing a bed bug population over time. PMID:28862646

  2. Diastereoselective synthesis and molecular docking studies of novel fused tetrahydropyridine derivatives as new inhibitors of HIV protease

    NASA Astrophysics Data System (ADS)

    Mohammadi, Ali A.; Taheri, Salman; Amouzegar, Ali; Ahdenov, Reza; Halvagar, Mohammad Reza; Sadr, Ahmad Shahir

    2017-07-01

    An efficient one-pot, catalyst-free, and four-components procedure for the synthesis of novel 10b-hydroxy-4-nitro-5-phenyl-2,3,5,5a-tetrahydro-1H-imidazo[1,2-a]indeno[2,1-e]pyridin-6(10bH)-one derivatives from corresponding diamine, nitro ketene dithioacetal, aldehydes and 1,3-indandione in ethanol has been achieved upon a Knoevenagel condensation-Michael addition-tautomerism-cyclisation sequence. All the newly synthesized compounds were screened for molecular docking studies. Molecular docking studies were carried out using the crystal structure of HIV protease enzyme. Some of the compounds obtain minimum binding energy and good affinity toward the active pocket of HIV protease enzyme in compare with Saquinavir as a standard HIV protease inhibitor.

  3. Locomotion Inhibition of Cimex lectularius L. Following Topical, Sublethal Dose Application of the Chitin Synthesis Inhibitor Lufenuron.

    PubMed

    Campbell, Brittany; Baldwin, Rebecca; Koehler, Philip

    2017-09-01

    To date, few studies have evaluated chitin synthesis inhibitors against bed bugs, although they would provide an alternative mode of action to circumvent insecticide resistance. Acute and sublethal effects of lufenuron were evaluated against two strains of the common bed bug. Combined acute and sublethal effects were used to calculate effective doses. The dose that was effective against 50% of Harlan strain bed bugs was 0.0081% (w/v), and was much higher against Bradenton strain bed bugs (1.11% w/v). Sublethal doses were chosen to determine the effect that leg abnormalities had on pulling force. Both Harlan and Bradenton strain bed bugs had significantly lower locomotion ability (p < 0.0001) following topical application of lufenuron. The observed sublethal effects that limit locomotion could prevent bed bugs from moving within a domicile and taking a blood meal, subsequently reducing a bed bug population over time.

  4. Synthesis and Evaluation of Derivatives of the Proteasome Deubiquitinase Inhibitor b-AP15

    PubMed Central

    Wang, Xin; D'Arcy, Pádraig; Caulfield, Thomas R.; Paulus, Aneel; Chitta, Kasyapa; Mohanty, Chitralekha; Gullbo, Joachim; Chanan-Khan, Asher; Linder, Stig

    2016-01-01

    The ubiquitin–proteasome system (UPS) is increasingly recognized as a therapeutic target for the development of anticancer therapies. The success of the 20S proteasome core particle (20S CP) inhibitor bortezomib in the clinical management of multiple myeloma has raised the possibility of identifying other UPS components for therapeutic intervention. We previously identified the small molecule b-AP15 as an inhibitor of 19S proteasome deubiquitinase (DUB) activity. Building upon our previous data, we performed a structure–activity relationship (SAR) study on b-AP15 and identified VLX1570 as an analog with promising properties, including enhanced potency and improved solubility in aqueous solution. In silico modeling was consistent with interaction of VLX1570 with key cysteine residues located at the active sites of the proteasome DUBs USP14 and UCHL5. VLX1570 was found to inhibit proteasome deubiquitinase activity in vitro in a manner consistent with competitive inhibition. Furthermore, using active-site-directed probes, VLX1570 also inhibited proteasome DUB activity in exposed cells. Importantly, VLX1570 did not show inhibitory activity on a panel of recombinant non-proteasome DUBs, on recombinant kinases, or on caspase-3 activity, suggesting that VLX1570 is not an overtly reactive general enzyme inhibitor. Taken together, our data shows the chemical and biological properties of VLX1570 as an optimized proteasome DUB inhibitor. PMID:25854145

  5. Synthesis and evaluation of tripeptidic plasmin inhibitors with nitrile as warhead.

    PubMed

    Teno, Naoki; Otsubo, Tadamune; Gohda, Keigo; Wanaka, Keiko; Sueda, Takuya; Ikeda, Kiyoshi; Hijikata-Okunomiya, Akiko; Tsuda, Yuko

    2012-10-01

    Plasmin is best known as the key molecule in the fibrinolytic system, which is critical for clot lysis and can initiate matrix metalloproteinase (MMP) activation cascade. Along with MMP, plasmin is suggested to be involved in physiological processes that are linked to the risk of carcinoma formation. Plasmin inhibitors could be perceived as a promising new principle in the treatment of diseases triggered by plasmin. On the basis of the peptidic sequence derived from the synthetic plasmin substrate, a series of peptidic plasmin inhibitors possessing nitrile as warhead were prepared and evaluated for their inhibitory activities against plasmin and other serine proteases, plasma kallikrein and urokinase. The most potent peptidic inhibitors with the nitrile warhead exhibit the potency toward plasmin (IC(50)  = 7.7-11 μM) and are characterized by their selectivity profile against plasma kallikrein and urokinase. The results and molecular modeling of the peptidic inhibitor complexed with plasmin reveal that the P2 residue makes favorable contacts with the open binding pocket comprising the S2 and S3 subsites of plasmin.

  6. Synthesis and evaluation of novel quinolinones as HIV-1 reverse transcriptase inhibitors.

    PubMed

    Patel, M; McHugh, R J; Cordova, B C; Klabe, R M; Bacheler, L T; Erickson-Viitanen, S; Rodgers, J D

    2001-07-23

    A series of 4,4-disubstituted quinolinones was prepared and evaluated as HIV-1 reverse transcriptase inhibitors. The C-3 substituted compound 9h displayed improved antiviral activity against clinically significant single (K103N) and double (K103N/L100I) mutant viruses.

  7. An Efficient Synthesis of 5-Amido-3-Hydroxy-4-Pyrones as Inhibitors of Matrix Metalloproteinases

    PubMed Central

    Yan, Yi-Long; Cohen, Seth M.

    2008-01-01

    3-Hydroxy-4-pyrones are a class of important metal chelators with versatile medicinal applications. An efficient pathway for the preparation of new 5-amido-3-hydroxy-4-pyrone derivatives has been developed. The synthesized 5-amido-3-hydroxy-4-pyrones have been evaluated as inhibitors of matrix metalloproteinases. PMID:17521196

  8. Cyanopeptide analogues: new lead structures for the design and synthesis of new thrombin inhibitors.

    PubMed

    Radau, G; Stürzebecher, J

    2002-11-01

    This contribution deals with the structure-based design and syntheses of the new serine protease inhibitors RA-1001 and RA-1002, which are analogues of the blue-green algae derived cyanopeptide aeruginosin 98-B. Both compounds inhibit thrombin with Ki values of 5.6 microM and 8.7 microM, respectively.

  9. New hydroxamate inhibitors of neurotensin-degrading enzymes. Synthesis and enzyme active-site recognition.

    PubMed

    Bourdel, E; Doulut, S; Jarretou, G; Labbe-Jullie, C; Fehrentz, J A; Doumbia, O; Kitabgi, P; Martinez, J

    1996-08-01

    Selective and mixed inhibitors of the three zinc metallopeptidases that degrade neurotensin (NT), e.g. endopeptidase 24-16 (EC 3.4.24.16), endopeptidase 24-11 (EC 3.4.24.11 or neutral endopeptidase, NEP) and endopeptidase 24-15 (EC 3.4.24.15), and leucine-aminopeptidase (type IV-S), that degrades the NT-related peptides, Neuromedin N (NN), are of great interest. On the structural basis of compound JMV 390-1 (N-[3-[(hydroxyamino)carbonyl]-1-oxo-2(R)-benzylpropyl]-L- isoleucyl-L-leucine), which was a full inhibitor of the major NT degrading enzymes, several hydroxamate inhibitors corresponding to the general formula HONHCO-CH2-CH(CH2-C6H5)CO-X-Y-OH (with X-Y = dipeptide) have been synthesized. Compound 7a (X-Y = Ile-Ala) was nearly 40-times more potent in inhibiting EC 24-16 than NEP and more than 800-times more potent than EC 24-15, with an IC50 (12 nM) almost equivalent to that of compound JMV 390-1. Therefore, this compound is an interesting selective inhibitor of EC 24-16, and should be an interesting probe to explore the physiological involvement of EC 24-16 in the metabolism of neurotensin.

  10. Synthesis and biological evaluation of analogues of AKT (protein kinase B) inhibitor-IV.

    PubMed

    Sun, Qi; Wu, Runzhi; Cai, Sutang; Lin, Yuan; Sellers, Llewlyn; Sakamoto, Kaori; He, Biao; Peterson, Blake R

    2011-03-10

    Inhibitors of the PI3-kinase/AKT (protein kinase B) pathway are under investigation as anticancer and antiviral agents. The benzimidazole derivative AKT inhibitor-IV (ChemBridge 5233705) affects this pathway and exhibits potent anticancer and antiviral activity. To probe its biological activity, we synthesized AKT inhibitor-IV and 21 analogues using a novel six-step route based on ZrCl(4)-catalyzed cyclization of 1,2-arylenediamines with α,β-unsaturated aldehydes. We examined effects on viability of HeLa carcinoma cells, viability of normal human cells (NHBE), replication of recombinant parainfluenza virus 5 (PIV5) in HeLa cells, and replication of the intracellular bacterium Mycobacterium fortuitum in HeLa cells. Replacement of the benzimidazole N-ethyl substitutent of AKT inhibitor-IV with N-hexyl and N-dodecyl groups enhanced antiviral activity and cytotoxicity against the cancer cell line, but these compounds showed substantially lower toxicity (from 6-fold to >20-fold) against NHBE cells and no effect on M. fortuitum, suggesting inhibition of one or more host protein(s) required for proliferation of cancer cells and PIV5. The key structural elements identified here may facilitate identification of targets of this highly biologically active scaffold.

  11. Synthesis of PSA Inhibitors as SPECT- and PET-Based Imaging Agents for Prostate Cancer

    DTIC Science & Technology

    2011-06-01

    for their ability to inhibit PSA and chymotrypsin. 15. SUBJECT TERMS Prostate cancer , PSA inhibitors, boronic acids, peptidomimetics, serine protease...prostate cancer . First, all men undergoing androgen ablation, eventually relapse and no longer respond to hormone treatment . Therefore, there is an...Imaging Agents for Prostate Cancer PRINCIPAL INVESTIGATOR: Maya Kostova, Ph.D. CONTRACTING ORGANIZATION: Johns Hopkins University

  12. Novel protein kinase C inhibitors: synthesis and PKC inhibition of beta-substituted polythiophene derivatives.

    PubMed

    Xu, W C; Zhou, Q; Ashendel, C L; Chang, C T; Chang, C J

    1999-08-02

    A series of beta-substituted polythiophene derivatives was synthesized through palladium-catalyzed coupling reaction. Their structure-protein kinase C (PKC) inhibitory activity relationship was studied. The carboxaldehyde and hydroxymethyl derivatives of alpha-terthiophene were potent PKC inhibitors (IC50 = 10(-7) M).

  13. Stereoselective synthesis of 3,3-diarylacrylonitriles as tubulin polymerization inhibitors.

    PubMed

    Fang, Zhenglai; Song, Yunlong; Sarkar, Taradas; Hamel, Ernest; Fogler, William E; Agoston, Gregory E; Fanwick, Phillip E; Cushman, Mark

    2008-06-06

    A series of 3,3-diarylacrylonitriles were synthesized stereoselectively as tubulin polymerization inhibitors for potential use in cancer chemotherapy. This synthetic route features stannylcupration and palladium-catalyzed Stille cross-coupling chemistry, allowing both E and Z isomers of 3,3-diarylacrylonitriles to be prepared in a very short sequence of reactions.

  14. Design and synthesis of potent, non-peptidic inhibitors of HPTPbeta.

    PubMed

    Amarasinghe, Kande K D; Evdokimov, Artem G; Evidokimov, Artem G; Xu, Kevin; Clark, Cynthia M; Maier, Matthew B; Srivastava, Anil; Colson, Anny-Odile; Gerwe, Gina S; Stake, George E; Howard, Brian W; Pokross, Matthew E; Gray, Jeffrey L; Peters, Kevin G

    2006-08-15

    The sulfamic acid phosphotyrosine mimetic was coupled with a previously known malonate template to obtain highly selective and potent inhibitors of HPTPbeta. Potentially hydrolyzable malonate ester functionalities were replaced with 1,2,4-oxadiazoles without a significant effect on HPTPbeta potency.

  15. AMPK activation protects cells from oxidative stress-induced senescence via autophagic flux restoration and intracellular NAD(+) elevation.

    PubMed

    Han, Xiaojuan; Tai, Haoran; Wang, Xiaobo; Wang, Zhe; Zhou, Jiao; Wei, Xiawei; Ding, Yi; Gong, Hui; Mo, Chunfen; Zhang, Jie; Qin, Jianqiong; Ma, Yuanji; Huang, Ning; Xiang, Rong; Xiao, Hengyi

    2016-06-01

    AMPK activation is beneficial for cellular homeostasis and senescence prevention. However, the molecular events involved in AMPK activation are not well defined. In this study, we addressed the mechanism underlying the protective effect of AMPK on oxidative stress-induced senescence. The results showed that AMPK was inactivated in senescent cells. However, pharmacological activation of AMPK by metformin and berberine significantly prevented the development of senescence and, accordingly, inhibition of AMPK by Compound C was accelerated. Importantly, AMPK activation prevented hydrogen peroxide-induced impairment of the autophagic flux in senescent cells, evidenced by the decreased p62 degradation, GFP-RFP-LC3 cancellation, and activity of lysosomal hydrolases. We also found that AMPK activation restored the NAD(+) levels in the senescent cells via a mechanism involving mostly the salvage pathway for NAD(+) synthesis. In addition, the mechanistic relationship of autophagic flux and NAD(+) synthesis and the involvement of mTOR and Sirt1 activities were assessed. In summary, our results suggest that AMPK prevents oxidative stress-induced senescence by improving autophagic flux and NAD(+) homeostasis. This study provides a new insight for exploring the mechanisms of aging, autophagy and NAD(+) homeostasis, and it is also valuable in the development of innovative strategies to combat aging.

  16. Synthesis of 11-Thialinoleic Acid and 14-Thialinoleic Acid, Inhibitors of Soybean and Human Lipoxygenases

    PubMed Central

    Jacquot, Cyril; McGinley, Chris M.; Plata, Erik; Holman, Theodore R.

    2010-01-01

    Lipoxygenases catalyse the oxidation of polyunsaturated fatty acids and have been invoked in many diseases including cancer, atherosclerosis and Alzheimer’s disease. Currently, no X-ray structures are available with substrate or substrate analogues bound in a productive conformation. Such structures would be very useful for examining interactions between substrate and active site residues. Reported here are the syntheses of linoleic acid analogues containing a sulphur atom at the 11 or 14 positions. The key steps in the syntheses were the incorporation of sulphur using nucleophilic attack of metallated alkynes on electrophilic sulphur compounds and the subsequent stereospecific tantalum-mediated reduction of the alkynylsulphide to the cis-alkenylsulphide. Kinetic assays performed with soybean lipoxygenase-1 showed that both 11-thialinoleic acid and 14-thialinoleic acid were competitive inhibitors with respect to linoleic acid with Ki values of 22 and 35 µM, respectively. On the other hand, 11-thialinoleic acid was a noncompetitive inhibitor with respect to arachidonic acid with Kis and Kii values of 48 and 36 µM, respectively. 11-Thialinoleic acid was also a noncompetitive inhibitor of human 15-lipoxygenase-1 with arachidonic acid (Kis = 11.4 µM, Kii = 18.1 µM) or linoleic acid as substrate (Kis = 20.1 µM, Kii = 20.0 µM), and a competitive inhibitor of human 12-lipoxygenase with arachidonic acid as substrate (Ki = 2.5 µM). The presence of inhibitor did not change the regioselectivity of soybean lipoxygenase-1, human 12- or 15-lipoxygenase-1. PMID:18972057

  17. Cinnamide Derivatives as Mammalian Arginase Inhibitors: Synthesis, Biological Evaluation and Molecular Docking

    PubMed Central

    Pham, Thanh-Nhat; Bordage, Simon; Pudlo, Marc; Demougeot, Céline; Thai, Khac-Minh; Girard-Thernier, Corine

    2016-01-01

    Arginases are enzymes that are involved in many human diseases and have been targeted for new treatments. Here a series of cinnamides was designed, synthesized and evaluated in vitro and in silico for their inhibitory activity against mammalian arginase. Using a microassay on purified liver bovine arginase (b-ARG I), (E)-N-(2-phenylethyl)-3,4-dihydroxycinnamide, also named caffeic acid phenylamide (CAPA), was shown to be slightly more active than our natural reference inhibitor, chlorogenic acid (IC50 = 6.9 ± 1.3 and 10.6 ± 1.6 µM, respectively) but it remained less active that the synthetic reference inhibitor Nω-hydroxy-nor-l-arginine nor-NOHA (IC50 = 1.7 ± 0.2 µM). Enzyme kinetic studies showed that CAPA was a competitive inhibitor of arginase with Ki = 5.5 ± 1 µM. Whereas the activity of nor-NOHA was retained (IC50 = 5.7 ± 0.6 µM) using a human recombinant arginase I (h-ARG I), CAPA showed poorer activity (IC50 = 60.3 ± 7.8 µM). However, our study revealed that the cinnamoyl moiety and catechol function were important for inhibitory activity. Docking results on h-ARG I demonstrated that the caffeoyl moiety could penetrate into the active-site pocket of the enzyme, and the catechol function might interact with the cofactor Mn2+ and several crucial amino acid residues involved in the hydrolysis mechanism of arginase. The results of this study suggest that 3,4-dihydroxycinnamides are worth being considered as potential mammalian arginase inhibitors, and could be useful for further research on the development of new arginase inhibitors. PMID:27690022

  18. A Two-Step Synthesis of Virstatin, a Virulence Inhibitor of "Vibrio cholerae"

    ERIC Educational Resources Information Center

    McDonald, Chriss E.

    2009-01-01

    Virstatin, an "N"-butanoic acid substituted naphthalimide, inhibits the ability of "Vibrio cholerae" to cause disease. A three-week experiment involving synthesis, purification, and spectral characterization of this compound is described. This experiment is appropriate for organic chemistry. It has been performed with three lab sections of about…

  19. Total synthesis and absolute stereochemistry of the proteasome inhibitors cystargolides A and B.

    PubMed

    Tello-Aburto, Rodolfo; Hallada, Liam P; Niroula, Doleshwar; Rogelj, Snezna

    2015-10-28

    The absolute stereochemistry of the cystargolides was determined by total synthesis. Evaluation of synthetic cystargolides and derivatives showed that the natural (2S,3R) stereochemistry is essential for activity. Moreover, benzyl esters (-)-10 and (-)-15 were found to be about 100 times more potent, and to selectively kill MCF-7 cancerous cells.

  20. A Two-Step Synthesis of Virstatin, a Virulence Inhibitor of "Vibrio cholerae"

    ERIC Educational Resources Information Center

    McDonald, Chriss E.

    2009-01-01

    Virstatin, an "N"-butanoic acid substituted naphthalimide, inhibits the ability of "Vibrio cholerae" to cause disease. A three-week experiment involving synthesis, purification, and spectral characterization of this compound is described. This experiment is appropriate for organic chemistry. It has been performed with three lab sections of about…

  1. Design, synthesis, and structure-activity relationship studies of new phenolic DNA gyrase inhibitors.

    PubMed

    Lübbers, Thomas; Angehrn, Peter; Gmünder, Hans; Herzig, Silvia

    2007-08-15

    Starting from a biased needle screening hit 3a, we report herein the design and synthesis of a series of novel 2,3-dihydroisoindol-1-ones structurally related to cyclothialidine 2 with DNA gyrase inhibitory activity. In this series, some compounds exhibited promising antibacterial activity against gram-positive bacterial strains.

  2. Total Synthesis and Absolute Stereochemistry of the Proteasome Inhibitors Cystargolides A and B

    PubMed Central

    Tello-Aburto, Rodolfo; Hallada, Liam P.; Niroula, Doleshwar; Rogelj, Snezna

    2015-01-01

    The absolute stereochemistry of the cystargolides was determined by total synthesis. Evaluation of synthetic cystargolides and derivatives showed that the natural (2S,3R) stereochemistry is essential for activity. Moreover, benzyl esters (−)-10 and (−)-15 were found to be about 100 times more potent, and to selectively kill MCF-7 cancerous cells. PMID:26400369

  3. General approach to glycosidase inhibitors. Enantioselective synthesis of deoxymannojirimycin and swainsonine.

    PubMed

    Martín, Rubén; Murruzzu, Caterina; Pericàs, Miquel A; Riera, Antoni

    2005-03-18

    [reaction: see text] Deoxymannojirimycin (2) and swainsonine (4) have been synthesized from each enantiomer of the same bicyclic carbamate precursor 7. The key intermediate was prepared by a simple and efficient three-step synthesis involving RCM of the diene 8, which in turn is easily accessible in any configuration from enantiomerically enriched 2,3-epoxy-4-penten-1-ol 9.

  4. Antidepressants may lead to a decrease in niacin and NAD in patients with poor dietary intake.

    PubMed

    Viljoen, Margaretha; Swanepoel, Annie; Bipath, Priyesh

    2015-03-01

    The term niacin is the generic name for the two compounds nicotinic acid and nicotinamide, the major dietary precursors for two important coenzymes, nicotinamide adenine dinucleotide (NAD) and its phosphorylated form, NADP. Niacin is important for the maintenance of cellular integrity and energy production and is involved in more than 500 intracellular reactions. Deficiencies of niacin may contribute to neuropsychiatric and neurodegenerative disorders. Patients who develop nutritional deficiencies as a result of poor dietary intake, especially inadequate intake of proteins and vitamins, could potentially suffer from niacin deficiency and NAD depletion. However, de novo synthesis of niacin and NAD in the kynurenine pathway of tryptophan metabolism may compensate for impaired dietary intake. The rate of synthesis of NAD and niacin from tryptophan oxidation depends on the induction of the enzyme indoleamine 2,3-dioxygenase (IDO) by pro-inflammatory cytokines such as interferon-gamma. Niacin synthesis is not limited by a decrease in tryptophan and excessive IDO activity may therefore lead to a decline in tryptophan levels. Antidepressants have an anti-inflammatory effect, including reduction of interferon-gamma and therefore inhibition of IDO, the rate-limiting enzyme of the kynurenine pathway. In theory, this could account for increased serotonin as more tryptophan becomes available for serotonin synthesis. However, the downside may be that less NAD and niacin are synthesised downstream, which could exacerbate common psychiatric problems. It is our hypothesis that patients with poor dietary intake, who are treated with antidepressants, are at risk of developing niacin/NAD deficiency with possible development of associated neuropsychiatric symptoms. We therefore propose that niacin supplementation be considered in patients with inadequate diets who are treated with antidepressants. We believe that if this does not happen, a subclinical niacin deficiency may result

  5. Selective n-butanol production by Clostridium sp. MTButOH1365 during continuous synthesis gas fermentation due to expression of synthetic thiolase, 3-hydroxy butyryl-CoA dehydrogenase, crotonase, butyryl-CoA dehydrogenase, butyraldehyde dehydrogenase, and NAD-dependent butanol dehydrogenase.

    PubMed

    Berzin, Vel; Tyurin, Michael; Kiriukhin, Michael

    2013-02-01

    Acetogen Clostridum sp. MT1962 produced 287 mM acetate (p < 0.005) and 293 mM ethanol (p < 0.005) fermenting synthesis gas blend 60% CO and 40% H₂ in single-stage continuous fermentation. This strain was metabolically engineered to the biocatalyst Clostridium sp. MTButOH1365. The engineered biocatalyst lost production of ethanol and acetate while initiated the production of 297 mM of n-butanol (p < 0.005). The metabolic engineering comprised Cre-lox66/lox71-based elimination of phosphotransacetylase and acetaldehyde dehydrogenase along with integration to chromosome synthetic thiolase, 3-hydroxy butyryl-CoA dehydrogenase, crotonase, butyryl-CoA dehydrogenase, butyraldehyde dehydrogenase, and NAD-dependent butanol dehydrogenase. This is the first report on elimination of acetate and ethanol production genes and expression of synthetic gene cluster encoding n-butanol biosynthesis pathway in acetogen biocatalyst for selective fuel n-butanol production with no antibiotic support for the introduced genes.

  6. 7 CFR 614.15 - Implementation of final NAD determinations.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 6 2014-01-01 2014-01-01 false Implementation of final NAD determinations. 614.15 Section 614.15 Agriculture Regulations of the Department of Agriculture (Continued) NATURAL RESOURCES... Implementation of final NAD determinations. (a) No later than 30 days after a NAD determination becomes a...

  7. 7 CFR 614.15 - Implementation of final NAD determinations.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 6 2013-01-01 2013-01-01 false Implementation of final NAD determinations. 614.15 Section 614.15 Agriculture Regulations of the Department of Agriculture (Continued) NATURAL RESOURCES... Implementation of final NAD determinations. (a) No later than 30 days after a NAD determination becomes a...

  8. Design, Synthesis and Structure-Activity Relationship Studies of Novel Survivin Inhibitors with Potent Anti-Proliferative Properties

    PubMed Central

    Xiao, Min; Wang, Jin; Lin, Zongtao; Lu, Yan; Li, Zhenmei; White, Stephen W.; Miller, Duane D.; Li, Wei

    2015-01-01

    The anti-apoptotic protein survivin is highly expressed in most human cancer cells, but has very low expression in normal differentiated cells. Thus survivin is considered as an attractive cancer drug target. Herein we report the design and synthesis of a series of novel survivin inhibitors based on the oxyquinoline scaffold from our recently identified hit compound UC-112. These new analogs were tested against a panel of cancer cell lines including one with multidrug-resistant phenotype. Eight of these new UC-112 analogs showed IC50 values in the nanomole range in anti-proliferative assays. The best three compounds among them along with UC-112 were submitted for NCI-60 cancer cell line screening. The results indicated that structural modification from UC-112 to our best compound 4g has improved activity by four folds (2.2 μM for UC-112 vs. 0.5 μM for 4g, average GI50 values over all cancer cell lines in the NCI-60 panel).Western blot analyses demonstrated the new compounds maintained high selectivity for survivin inhibition over other members in the inhibition of apoptosis protein family. When tested in an A375 human melanoma xenograft model, the most active compound 4g effectively suppressed tumor growth and strongly induced cancer cell apoptosis in tumor tissues. This novel scaffold is promising for the development of selective survivin inhibitors as potential anticancer agents. PMID:26070194

  9. Design, Synthesis, and Biological Evaluation of Tetra-Substituted Thiophenes as Inhibitors of p38α MAPK

    PubMed Central

    Vinh, Natalie B; Devine, Shane M; Munoz, Lenka; Ryan, Renae M; Wang, Bing H; Krum, Henry; Chalmers, David K; Simpson, Jamie S; Scammells, Peter J

    2015-01-01

    p38α mitogen-activated protein kinase (MAPK) plays a role in several cellular processes and consequently has been a therapeutic target in inflammatory diseases, cancer, and cardiovascular disease. A number of known p38α MAPK inhibitors contain vicinal 4-fluorophenyl/4-pyridyl rings connected to either a 5- or 6-membered heterocycle. In this study, a small library of substituted thiophene-based compounds bearing the vicinal 4-fluorophenyl/4-pyridyl rings was designed using computational docking as a visualisation tool. Compounds were synthesised and evaluated in a fluorescence polarisation binding assay. The synthesised analogues had a higher binding affinity to the active phosphorylated form of p38α MAPK than the inactive nonphosphorylated form of the protein. 4-(2-(4-fluorophenyl)thiophen-3-yl)pyridine had a Ki value of 0.6 μm to active p38α MAPK highlighting that substitution of the core ring to a thiophene retains affinity to the enzyme and can be utilised in p38α MAPK inhibitors. This compound was further elaborated using a substituted phenyl ring in order to probe the second hydrophobic pocket. Many of these analogues exhibited low micromolar affinity to active p38α MAPK. The suppression of neonatal rat fibroblast collagen synthesis was also observed suggesting that further development of these compounds may lead to potential therapeutics having cardioprotective properties. PMID:25861571

  10. Synthesis of D-lyxitol and D-ribitol analogues of the naturally occurring glycosidase inhibitor salacinol.

    PubMed

    Kumar, Nag S; Pinto, B Mario

    2005-12-12

    The synthesis of analogues of the naturally occurring glycosidase inhibitor, salacinol, in which the D-arabinitol ring has been replaced by D-lyxitol or D-ribitol, is described. Salacinol is one of the active principles in the aqueous extracts of Salacia reticulata, which are traditionally used in India and Sri Lanka for the treatment of Type II diabetes. The synthetic strategy relies on the nucleophilic attack of 1,4-anhydro-2,3,5-tri-O-p-methoxybenzyl-4-thio-D-lyxitol or 1,4-anhydro-2,3,5-tri-O-p-methoxybenzyl-4-thio-D-ribitol at the least hindered carbon of the benzylidene-protected L-cyclic sulfate derived from L-erythritol. Screening of these compounds against recombinant human maltase glucoamylase (MGA), a critical intestinal glucosidase involved in the processing of oligosaccharides of glucose into glucose itself, shows that they are not effective inhibitors of MGA and demonstrates the importance of the d-arabinitol configuration in the heterocyclic ring for effective inhibition.

  11. Affinity capture of a mammalian DNA polymerase beta by inhibitors immobilized to resins used in solid-phase organic synthesis.

    PubMed

    Kuramochi, Kouji; Haruyama, Tetsuya; Takeuchi, Ryo; Sunoki, Takashi; Watanabe, Madoka; Oshige, Masahiko; Kobayashi, Susumu; Sakaguchi, Kengo; Sugawara, Fumio

    2005-01-01

    The application of resins normally used in solid-phase organic synthesis to the affinity capture of a mammalian DNA polymerase beta (pol beta) is reported. Lithocholic acid (LCA), an inhibitor of pol beta, was immobilized on various solid supports, and the batch affinity purification of pol beta from a mixture of proteins using these LCA-immobilized resins was examined. Of the resins tested, TentaGel was the most effective at purifying pol beta and at resisting nonspecific absorption of proteins. The immobilized LCA recognized pol beta specifically, which resulted in pol beta binding to the resin. Using the LCA-immobilized resin, it was possible to purify pol beta from a mixture of proteins. Furthermore, it was possible to concentrate pol beta from a crude nuclear extract of human T lymphoma Molt4 cells. To facilitate the immobilization of compounds on TentaGel resins, we also designed and prepared photoaffinity beads containing a photoreactive group at the free termini of the TentaGel resin. The pol beta inhibitors LCA, C18-beta-SQDG, and epolactaene were immobilized on the photoaffinity beads by photoreaction. The batch affinity purification of pol beta from a protein mixture could be also achieved with these beads.

  12. Design, Synthesis and Biological Evaluation of New 1, 4-Dihydropyridine (DHP) Derivatives as Selective Cyclooxygenase-2 Inhibitors.

    PubMed

    Sabakhi, Iman; Topuzyan, Vigen; Hajimahdi, Zahra; Daraei, Bahram; Arefi, Hadi; Zarghi, Afshin

    2015-01-01

    As a continuous research for discovery of new COX-2 inhibitors, chemical synthesis, in vitro biological activity and molecular docking study of a new group of 1, 4-dihydropyridine (DHP) derivatives were presented. Novel synthesized compounds possessing a COX-2 SO2Me pharmacophore at the para position of C-4 phenyl ring, different hydrophobic groups (R1) at C-2 position and alkoxycarbonyl groups (COOR2) at C-3 position of 1, 4-dihydropyridine, displayed selective inhibitory activity against COX-2 isozyme. Among them, compound 5e was identified as the most potent and selective COX-2 inhibitor with IC50 value of 0.30 μM and COX-2 selectivity index of 92. Molecular docking study was performed to determine probable binding models of compound 5e. The study showed that the p-SO2Me-phenyl fragment of 5e inserted inside secondary COX-2 binding site (Arg(513), Phe(518), Gly(519), and His(90)). The structure-activity relationships acquired reveal that compound 5e with methyl and ethoxycarbonyl as R1 and COOR2 substitutions has the necessary geometry to provide selective inhibition of the COX-2 isozyme and it can be a good basis for the development of new hits.

  13. Rosuvastatin prevents angiotensin II-induced vascular changes by inhibition of NAD(P)H oxidase and COX-1

    PubMed Central

    Colucci, Rocchina; Fornai, Matteo; Duranti, Emiliano; Antonioli, Luca; Rugani, Ilaria; Aydinoglu, Fatma; Ippolito, Chiara; Segnani, Cristina; Bernardini, Nunzia; Taddei, Stefano; Blandizzi, Corrado; Virdis, Agostino

    2013-01-01

    Background and Purpose NAD(P)H oxidase and COX-1 participate in vascular damage induced by angiotensin II. We investigated the effect of rosuvastatin on endothelial dysfunction, vascular remodelling, changes in extracellular matrix components and mechanical properties of small mesenteric arteries from angiotensin II-infused rats. Experimental Approach Male rats received angiotensin II (120 ng·kg−1·min−1, subcutaneously) for 14 days with or without rosuvastatin (10 mg·kg−1·day−1, oral gavage) or vehicle. Vascular functions and morphological parameters were assessed by pressurized myography. Key Results In angiotensin II-infused rats, ACh-induced relaxation was attenuated compared with controls, less sensitive to L-NAME, enhanced by SC-560 (COX-1 inhibitor) or SQ-29548 (prostanoid TP receptor antagonist), and normalized by the antioxidant ascorbic acid or NAD(P)H oxidase inhibitors. After rosuvastatin, relaxations to ACh were normalized, fully sensitive to L-NAME, and no longer affected by SC-560, SQ-29548 or NAD(P)H oxidase inhibitors. Angiotensin II enhanced intravascular superoxide generation, eutrophic remodelling, collagen and fibronectin depositions, and decreased elastin content, resulting in increased vessel stiffness. All these changes were prevented by rosuvastatin. Angiotensin II increased phosphorylation of NAD(P)H oxidase subunit p47phox and its binding to subunit p67phox, effects inhibited by rosuvastatin. Rosuvastatin down-regulated vascular Nox4/NAD(P)H isoform and COX-1 expression, attenuated the vascular release of 6-keto-PGF1α, and enhanced copper/zinc-superoxide dismutase expression. Conclusion and Implications Rosuvastatin prevents angiotensin II-induced alterations in resistance arteries in terms of function, structure, mechanics and composition. These effects depend on restoration of NO availability, prevention of NAD(P)H oxidase-derived oxidant excess, reversal of COX-1 induction and its prostanoid production, and stimulation of

  14. Flexible Cyclic Ethers/Polyethers as Novel P2-Ligands for HIV-1 Protease Inhibitors: Design, Synthesis, Biological Evaluation and Protein-ligand X-ray Studies

    PubMed Central

    Ghosh, Arun K.; Gemma, Sandra; Baldridge, Abigail; Wang, Yuan-Fang; Kovalevsky, Andrey Yu.; Koh, Yashiro; Weber, Irene T.; Mitsuya, Hiroaki

    2009-01-01

    We report the design, synthesis and biological evaluation of a series of novel HIV-1 protease inhibitors. The inhibitors incorporate stereochemically defined flexible cyclic ethers/polyethers as the high affinity P2-ligands. Inhibitors containing small ring 1,3-dioxacycloalkanes have shown potent enzyme inhibitory and antiviral activity. Inhibitors 3d and 3h are the most active inhibitors. Inhibitor 3d maintains excellent potency against a variety of multi-PI-resistant clinical strains. Our structure-activity studies indicate that the ring size, stereochemistry, and position of oxygens are important for the observed activity. Optically active synthesis of 1,3-dioxepan-5-ol along with the syntheses of various cyclic ether and polyether ligands have been described. A protein-ligand X-ray crystal structure of 3d-bound HIV-1 protease was determined. The structure revealed that the P2-ligand makes extensive interactions including hydrogen bonding with the protease backbone in the S2-site. In addition, the P2-ligand in 3d forms a unique water-mediated interaction with the NH of Gly-48. PMID:18783203

  15. Flexible Cyclic Ethers/Polyethers as Novel P2-Ligands for HIV-1 Protease Inhibitors: Design, Synthesis, Biological Evaluation, and Protein-Ligand X-Ray Studies

    SciTech Connect

    Ghosh, Arun; Gemma, Sandra; Baldridge, Abigal; Wang, Yuan-Fang; Kovalevsky, Andrey; Koh, Yashiro; Weber, Irene; Mitsuya, Hiroaki

    2008-12-05

    We report the design, synthesis, and biological evaluation of a series of novel HIV-1 protease inhibitors. The inhibitors incorporate stereochemically defined flexible cyclic ethers/polyethers as high affinity P2-ligands. Inhibitors containing small ring 1,3-dioxacycloalkanes have shown potent enzyme inhibitory and antiviral activity. Inhibitors 3d and 3h are the most active inhibitors. Inhibitor 3d maintains excellent potency against a variety of multi-PI-resistant clinical strains. Our structure-activity studies indicate that the ring size, stereochemistry, and position of oxygens are important for the observed activity. Optically active synthesis of 1,3-dioxepan-5-ol along with the syntheses of various cyclic ether and polyether ligands have been described. A protein-ligand X-ray crystal structure of 3d-bound HIV-1 protease was determined. The structure revealed that the P2-ligand makes extensive interactions including hydrogen bonding with the protease backbone in the S2-site. In addition, the P2-ligand in 3d forms a unique water-mediated interaction with the NH of Gly-48.

  16. Lupin nad9 and nad6 genes and their expression: 5' termini of the nad9 gene transcripts differentiate lupin species.

    PubMed

    Rurek, Michał; Nuc, Katarzyna; Raczyńska, Katarzyna Dorota; Augustyniak, Halina

    2003-10-02

    The mitochondrial nad9 and nad6 genes were analyzed in four lupin species: Lupinus luteus, Lupinus angustifolius, Lupinus albus and Lupinus mutabilis. The nucleotide sequence of these genes confirmed their high conservation, however, higher number of nucleotide substitution was observed in the L. albus genes. Southern hybridizations confirmed the presence of single copy number of these genes in L. luteus, L. albus and L. angustifolius. The expression of nad9 and nad6 genes was analyzed by Northern in different tissue types of analyzed lupin species. Transcription analyses of the two nad genes displayed single predominant mRNA species of about 0.6 kb in L. luteus and L. angustifolius. The L. albus transcripts were larger in size. The nad9 and nad6 transcripts were modified by RNA editing at 8 and 11 positions, in L. luteus and L. angustifolius, respectively. The gene order, rps3-rpl16-nad9, found in Arabidopsis thaliana is also conserved in L. luteus and L. angustifolius mitochondria. L. luteus and L. angustifolius showed some variability in the sequence of the nad9 promoter region. The last feature along with the differences observed in nad9 mRNA 5' termini of two lupins differentiate L. luteus and L. angustifolius species.

  17. Artepillin C isoprenomics: design and synthesis of artepillin C isoprene analogues as lipid peroxidation inhibitor having low mitochondrial toxicity.

    PubMed

    Uto, Yoshihiro; Ae, Shutaro; Koyama, Daisuke; Sakakibara, Mitsutoshi; Otomo, Naoki; Otsuki, Mamoru; Nagasawa, Hideko; Kirk, Kenneth L; Hori, Hitoshi

    2006-08-15

    We designed and synthesized isoprene analogues of artepillin C, a major component of Brazilian propolis, and investigated the inhibitory activity on lipid peroxidation of rat liver mitochondria (RLM) and RLM toxicity based on isoprenomics. We succeeded in the synthesis of artepillin C isoprene analogues using regioselective prenylation within the range from 22% to 53% total yield. Reactivity of artepillin C and its isoprene analogues with ABTS (2,2'-Azinobis(3-ethylbenzothiazoline-6-sulfonate)) radical cations showed only a slight difference among the molecules. The isoprene side-chain elongation analogues of artepillin C showed almost the same inhibitory activity against RLM lipid peroxidation as artepillin C. Artepillin C and its isoprene analogues had very weak RLM uncoupling activity. Moreover, artepillin C and its isoprene analogues exhibited a lower inhibitory activity against adenosine 5'-triphosphate (ATP) synthesis by about two orders of magnitude than the effective inhibitory activity against RLM lipid peroxidation. From these results we conclude that artepillin C isoprene analogues could be potent lipid peroxidation inhibitors having low mitochondrial toxicity. We also conclude that elongation of the isoprene side chain of artepillin C to increase lipophilicity had little influence on the inhibitory activity toward RLM lipid peroxidation.

  18. Regulatory substances produced by lymphocytes. VI. Cell cycle specificity of inhibitor of DNA synthesis action in L cells.

    PubMed

    Wagshal, A B; Jegasothy, B V; Waksman, B H

    1978-01-01

    IDS inhibits DNA synthesis and mitosis of L cells only when present during the late G1 phase of the cell cycle, as shown with L cells synchronized by a variety of methods. This corresponds well with earlier findings that IDS inhibits DNA synthesis in mitogen-stimulated lymphocytes when present between 16 and 24 h after adding mitogen. In both cell types, the inhibition produced by IDS appears to be totally the result of elevation of cAMP level. Thus, inhibitors of cAMP phosphodiesterase work synergistically with IDS, and activators of cAMP phosphodiesterase overcome the inhibition by IDS. This paper shows that IDS raises cAMP levels in L cells only within a narrow interval of the cell cycle, around 6-8 h after mitosis. This cell cycle specificity, which may be related to appearance of receptors for IDS only at discrete times, may be important in limiting IDS action to suppression, as elevated cAMP levels have a variety of other effects during other phases of the cell cycle.

  19. Synthesis, biological evaluation and molecular modeling studies on novel quinonoid inhibitors of CDC25 phosphatases.

    PubMed

    Evain-Bana, Emilie; Schiavo, Lucie; Bour, Christophe; Lanfranchi, Don Antoine; Berardozzi, Simone; Ghirga, Francesca; Bagrel, Denyse; Botta, Bruno; Hanquet, Gilles; Mori, Mattia

    2017-12-01

    The cell division cycle 25 phosphatases (CDC25A, B, and C; E.C. 3.1.3.48) are key regulator of the cell cycle in human cells. Their aberrant expression has been associated with the insurgence and development of various types of cancer, and with a poor clinical prognosis. Therefore, CDC25 phosphatases are a valuable target for the development of small molecule inhibitors of therapeutic relevance. Here, we used an integrated strategy mixing organic chemistry with biological investigation and molecular modeling to study novel quinonoid derivatives as CDC25 inhibitors. The most promising molecules proved to inhibit CDC25 isoforms at single digit micromolar concentration, becoming valuable tools in chemical biology investigations and profitable leads for further optimization. [Formula: see text].

  20. Design, synthesis, and biological activity of diaryl ether inhibitors of Toxoplasma gondii enoyl reductase

    PubMed Central

    Cheng, Gang; Muench, Stephen P.; Zhou, Ying; Afanador, Gustavo A.; Mui, Ernest J.; Fomovska, Alina; Lai, Bo Shiun; Prigge, Sean T.; Woods, Stuart; Roberts, Craig W.; Hickman, Mark R.; Lee, Patty J.; Leed, Susan E.; Auschwitz, Jennifer M.; Rice, David W.; McLeod, Rima

    2013-01-01

    Triclosan is a potent inhibitor of Toxoplasma gondii enoyl reductase (TgENR), which is an essential enzyme for parasite survival. In view of triclosan’s poor druggability, which limits its therapeutic use, a new set of B-ring modified analogs were designed to optimize its physico-chemical properties. These derivatives were synthesized and evaluated by in vitro assay and TgENR enzyme assay. Some analogs display improved solubility, permeability and a comparable MIC50 value to that of triclosan. Modeling of these inhibitors revealed the same overall binding mode with the enzyme as triclosan, but the Bring modifications have additional interactions with the strongly conserved Asn130. PMID:23453069