Novel nicotinamide analog as inhibitor of nicotinamide N-methyltransferase

Nicotinamide N-methyltransferase (NNMT) has been linked to obesity and diabetes. We have identified a novel nicotinamide (NA) analog, compound 12 that inhibited NNMT enzymatic activity and reduced the formation of 1-methyl-nicotinamide (MNA), the primary metabolite of NA by ～80% at 2?h when dosed in mice orally at 50?mg/kg.     

2,4-Diamino-8-quinazoline carboxamides as novel,potent inhibitors of the NAD hydrolyzing enzyme CD38: Exploration of the 2-position structure-activity relationships

Starting from 4-amino-8-quinoline carboxamide lead 1a and scaffold hopping to the chemically more tractable quinazoline, a systematic exploration of the 2-substituents of the quinazoline ring, utilizing structure activity relationships and conformational constraint, resulted in the identification of 39 novel CD38 inhibitors. Eight of these analogs were 10–100-fold more potent human CD38 inhibitors, including the single digit nanomolar inhibitor 1am. Several of these molecules also exhibited improved therapeutic indices relative to hERG activity. A representative analog 1r exhibited suitable pharmacokinetic parameters for in vivo animal studies, including moderate clearance and good oral bioavailability. These inhibitor compounds will aid in the exploration of the enzymatic functions of CD38, as well as furthering the study of the therapeutic implications of NAD enhancement in metabolic disease models.     

Effects of pressure on deuterium isotope effects of yeast alcohol dehydrogenase using alternative substrates

Hydrostatic pressure causes biphasic effects on the oxidation of alcohols by yeast alcohol dehydrogenase as expressed on the kinetic parameter V/K which measures substrate capture. Moderate pressure increases capture by activating hydride transfer, whose transition-state must therefore have a smaller volume than the free alcohol plus the capturing form of enzyme, with DeltaV(double dagger)=-30 mL mol(-1) for isopropanol. A comparison of these effects with those on the oxidation of deutero-isopropanol generates a monophasic decrease in the intrinsic isotope effect; therefore, the volume of activation for the transition-state of deuteride transfer must be even more negative, by 7.6 mL mol(-1). The pressure data extrapolate and factor the kinetic isotope effect into a semi-classical reactant-state component, with a null value of k(H)/k(D)=1, and a transition-state component of Q(H)/Q(D)=4, suggestive of hydrogen tunneling. Pressures above 1.5 kbar decrease capture by favoring a minor conformation of enzyme which binds nicotinamide adenine dinucleotide (NAD(+)) less tightly. This inactive conformation has a smaller volume than active E-NAD(+), with a difference of 74 mL mol(-1) and an equilibrium constant of 93 between them, at one atmosphere of pressure. These results are virtually identical to those obtained with benzyl alcohol and give credence to this method of analysis. Moreover, qualitatively similar results with greater pressure sensitivity but less precision are obtained using ethanol as a substrate, only with pressure driving the value of the isotope effect to a value less than (D)k=1.03 directly, without extrapolation. The ethanol data verify the most surprising finding of these studies, namely that the entire kinetic isotope effect arises from a transition-state phenomenon.     

NAD+ accumulation as a metabolic off switch for orthodox pollen

Terrestrial plant pollen is classified into two categories based on its metabolic status: pollen with low-metabolism are termed “orthodox” and pollen with high-metabolism are termed “recalcitrant.” Nicotinamide adenine dinucleotide (NAD) is crucial for a number of metabolisms in all extant organisms. It has recently been shown that NAD homeostasis plays an important role in a broad range of developmental processes and responses to environment. Recently, a reverse genetic approach shed light on the significance of NAD biosynthesis on pollen fate. In orthodox Arabidopsis pollen, NAD⁺ that was accumulated in excess at dispersal dramatically decreased on rehydration. The lack of a key gene that is involved in NAD biosynthesis compromised the excess accumulation. Moreover, absence of the excess accumulation phenocopied the so-called recalcitrant pollen, as demonstrated by the germination inside anthers and the loss of desiccation tolerance. Upon rehydration, NAD⁺-consuming inhibitors impaired tube germination. Taken together, our results suggest that accumulation of NAD⁺ functions as a physiochemical molecular switch for suspended metabolism and that the decrease of NAD⁺ plays a very important role during transitions in metabolic states. Shifting of the redox state to an oxidizing environment may efficiently control the comprehensive metabolic network underlying the onset of pollen germination.     

Structural Uniformity of Pullulan Produced by Several Strains of Pullularia pullulans

Extracellular polysaccharides produced by 3 strains of Pullularia pullulans were fractionated by treating with cetyl trimethyl ammonium hydroxide into soluble and insoluble fractions, and the structure of the former fraction, i.e., pullulan, was studied. The yield and the ratio of 2 fractions varied widely according to the strains. But the structure of pullulan was found to be uniform irrespective of the strains used. All 3 samples of pullulan gave only glucose on complete acid hydrolysis, and 93~95% maltotriose and 5~7% maltotetraose after isoamylase (pullulanase) action. The ratio of α-1,4- to α-1,6-glucosidic linkages calculated from periodate oxidation data coincided very well with the value expected from the ratio of maltotriose to maltotetraose units. An evidence for the complete absence of branch structure in pullulan was presented from the results of hydrolysis by pullulan 4-glucanohydrolase.     

Inhibition of Nicotinamide Phosphoribosyltransferase (NAMPT), an Enzyme Essential for NAD+ Biosynthesis,Leads to Altered Carbohydrate Metabolism in Cancer Cells

Nicotinamide phosphoribosyltransferase (NAMPT) has been extensively studied due to its essential role in NAD⁺ biosynthesis in cancer cells and the prospect of developing novel therapeutics. To understand how NAMPT regulates cellular metabolism, we have shown that the treatment with FK866, a specific NAMPT inhibitor, leads to attenuation of glycolysis by blocking the glyceraldehyde 3-phosphate dehydrogenase step (Tan, B., Young, D. A., Lu, Z. H., Wang, T., Meier, T. I., Shepard, R. L., Roth, K., Zhai, Y., Huss, K., Kuo, M. S., Gillig, J., Parthasarathy, S., Burkholder, T. P., Smith, M. C., Geeganage, S., and Zhao, G. (2013) Pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT), an enzyme essential for NAD⁺ biosynthesis, in human cancer cells: metabolic basis and potential clinical implications. J. Biol. Chem. 288, 3500–3511). Due to technical limitations, we failed to separate isotopomers of phosphorylated sugars. In this study, we developed an enabling LC-MS methodology. Using this, we confirmed the previous findings and also showed that NAMPT inhibition led to accumulation of fructose 1-phosphate and sedoheptulose 1-phosphate but not glucose 6-phosphate, fructose 6-phosphate, and sedoheptulose 7-phosphate as previously thought. To investigate the metabolic basis of the metabolite formation, we carried out biochemical and cellular studies and established the following. First, glucose-labeling studies indicated that fructose 1-phosphate was derived from dihydroxyacetone phosphate and glyceraldehyde, and sedoheptulose 1-phosphate was derived from dihydroxyacetone phosphate and erythrose via an aldolase reaction. Second, biochemical studies showed that aldolase indeed catalyzed these reactions. Third, glyceraldehyde- and erythrose-labeling studies showed increased incorporation of corresponding labels into fructose 1-phosphate and sedoheptulose 1-phosphate in FK866-treated cells. Fourth, NAMPT inhibition led to increased glyceraldehyde and erythrose levels in the cell. Finally, glucose-labeling studies showed accumulated fructose 1,6-bisphosphate in FK866-treated cells mainly derived from dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. Taken together, this study shows that NAMPT inhibition leads to attenuation of glycolysis, resulting in further perturbation of carbohydrate metabolism in cancer cells. The potential clinical implications of these findings are also discussed.     

Temporal Patterns of Poly(ADP-Ribose) Polymerase Activation in the Cortex Following Experimental Brain Injury in the Rat

The activation of poly(ADP-ribose) polymerase, a DNA base excision repair enzyme, is indicative of DNA damage. This enzyme also undergoes site-specific proteolysis during apoptosis. Because both DNA fragmentation and apoptosis are known to occur following experimental brain injury, we investigated the effect of lateral fluid percussion brain injury on poly(ADP-ribose) polymerase activity and cleavage. Male Sprague-Dawley rats (n = 52) were anesthetized, subjected to fluid percussion brain injury of moderate severity (2.5-2.8 atm), and killed at 30 min, 2 h, 6 h, 24 h, 3 days, or 7 days postinjury. Genomic DNA from injured cortex at 24 h, but not at 30 min, was both fragmented and able to stimulate exogenous poly(ADP-ribose) polymerase. Endogenous poly(ADP-ribose) polymerase activity, however, was enhanced in the injured cortex at 30 min but subsequently returned to baseline levels. Slight fragmentation of poly(ADP-ribose) polymerase was detected in the injured cortex in the first 3 days following injury, but significant cleavage was detected at 7 days postinjury. Taken together, these data suggest that poly(ADP-ribose) polymerase-mediated DNA repair is initiated in the acute posttraumatic period but that subsequent poly(ADP-ribose) polymerase activation does not occur, possibly owing to delayed apoptosis-associated proteolysis, which may impair the repair of damaged DNA.     

烟酰胺腺嘌呤二核苷酸和Sirtuins在衰老和疾病中的作用

烟酰胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide, NAD⁺)作为氧化还原反应的重要辅酶,是能量代谢的核心。NAD⁺也是非氧化还原NAD⁺依赖性酶的共底物,包括沉默信息调节因子(Sirtuins)、聚ADP-核糖聚合酶(poly ADP-ribose polymerases, PARPs)、CD38/CD157胞外酶等。NAD⁺已成为细胞信号转导和细胞存活的关键调节剂。最近的研究表明,Sirtuins催化多种NAD⁺依赖性反应,包括去乙酰化、脱酰基化和ADP-核糖基化。Sirtuins催化活性取决于NAD⁺水平的高低。因此,Sirtuins是细胞代谢和氧化还原状态关键传感器。哺乳动物中已经鉴定并表征了7个Sirtuins家族成员(SIRT1-7),其参与炎症、细胞生长、生理节律、能量代谢、神经元功能、应激反应和健康衰老等多种生理过程。本文归纳了NAD⁺的生理浓度及状态、NAD⁺     

烟酰胺腺嘌呤二核苷酸合成相关酶有助于1,4-丁二醇转化为4-羟基丁酸

4-羟基丁酸(4-HB)不仅具有医学应用价值,而且是合成生物材料P3HB4HB的重要前体.在烟酰胺腺嘌呤二核苷酸(NAD)参与情况下,大肠杆菌Escherichia coli S17-1(pZL-dhaT-aldD)可以把1,4-丁二醇(1,4-BD)转化为4HB.为提高4HB产率,通过过表达烟酸磷酸核糖转移酶(PncB)和烟酰胺腺嘌呤二核苷酸合成酶(NadE)增加胞内NAD含量,从而加速1,4-BD转化反应的进行.结果表明,PncB-NadE的表达使1,4-BD转化率比对照组增加13.03％,由10g/L的1,4-BD得到4.87 g/L的4HB,单位细胞的4HB产量由1.32 g/g提高40.91％至1.86 g/g.因此PncB和NadE可用于促进1,4-BD转化为4HB.     

Direct evidence for the presence of a rotenone-resistant NADH dehydrogenase on the inner surface of the inner membrane of plant mitochondria

Submitochondrial particles (SMP) were produced from Jerusalem artichoke (Helianthus tuberosus L.) mitochondria by sonication and differential centrifugation. The SMP were about 50% inside-out as measured by the access of reduced cytochrome c to cytochrome c oxidase. Uncoupled NADH oxidation (1 mM NADH) by the SMP was 120 nmol O₂ min^?1mg^?1, which was reduced to 98 nmol O₂ min^?1 (mg mitochondrial protein)^?1 in the presence of EGTA. In contrast, the oxidation of NADH by intact mitochondria was completely inhibited by EGTA (from 182 to 14 nmol O₂ min^?1mg^?1). The EGTA-resistant NADH oxidation by the SMP is ascribed to the NADH dehydrogenase(s) on the inside of the inner membrane and exposed to the medium in the inside-out SMP. In the presence of EGTA it could be shown that two NADH dehydrogenase activities were present in the SMP. One had an apparent K_m of 7 μM for NADH, a V_max of 80 nmol NADH min^?1mg^?1, and was rotenone-sensitive. This dehydrogenase is equivalent to the mammalian Complex I NADH dehydrogenase. The other dehydrogenase, which was rotenone-resistant, had a K_m of 80 μM and a V_max of 131 nmol NADH min^?1mg^?1; it is probably responsible for the rotenone-resistant oxidation of organic acids often observed in plant mitochondria. The redox poise of the pyridine nucleotides had only a small effect on the relative rates of the two internal dehydrogenases. Electron flow through these dehydrogenases appears, therefore, to be regulated mainly by the concentration of NADH in the matrix of the mitochondria.     

Iron uptake mechanism in the chrysophyte microalga Dinobryon

The mechanism of iron uptake in the chrysophyte microalga Dinobryon was studied. Previous studies have shown that iron is the dominant limiting elements for growth of Dinobryon in the Eshkol reservoir in northern Israel, which control its burst of bloom. It is demonstrated that Dinobryon has a light-stimulated ferrireductase activity, which is sensitive to the photosynthetic electron transport inhibitor DCMU and to the uncoupler CCCP. Iron uptake is also light-dependent, is inhibited by DCMU and by CCCP and also by the ferrous iron chelator BPDS. These results suggest that ferric iron reduction by ferrireductase is involved in iron uptake in Dinobryon and that photosynthesis provides the major reducing power to energize iron acquisition. Iron deprivation does not enhance but rather inhibits iron uptake contrary to observations in other algae.     

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

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.     

Sirt1与生物钟的相互调控

生物钟调控机制广泛存在于各种类型的细胞中,控制着细胞代谢的节律性变化.最近的研究发现,NAD+依赖的组蛋白去乙酰化酶Sirt1参与了生物钟调控过程,对维持正常的生物钟节律具有重要作用;另一方面,Sirt1的表达也受到生物钟系统的调控,呈现出昼夜节律性的表达.因此Sirt1能与生物钟进行相互调控,并且这一作用机制很可能广泛参与了不同类型细胞内的信号转导和能量代谢过程.本文总结了Sirt1与生物钟之间相互调控的一些研究进展,对它们之间的分子调控机制进行了概述.     

Distribution and colocalization of nitric oxide synthase and NADPH-diaphorase in adrenal gland of developing,adult and aging Sprague-Dawley rats

The distribution and colocalization of nitric oxide synthase and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-diaphorase) was investigated in the adrenal gland of developing, adult and aging rats with the use of immunohistochemical and histochemical techniques. Nitric oxide synthase-immunoreactive neurons within the adrenal gland were found from the 20th day of gestation onwards. During early development the neurons were found as small clusters of smaller-size cells compared to those observed in the adult gland. Their number reached that of adult level by the 4th day after birth, and in the glands from aging rats a 28.6% increase was observed. Whilst no immunofluorescence was seen in chromaffin cells during early development, some cells from glands of aging rats showed nitric oxide synthase-immunoreactivity with varying intensity. The immunoreactive neurons from postnatal rat adrenals were also positive for NADPH-diaphorase, whilst those in prenatal rats were negative or lightly stained. Nitric oxide synthase-immunoreactive nerve fibres were present in all adrenal glands examined from the 16th day of gestation onwards. A considerable degree of variation in the distribution of immunoreactive fibres both in medulla and outer region of cortex at the different age groups was observed and described. Most, but not all, nitric oxide synthase-immunoreactive nerve fibres also showed NADPH-diaphorase staining.     

Critical Role for NAD Glycohydrolase in Regulation of Erythropoiesis by Hematopoietic Stem Cells through Control of Intracellular NAD Content

NAD glycohydrolases (NADases) catalyze the hydrolysis of NAD to ADP-ribose and nicotinamide. Although many members of the NADase family, including ADP-ribosyltransferases, have been cloned and characterized, the structure and function of NADases with pure hydrolytic activity remain to be elucidated. Here, we report the structural and functional characterization of a novel NADase from rabbit reticulocytes. The novel NADase is a glycosylated, glycosylphosphatidylinositol-anchored cell surface protein exclusively expressed in reticulocytes. shRNA-mediated knockdown of the NADase in bone marrow cells resulted in a reduction of erythroid colony formation and an increase in NAD level. Furthermore, treatment of bone marrow cells with NAD, nicotinamide, or nicotinamide riboside, which induce an increase in NAD content, resulted in a significant decrease in erythroid progenitors. These results indicate that the novel NADase may play a critical role in regulating erythropoiesis of hematopoietic stem cells by modulating intracellular NAD.