Cellular Nicotinamide Adenine Dinucleotide Research Articles

Apollo-NADP+ in 3D: Fluorescence Anisotropy Imaging of a Homofret-Based Biosensor for NADP+ in Living Tissue

We developed a genetically encoded homoFRET biosensor to measure cellular nicotinamide adenine dinucleotide phosphate redox state (Apollo-NADP+). This sensor is based on the homo-dimerization of glucose-6-phosphate dehydrogenase and responds to NADPH/NADP+ redox state with changes in steady-state fluorescence anisotropy associated with homologous Forster resonance energy transfer (homoFRET). We now aim to use this sensor in a tissue. However, tissue scatter has a significant affect on steady-state anisotropy imaging, which may prohibit use of the sensor. To determine whether Apollo-NADP+ is suited for imaging in 3D tissues, we are investigating the effect of scatter on fluorescence anisotropy using tissue phantoms and living tissue. Thus far, we imaged monomeric and tandem-dimer fluorescent protein constructs suspended in a light scattering solution and showed these controls can be distinguished despite a significant affect on their fluorescence intensity. We also expressed our biosensor in living neural-progenitor cell colonies to discern whether stimulant responses in 3D cell cultures are consistent with those observed in monolayer cell cultures. We will subsequently explore the use of our biosensor in in vivo tissue imaging by expressing our sensor in zebrafish. Overall, this project aims to determine whether anisotropy-based sensors such as Apollo-NADP+ can be translated to study living tissue.

Purification and characterization of the enzymes involved in nicotinamide adenine dinucleotide degradation by Penicillium brevicompactum NRC 829.

The present study was conducted to investigate a new pathway for the degradation of nicotinamide adenine dinucleotide (NAD) by Penicillium brevicompactum NRC 829 extracts. Enzymes involved in the hydrolysis of NAD, i.e. alkaline phosphatase, aminohydrolase and glycohydrolase were determined. Alkaline phosphatase was found to catalyse the sequential hydrolysis of two phosphate moieties of NAD molecule to nicotinamide riboside plus adenosine. Adenosine was then deaminated by aminohydrolase to inosine and ammonia. While glycohydrolase catalyzed the hydrolysis of the nicotinamide-ribosidic bond of NAD+ to produce nicotinamide and ADP-ribose in equimolar amounts, enzyme purification through a 3-step purification procedure revealed the existence of two peaks of alkaline phosphatases, and one peak contained deaminase and glycohydrolase activities. NAD deaminase was purified to homogeneity as estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis with an apparent molecular mass of 91 kDa. Characterization and determination of some of NAD aminohydrolase kinetic properties were conducted due to its biological role in the regulation of cellular NAD level. The results also revealed that NAD did not exert its feedback control on nicotinamide amidase produced by P. brevicompactum.

NAD+ in Cancer Prevention and Treatment: Pros and Cons

Abstract Oxidized form of cellular nicotinamide adenine dinucleotide (NAD+) is currently intensively investigated topic in longevity science. However, if ageing is considered a defense mechanism against cancer, caution should be implemented regarding the use of NAD+ and its precursors. In the hypothesis presented NAD+ is shown as an important factor related to cancer formation and prevention. NAD+ depletion with age may play a major role in the process of cancer formation by limiting (1) energy production, (2) DNA repair, (3) genomic stability and signaling. Disruption of any of these processes could increase the cancer risk due to impaired genomic stability. NAD+ content is a critical protective factor in early carcinogenesis and can become detrimental factor later in cancer progression and promotion phase. Namely, NAD+ restoration could prevent or reverse the phenotype of malignant cells at early stages by inducing cellular repair and stress adaptive response as well as regulate cell cycle arrest and apoptotic removal of damaged cells. Contrary, during cancer promotion, progression and treatment increased NAD+ levels could have deleterious effects on the malignancy process due to growth advantage, increased resistance and greater cell survival. NAD+ levels can be increased with exercise, caloric restriction and ingestion of NAD+ precursors and intermediates or could be increased by using PARP and CD 38 inhibitors. The evidence indicating that modulation of NAD+ levels could be important in cancer prevention, initiation and progression phase is presented.

Functional Characterization of Sirtuin-like Protein in Mycobacterium smegmatis.

Nicotinamide adenine dinucleotide (NAD)-dependent deacetylases (sirtuins) are well conserved from prokaryotes to eukaryotes. Functions and regulations of mammalian sirtuins have been extensively studied and indicate that sirtuins play an important role in regulation of biological processes, whereas functions of mycobacterial sirtuins were less explored. To examine functions of the sirtuin-like protein in mycobacteria, a Mycobacterium smegmatis sirtuin, MSMEG_5175, was overexpressed in a M. smegmatis strain mc(2)155 to generate an MSMEG_5175-overexpression strain (mc(2)155-MS5175) in the present study. The physiological aspects of mc(2)155-MS5175 strain were characterized showing that they had a lower intracellular NAD level and a higher resistance to isoniazid (INH) as compared to mc(2)155 containing empty pMV261 plasmid (mc(2)155-pMV261). Quantitative proteomic analysis was carried out to determine differentially expressed proteins between mc(2)155-pMV261 and mc(2)155-MS5175. Among 3032 identified proteins, overexpression of MSMEG_5175 results in up-regulation of 34 proteins and down-regulation of 72 proteins, which involve in diverse cellular processes including metabolic activation, transcription and translation, antioxidant, and DNA repair. Down-regulation of catalase peroxidase (KatG) expression in both mRNA and protein levels were observed in mc(2)155-MS5175 strain, suggesting that a decrease in cellular NAD content and down-regulation of KatG expression contribute to the higher resistance to INH in mc(2)155-MS5175. Using a combination of immunoprecipitation and proteomic analysis, we found that acetylation in 27 proteins was decreased in mc(2)155-MS5175 as compared to those in mc(2)155-pMV261, suggesting that these proteins including the beta prime subunit of RNA polymerase (rpoC), ribosomal proteins, and metabolic enzymes were substrates of MSMEG_5175. Acetylation changes in rpoC may affect its function and cause changes in global gene transcription. Taken together, these results suggest that MSMEG_5175 regulates diverse cellular processes resulting in an increase in INH resistance in mycobacteria, and provide a useful resource to further biological exploration into functions of protein acetylation in mycobacteria.

Effect of nicotinamide mononucleotide on brain mitochondrial respiratory deficits in an Alzheimer's disease-relevant murine model.

BackgroundMitochondrial dysfunction is a hallmark of neurodegenerative diseases including Alzheimer’s disease (AD), with morphological and functional abnormalities limiting the electron transport chain and ATP production. A contributing factor of mitochondrial abnormalities is loss of nicotinamide adenine dinucleotide (NAD), an important cofactor in multiple metabolic reactions. Depletion of mitochondrial and consequently cellular NAD(H) levels by activated NAD glycohydrolases then culminates in bioenergetic failure and cell death. De Novo NAD+ synthesis from tryptophan requires a multi-step enzymatic reaction. Thus, an alternative strategy to maintain cellular NAD+ levels is to administer NAD+ precursors facilitating generation via a salvage pathway. We administered nicotinamide mononucleotide (NMN), an NAD+ precursor to APP(swe)/PS1(ΔE9) double transgenic (AD-Tg) mice to assess amelioration of mitochondrial respiratory deficits. In addition to mitochondrial respiratory function, we examined levels of full-length mutant APP, NAD+-dependent substrates (SIRT1 and CD38) in homogenates and fission/fusion proteins (DRP1, OPA1 and MFN2) in mitochondria isolated from brain. To examine changes in mitochondrial morphology, bigenic mice possessing a fluorescent protein targeted to neuronal mitochondria (CaMK2a-mito/eYFP), were administered NMN.MethodsMitochondrial oxygen consumption rates were examined in N2A neuroblastoma cells and non-synaptic brain mitochondria isolated from mice (3 months). Western blotting was utilized to assess APP, SIRT1, CD38, DRP1, OPA1 and MFN2 in brain of transgenic and non-transgenic mice (3–12 months). Mitochondrial morphology was assessed with confocal microscopy. One-way or two-way analysis of variance (ANOVA) and post-hoc Holm-Sidak method were used for statistical analyses of data. Student t-test was used for direct comparison of two groups.ResultsWe now demonstrate that mitochondrial respiratory function was restored in NMN-treated AD-Tg mice. Levels of SIRT1 and CD38 change with age and NMN treatment. Furthermore, we found a shift in dynamics from fission to fusion proteins in the NMN-treated mice.ConclusionsThis is the first study to directly examine amelioration of NAD+ catabolism and changes in mitochondrial morphological dynamics in brain utilizing the immediate precursor NMN as a potential therapeutic compound. This might lead to well-defined physiologic abnormalities that can serve an important role in the validation of promising agents such as NMN that target NAD+ catabolism preserving mitochondrial function.

TP53-induced glycolysis and apoptosis regulator promotes proliferation and invasiveness of nasopharyngeal carcinoma cells.

The TP53-induced glycolysis and apoptosis regulator (TIGAR) is the protein product of the p53 target gene, C12orf5. TIGAR blocks glycolysis and promotes cellular metabolism via the pentose phosphate pathway; it promotes the production of cellular nicotinamide adenine dinucleotide phosphate (NADPH), which leads to enhanced scavenging of intracellular reactive oxygen species, and inhibition of oxidative stress-induced apoptosis in normal cells. Our previous study identified a novel nucleoside analog that inhibited cellular growth and induced apoptosis in nasopharyngeal carcinoma (NPC) cell lines via downregulation of TIGAR expression. Furthermore, the growth inhibitory effects of c-Met tyrosine kinase inhibitors were ameliorated by the overexpression of TIGAR in the NPC cell lines. These results indicate a significant role for TIGAR expression in the survival of NPCs. The present study aimed to further define the function of TIGAR expression in NPC cells. In total, 36 formalin-fixed, paraffin-embedded NPC tissue samples were obtained for the immunohistochemical determination of TIGAR expression. The effects of TIGAR expression on cell proliferation, NADPH production and cellular invasiveness were also assessed in NPC cell lines. Overall, TIGAR was overexpressed in 27/36 (75%) of the NPC tissues compared with the adjacent non-cancer epithelial cells. Similarly, TIGAR overexpression was also observed in a panel of six NPC cell lines compared with normal NP460 hTert and Het1A cell lines. TIGAR overexpression led to increased cellular growth, NADPH production and invasiveness of the NPC cell lines, whereas a knockdown of TIGAR expression resulted in significant inhibition of cellular growth and invasiveness. The expression of the two mesenchymal markers, fibronectin and vimentin, was increased by TIGAR overexpression, but reduced following TIGAR-knockdown. The present study revealed that TIGAR overexpression led to increased cellular growth, NADPH production and invasiveness, and the maintenance of a mesenchymal phenotype, in NPC tissues.

Bioluminescent cell-based NAD(P)/NAD(P)H assays for rapid dinucleotide measurement and inhibitor screening.

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.

Abstract 5003: Functional significance of TIGAR expression in nasopharyngeal carcinoma

Abstract Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus associated cancer of the nasopharynx. Annual incidence in China is high, reaching up to 25 per 100,000. NPC is a highly invasive and metastatic disease. At diagnosis, over 60% of patients have advanced disease where treatment failure due to distant recurrence or metastasis is common. More effective forms of therapy to treat this deadly disease are therefore urgently needed. The TP53 induced glycolysis and apoptosis regulator (TIGAR) is a p53 target gene which blocks glycolysis and promotes cellular metabolism via the pentose phosphate pathway. TIGAR promotes production of cellular nicotinamide adenine dinucleotide phosphate (NADPH), leading to enhanced scavenging of intracellular reactive oxygen species (ROS) and inhibition of oxidative-stress induced apoptosis in normal cells. Previously, we have found that a novel nucleoside analog inhibited cell growth and induced apoptosis in NPC cell lines via down-regulation of TIGAR expression. Moreover, the growth inhibitory effect of c-Met tyrosine kinase inhibitors was ameliorated by over-expression of TIGAR in NPC cell lines. These results are indicative of a pivotal role for TIGAR expression in maintaining NPC cell survival. In this study, we investigated the expression pattern of TIGAR in NPC tumor tissues and the consequences of TIGAR overexpression and knockdown on NPC cell growth and invasion. It was found that TIGAR is overexpressed in 27 out of 36 (75%) NPC cases in the tumor cells compared to their respective normal epithelial cells. Overexpression of TIGAR in NPC cell lines resulted in increased proliferation, invasion, NADPH production and up-regulation of mesenchymal markers including fibronectin and vimentin. These changes were reversed when TIGAR expression was knocked down by transfection with siRNA, demonstrating specificity of the observed effects. Together, our results indicate that TIGAR expression promotes proliferation, invasiveness and expression of mesenchymal markers in NPC cells. These findings may form the basis for development of a new therapeutic target for treatment of NPC in the future. Citation Format: Elaine YL Wong, SC Cesar Wong, Charles ML Chan, Emily KY Lam, Louisa Ho, Cecilia PY Lau, Thomas CC Au, Amanda KC Chan, CM Tsang, George SW Tsao, Vivian WY Lui, Anthony TC Chan. Functional significance of TIGAR expression in nasopharyngeal carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5003. doi:10.1158/1538-7445.AM2014-5003

AMPK is involved in mediation of erythropoietin influence on metabolic activity and reactive oxygen species production in white adipocytes

Erythropoietin, discovered for its indispensable role during erythropoiesis, has been used in therapy for selected red blood cell disorders in erythropoietin-deficient patients. The biological activities of erythropoietin have been found in animal models to extend to non-erythroid tissues due to the expression of erythropoietin receptor. We previously demonstrated that erythropoietin promotes metabolic activity and white adipocytes browning to increase mitochondrial function and energy expenditure via peroxisome proliferator-activated receptor alpha and Sirtuin1. Here we report that AMP-activated protein kinase was activated by erythropoietin possibly via Ca2+/calmodulin-dependent protein kinase kinase in adipocytes as well as in white adipose tissue from diet induced obese mice. Erythropoietin increased cellular nicotinamide adenine dinucleotide via increased AMP-activated protein kinase activity, possibly leading to Sirtuin1 activation. AMP-activated protein kinase knock down reduced erythropoietin mediated increase in cellular oxidative function including the increased oxygen consumption rate, fatty acid utilization and induction of key metabolic genes. Under hypoxia, adipocytes were found to generate more reactive oxygen species, and erythropoietin reduced the reactive oxygen species and increased antioxidant gene expression, suggesting that erythropoietin may provide protection from oxidative stress in adipocytes. Erythropoietin also reversed increased nicotinamide adenine dinucleotide by hypoxia via increased AMP-activated protein kinase. Additionally, AMP-activated protein kinase is found to be involved in erythropoietin stimulated increase in oxygen consumption rate, fatty acid oxidation and mitochondrial gene expression. AMP-activated protein kinase knock down impaired erythropoietin stimulated increases in antioxidant gene expression. Collectively, our findings identify the AMP-activated protein kinase involvement in erythropoietin signaling in regulating adipocyte cellular redox status and metabolic activity.

Revealing CD38 Cellular Localization Using a Cell Permeable, Mechanism-Based Fluorescent Small-Molecule Probe

Nicotinamide adenine dinucleotide (NAD) is increasingly recognized as an important signaling molecule that affects numerous biological pathways. Thus, enzymes that metabolize NAD can have important biological functions. One NAD-metabolizing enzyme in mammals is CD38, a type II transmembrane protein that converts NAD primarily to adenosine diphosphate ribose (ADPR) and a small amount of cyclic adenosine diphosphate ribose (cADPR). Localization of CD38 was originally thought to be only on the plasma membrane, but later reports showed either significant or solely, intracellular CD38. With the efficient NAD-hydrolysis activity, the intracellular CD38 may lead to depletion of cellular NAD, thus producing harmful effects. Therefore, the intracellular localization of CD38 needs to be carefully validated. Here, we report the synthesis and application of a cell permeable, fluorescent small molecule (SR101–F-araNMN) that can covalently label enzymatically active CD38 with minimal perturbation of live cells. Using this fluorescent probe, we revealed that CD38 is predominately on the plasma membrane of Raji and retinoic acid (RA)-treated HL-60 cells. Additionally, the probe revealed no CD38 expression in K562 cells, which was previously reported to have solely intracellular CD38. The finding that very little intracellular CD38 exists in these cell lines suggests that the major enzymatic function of CD38 is to hydrolyze extracellular rather than intracellular NAD. The fluorescent activity-based probes that we developed allow the localization of CD38 in different cells to be determined, thus enabling a better understanding of the physiological function.

Modulation of poly(ADP-ribose) polymerase-1 (PARP-1)-mediated oxidative cell injury by ring finger protein 146 (RNF146) in cardiac myocytes.

Poly(ADP-ribose) polymerase-1 (PARP-1) activation is a hallmark of oxidative stress-induced cellular injury that can lead to energetic failure and necrotic cell death via depleting the cellular nicotinamide adenine dinucleotide (NAD(+)) and ATP pools. Pharmacological PARP-1 inhibition or genetic PARP-1 deficiency exert protective effects in multiple models of cardiomyocyte injury. However, the connection between nuclear PARP-1 activation and depletion of the cytoplasmic and mitochondrial energy pools is poorly understood. By using cultured rat cardiomyocytes, here we report that ring finger protein 146 (RNF146), a cytoplasmic E3-ubiquitin ligase, acts as a direct interactor of PARP-1. Overexpression of RNF146 exerts protection against oxidant-induced cell death, whereas PARP-1-mediated cellular injury is augmented after RNF146 silencing. RNF146 translocates to the nucleus upon PARP-1 activation, triggering the exit of PARP-1 from the nucleus, followed by rapid degradation of both proteins. PARP-1 and RNF146 degradation occurs in the early phase of myocardial ischemia-reperfusion injury; it precedes the induction of heat shock protein expression. Taken together, PARP-1 release from the nucleus and its rapid degradation represent newly identified steps of the necrotic cell death program induced by oxidative stress. These steps are controlled by the ubiquitin-proteasome pathway protein RNF146. The current results shed new light on the mechanism of necrotic cell death. RNF146 may represent a distinct target for experimental therapeutic intervention of oxidant-mediated cardiac injury.

In Vivo Detection of Rat Colorectal Cancers by using a Dual-Wavelength Excitation Method.

Hypoxia is a characteristic feature of solid neoplasms, and insufficient oxygen supply increases cellular nicotinamide adenine dinucleotide (NADH) fluorescence, which is a main component of autofluorescence of the colorectal mucosa. We investigated whether a dual-wavelength excitation method which is optimized for sensing mucosal NADH fluorescence could be applicable to the detection of rat colorectal cancers in vivo. Rat colorectal adenocarcinomas were studied by using fluorescence stereomicroscopy. After autofluorescence images at 470 nm irradiated with dual-wavelength excitation at 365 nm (F365ex) and 405 nm (F405ex) were acquired, ratio images were produced by dividing F365ex by F405ex: The excitation-emission wavelength pairs in F365ex and F405ex were adjusted for acquisition of NADH fluorescence and reference fluorescence. Based on observations from the luminal surface in vivo, F365ex/F405ex ratio images indicated a 1.57-fold higher signal value in the cancers than in the surrounding normal mucosa. The signal values in F365ex/F405ex ratio images were less mutually related with the hemoglobin concentration index. Small adenocarcinomas (less than 4 mm) could be detected on F365ex/F405ex ratio images. The results showed that NADH fluorescence measurement with little interference from tissue hemoglobin is efficient for visualizing rat colorectal cancers in vivo, suggesting that the dual-wavelength excitation method has potential for label-free endoscopic detection of diminutive colorectal neoplasms.

Overexpression of CD38 Decreases Cellular NAD Levels and Alters the Expression of Proteins Involved in Energy Metabolism and Antioxidant Defense

Nicotinamide adenine dinucleotide (NAD) is a coenzyme found in all living cells and mediates multiple cellular signaling pathways. In the present study, a 35% decrease of cellular NAD level is achieved by stable expression of the N-terminal truncated CD38, a NAD hydrolase. CD38-expressing (CD38(+)) cells have the lower growth rate and are more susceptive to oxidative stress than the wild type cells and empty vector-transfected (CD38(-)) cells. Quantitative proteomic analysis shows that 178 proteins are down-regulated in CD38(+) cells, which involve in diverse cellular processes including glycolysis, RNA processing and protein synthesis, antioxidant, and DNA repair. Down regulation of six selected proteins is confirmed by Western blotting. However, down-regulation of mRNA expressions of genes associated with glycolysis, antioxidant, and DNA repair is less significant than the corresponding change in protein expression, suggesting the low NAD level impairs the protein translational machinery in CD38(+) cells. Down-regulation of antioxidant protein and DNA-repair protein expression contributes to the susceptibility of CD38(+) cells to oxidative stress. Taken together, these results demonstrate that CD38(+) cells are a useful model to study effects of the cellular NAD levels on cellular processes and establish a new linker between cellular NAD levels and oxidative stress.

NADPH fluorescence as an indicator of hydrogen production in the green algae Chlamydomonas reinhardtii

This study investigated cellular Nicotinamide Adenine Dinucleotide Phosphate (NADPH) fluorescence as a potential indicator of biohydrogen production in Chlamydomonas reinhardtii and a β-NADPH standard. NADPH fluorescence profiles of cultures grown in TAP-S (Tris-acetate phosphate minus sulphur) media, TAP (Tris-acetate phosphate) media and TAP + 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) were subsequently compared. Hydrogen production induced from sulphur depletion was found to correlate directly (r = 0.941) with NADPH over the ten day period. The addition of leachate was used to increase hydrogen yields, and subsequently increased the NADPH concentration by 50%–70%. A direct correlation was observed (r = 0.929) between NADPH and hydrogen when the leachate supplemented media was used. As NADPH is the terminal electron acceptor in the photosynthetic chain, results show that NADPH has a pivotal role in hydrogen production as a carrier molecule. Under sulphur depletion, cellular NADPH fluorescence can be used as an indicator of hydrogen production.

Abstract B210: Novel inhibitors of nicotinamide phosphoribosyl transferase and their evaluation in combination with bortezomib.

Abstract Nicotinamide phosphoribosyl transferase (NAMPT) is the enzyme that catalyzes the rate limiting step in the salvage pathway of Nicotinamide Adenine Dinucleotide (NAD) biosynthesis. NAMPT is reported to be overexpressed in a number of cancer and inflammatory indications. Because of the requirement of NAD for a number of key biochemical pathways, inhibition of NAMPT has been shown to result in antitumor efficacy in preclinical models. NAMPT inhibitors have also been reported to enhance sensitivity to a number of targeted agents and overcome resistance to available therapies such as bortezomib resistance in multiple myeloma. In view of the sub-optimal profile of the reported first generation NAMPT inhibitors with respect to pharmacokinetics and drug-drug interaction, we sought to develop NAMPT inhibitors with the "best-in-class" profile for overcoming mechanism-based toxicities and/or resistance to current therapies. Utilizing structure-guided drug design and SAR-based approaches, we have optimized two chemical series of inhibitors of NAMPT. Determination of co-crystal structures with several de novo designed hits greatly aided in the identification of lead compounds that exhibited potent inhibition of NAMPT. Lead compounds were highly active in inhibiting proliferation that correlated well with cellular NAD depletion of cell lines derived from multiple myeloma, prostate and breast cancers. The anti-proliferative activities were rescued in NAPRT- proficient cell lines with the addition of NA due to the NAMPT independent salvage pathway for biosynthesis of NAD, confirming the mechanism of action through NAD depletion. Lead compounds exhibited desirable drug-like properties including solubility, permeability, metabolic stability, lack of CYP inhibition and pharmacokinetic exposure. In a xenograft model of pancreatic cancer, treatment with lead compounds resulted in regression of tumors with no signs of toxicity. Recent reports demonstrating the overexpression of NAMPT in bortezomib-resistant cells and a synergistic efficacy with a combination of NAMPT inhibitor and bortezomib against the resistant cells prompted us to evaluate the combination with our optimized leads. The combination of lead NAMPT inhibitors with bortezomib showed synergistic killing of cultured multiple myeloma cell lines. Evaluation of efficacy of the lead compounds as a single agent or in combination with bortezomib in xenograft models of multiple myeloma is currently underway. Modulation of NAD levels at lower doses of NAMPT inhibitors in combination with bortezomib could overcome the limitations such as mechanism-based toxicity and/or resistance of both these therapies and provide an effective treatment option for multiple myeloma. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B210. Citation Format: Dinesh Chikkanna, Anirudha Lakshminarasimhan, Vinayak Khairnar, Sunil Panigrahi, Anuradha Ramanathan, Aparna Satyanandan, Narasimha Rao, S Karthikeyan, Kishore Narayanan, Sreevalsam Gopinath, Raghuveer Ramachandra, Hosahalli Subramanya, Chetan Pandit, Murali Ramachandra. Novel inhibitors of nicotinamide phosphoribosyl transferase and their evaluation in combination with bortezomib. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B210.

Cellular NAD depletion and decline of SIRT1 activity play critical roles in PARP-1-mediated acute epileptic neuronal death in vitro

Intense poly(ADP-ribose) polymerase-1 (PARP-1) activation was implicated as a major cause of caspase-independent cell death in the hippocampal neuronal culture (HNC) model of acute acquired epilepsy (AE). The molecular mechanisms are quite complicated. The linkage among neuronal death, cellular nicotinamide adenine dinucleotide (NAD) levels, apoptosis-inducing factor (AIF) translocation, SIRT1 expression and activity were investigated here. The results showed that PARP-1 over-activation caused by Mg2+-free stimuli led to cellular NAD depletion which could block AIF translocation from mitochondria to nucleus and attenuate neuronal death. Also, SIRT1 deacetylase activity was reduced by Mg2+-free treatment, accompanied by elevated ratio of neuronal death, which could be rescued by NAD repletion. These data demonstrated that cellular NAD depletion and decline of SIRT1 activity play critical roles in PARP-1-mediated epileptic neuronal death in the HNC model of acute AE.

Abstract 5389: Novel inhibitors of nicotinamide phosphoribosyl transferase (NAMPT).

Abstract Nicotinamide phosphoribosyl transferase (NAMPT) is the enzyme that catalyzes the rate limiting step in the salvage pathway of Nicotinamide Adenine Dinucleotide (NAD) biosynthesis. NAMPT is reported to be overexpressed in a number of cancer and inflammatory indications. Because of the requirement of NAD as a co-factor or substrate for a number of key biochemical pathways including those catalyzed by PARP1, Sirtuins and ADP-ribosyl cyclase, inhibition of NAMPT has been shown to result in anti-tumor efficacy in preclinical models. Two NAMPT Inhibitors FK866/APO866 and GMX1778 are currently in clinical trials for oncology indications. In the presence of these clinical agents, cultured cell lines show development of resistance due to mutations underscoring the potential need for inhibitors from distinct chemical series. Here, we report a structure-guided drug design based approach for identification of lead compounds from two chemical series selectively targeting NAMPT. Determination of co-crystal structures with several de novo designed hits greatly aided in the identification of lead compounds that exhibited potent inhibition of NAMPT against both wild type and resistance mutants (G217R and H191R) Lead compounds were highly active in inhibiting proliferation that correlated well with cellular NAD depletion in several cancer cell lines. Normal cells and selected cancer cell lines have an NAMPT independent salvage pathway for biosynthesis of NAD, which is dependent on nicotinic acid phosphoribosyltransferase (NAPRT) and Nicotinic acid (NA). The anti-proliferative activities were fully rescued in NAPRT- proficient cell lines with the addition of NA, confirming the mechanism of action through specific NAD depletion. Lead compounds from both series exhibited excellent drug-like properties including solubility, metabolic stability and permeability, and desired exposure in pharmacokinetic studies. Anti-tumor activities of these compounds including NA rescue in NAPRT-proficient tumor models are currently being evaluated in preclinical models. Citation Format: Murali Ramachandra, Chetan Pandit, Hosahalli Subramanya, Dinesh Chikkanna, Anirudha Lakshminarasimhan, Vinayak Khairnar, Sunil Panigrahi, Anuradha Ramanathan, Aparna Satyanandan, Narasimha Rao, Arnab Bera, Kishore Narayanan, Sreevalsam Gopinath, Raghuveer Ramachandra. Novel inhibitors of nicotinamide phosphoribosyl transferase (NAMPT). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5389. doi:10.1158/1538-7445.AM2013-5389

Label-free in vivo flow cytometry in zebrafish using two-photon autofluorescence imaging

We demonstrate a label-free in vivo flow cytometry in zebrafish blood vessels based on two-photon excited autofluorescence imaging. The major discovery in this work is the strong autofluorescence emission from the plasma in zebrafish blood. The plasma autofluorescence provides excellent contrast for visualizing blood vessels and counting blood cells. In addition, the cellular nicotinamide adenine dinucleotide autofluorescence enables in vivo imaging and counting of white blood cells (neutrophils).

Impact of high glucose and transforming growth factor–β on bioenergetic profiles in podocytes

Diabetic nephropathy is the most common cause of chronic renal failure in industrialized countries. Depletion of podocytes plays an important role in the progression of diabetic glomerulopathy. Various factors in the diabetic milieu lead to serious podocyte stress driving the cells toward cell cycle arrest (p27Kip1), hypertrophy, detachment, and apoptosis. Mitochondria are responsible for oxidative phosphorylation and energy supply in podocytes. Recent studies indicated that mitochondrial dysfunction is a key factor in diabetic nephropathy. In the present study, we investigated metabolic profiles of podocytes under diabetic conditions. We examined oxygen consumption rates (OCRs) and oxidative phosphorylation complex activities in murine podocytes. Cells were exposed to high glucose for 48 hours, cultured for 10 passages under high-glucose conditions (30 mmol/L), or incubated with transforming growth factor–β (5 ng/mL) for 24 hours. After prolonged exposure to high glucose, podocytes showed a significantly increased OCR at baseline and also a higher OCR after addition of oligomycin, indicating significant changes in mitochondrial energy metabolism. Higher OCRs after inhibition of respiration by rotenone also indicated changes in nonmitochondrial respiration. Podocytes stimulated with a proapoptotic concentration of transforming growth factor–β displayed similar bioenergetic profiles, even with decreased citrate synthase activity. In all tested conditions, we found a higher cellular nicotinamide adenine dinucleotide content and changes in activities of respiratory chain complexes. In summary, we provide for the first time evidence that key factors of the diabetic milieu induce changes in glucose metabolism and mitochondrial function in podocytes.

NAMPT/PBEF Enzymatic Activity Is Indispensable for Myeloma Cell Growth and Osteoclast Activity

Abstract 886Clinical MRI and PET-CT analyses implicated hypermetabolic activity of multiple myeloma (MM) cells in focal lesions with tumor burden, metastasis and survival. Osteoclasts induced by MM are also naturally hypermetabolic suggesting that targeting cellular metabolism may contribute to controlling MM and associated bone disease. The coenzyme nicotinamide adenine dinucleotide (NAD) not only is essential for proper cellular metabolism but also affects activity of NAD-dependent enzymes, such as PARP1 and sirtuins. Nicotinamide phosphoribosyl transferase (NAMPT/PBEF/Visfatin) is a rate-limiting enzyme in NAD biosynthesis from nicotinamide and also functions extracellularly. APO866 (TopoTarget, Switzerland), an inhibitor of NAMPT, depletes cellular NAD particularly in cancer cells. The aims of the study were to evaluate expression of NAMPT (PBEF1) in clinical and experimental MM samples using global gene expression profiling (GEP) and investigate the effect of APO866 on MM cell growth and osteoclastogenesis. Our molecular classification data revealed elevated expression of PBEF1 in the PR, MAF and LB subtypes of newly diagnosed MM patients (n=796) and in MM cell lines (n=41). Based on our GEP70 risk model, PBEF1 expression was higher in high risk of the CD-1, HY, and MS subtypes relative to their low risk counterparts, and in focal lesions at relapse than at diagnosis. PBEF1 expression was 2.5 fold higher in myelomatous versus nonmyelomatous human bones from SCID-hu mice (p<0.0001). In primary MM cell-osteoclast cocultures (n=8) PBEF1 expression was upregulated by >3 folds in cocultured MM cells or osteoclasts compared to baseline MM cells (p<0.02) or control osteoclasts (p<0.04). APO866 dose dependently inhibited growth of 6 MM cell lines at low nanomolar concentrations (IC50 at 0.1–5 nM, 72 hrs) and similarly inhibited growth of MM cells in coculture with stromal cells or osteoclasts. NAD depletion by APO866 (5 nM) in 3 MM cell lines was consistently associated with reduced enzymatic activities of PARP1 by 73 ± 5% (p<0.001) and of SIRT1 by 49 ± 3% (p<0.005). In vivo, SCID-rab mice engrafted with our DAS MM cell line (MF subtype) were intraperitoneally treated with saline or APO866 (10 mice/group, 20 mg/kg, twice weekly) for 3 weeks. At experiment’s end circulating human Ig levels in control and APO866 groups were 43 ± 15 μg/ml and 1 ± 0.6 μg/ml, respectively (p<0.01). APO866 prevented bone loss as assessed by X-rays and measurement of bone mineral density (p<0.05). Histological examination confirmed high infiltration of MM cells in myelomatous bones of control hosts while in APO866-treated hosts the implanted bones reduced numbers of osteoclasts by 53% (p<0.0007). In vitro, at concentration of ≥0.1 nM, APO866 significantly suppressed osteoclast differentiation and bone resorption activity of mature osteoclasts on dentine slices (p<0.007). We conclude that NAMPT/PBEF/Visfatin is a potential target in certain molecularly classified and high risk MM patients, and that MM cells and osteoclasts are highly sensitive to NAD depletion. Disclosures:Barlogie:Celgene, Genzyme, Novartis, Millennium: Consultancy, Honoraria, Patents & Royalties. Shaughnessy:Myeloma Health, Celgene, Genzyme, Novartis: Consultancy, Employment, Equity Ownership, Honoraria, Patents & Royalties.