Photo-induced mitochondrial DNA damage and NADH depletion by -NO2 modified Ru(II) complexes.

Two mitochondria-localized Ru(ii) complexes with photo-labile ligands were reported to exert one- and two-photon activatable anticancer activity through a dual-function mechanism, i.e. mitochondrial DNA covalent binding after photo-induced ligand dissociation and photo-catalyzed NADH depletion, thus displaying good activity towards cisplatin-resistant cancer cells under both normoxic and hypoxic conditions.

Targeting NAD+ Synthesis to Potentiate CD38-Based Immunotherapy of Multiple Myeloma.

Antibodies targeting CD38, a NAD+-degrading enzyme, have emerged as a promising immunotherapy against multiple myeloma (MM). Currently, the mechanisms by which anti-CD38 antibodies establish their therapeutic effects are poorly understood. Here, we advocate for the depletion of NAD+ to enhance the efficacy of anti-CD38-based immunotherapies in MM.

Nicotinamide phosphoribosyltransferase­related signaling pathway in early Alzheimer's disease mouse models.

Alzheimer's disease (AD) is a neurodegenerative disease of the central nervous system that is characterized by progressive cognitive dysfunction and which ultimately leads to dementia. Studies have shown that energy dysmetabolism contributes significantly to the pathogenesis of a variety of aging­associated diseases and degenerative diseases of the nervous system, including AD. One focus of research thus has been how to regulate the expression of nicotinamide phosphoribosyltransferase (NAMPT) to prevent against neurodegenerative diseases. Therefore, the present study used 6­month­old APPswe/PS1ΔE9 (APP/PS1) transgenic mice as early AD mouse models and sought to evaluate nicotinamide adenine dinucleotide (NAD+) and FK866 (a NAMPT inhibitor) treatment in APP/PS1 mice to study NAMPT dysmetabolism in the process of AD and elucidate the underlying mechanisms. As a result of this treatment, the expression of NAMPT decreased, the synthesis of ATP and NAD+ became insufficient and the NAD+/NADH ratio was reduced. The administration of NAD+ alleviated the spatial learning and memory of APP/PS1 mice and reduced senile plaques. Administration of NAD+ may also increase the expression of the key protein NAMPT and its related protein sirtuin 1 as well as the synthesis of NAD+. Therefore, increasing NAMPT expression levels may promote NAD+ production. Their regulation could form the basis for a new therapeutic strategy.

Targeted photoredox catalysis in cancer cells.

Hypoxic tumours are a major problem for cancer photodynamic therapy. Here, we show that photoredox catalysis can provide an oxygen-independent mechanism of action to combat this problem. We have designed a highly oxidative Ir(III) photocatalyst, [Ir(ttpy)(pq)Cl]PF6 ([1]PF6, where 'ttpy' represents 4'-(p-tolyl)-2,2':6',2''-terpyridine and 'pq' represents 3-phenylisoquinoline), which is phototoxic towards both normoxic and hypoxic cancer cells. Complex 1 photocatalytically oxidizes 1,4-dihydronicotinamide adenine dinucleotide (NADH)-an important coenzyme in living cells-generating NAD• radicals with a high turnover frequency in biological media. Moreover, complex 1 and NADH synergistically photoreduce cytochrome c under hypoxia. Density functional theory calculations reveal π stacking in adducts of complex 1 and NADH, facilitating photoinduced single-electron transfer. In cancer cells, complex 1 localizes in mitochondria and disrupts electron transport via NADH photocatalysis. On light irradiation, complex 1 induces NADH depletion, intracellular redox imbalance and immunogenic apoptotic cancer cell death. This photocatalytic redox imbalance strategy offers a new approach for efficient cancer phototherapy.

Novel Antimycobacterial Compounds Suppress NAD Biogenesis by Targeting a Unique Pocket of NaMN Adenylyltransferase.

Conventional treatments to combat the tuberculosis (TB) epidemic are falling short, thus encouraging the search for novel antitubercular drugs acting on unexplored molecular targets. Several whole-cell phenotypic screenings have delivered bioactive compounds with potent antitubercular activity. However, their cellular target and mechanism of action remain largely unknown. Further evaluation of these compounds may include their screening in search for known antitubercular drug targets hits. Here, a collection of nearly 1400 mycobactericidal compounds was screened against Mycobacterium tuberculosis NaMN adenylyltransferase ( MtNadD), a key enzyme in the biogenesis of NAD cofactor that was recently validated as a new drug target for dormant and active tuberculosis. We found three chemotypes that efficiently inhibit MtNadD in the low micromolar range in vitro. SAR and cheminformatics studies of commercially available analogues point to a series of benzimidazolium derivatives, here named N2, with bactericidal activity on different mycobacteria, including M. abscessus, multidrug-resistant M. tuberculosis, and dormant M. smegmatis. The on-target activity was supported by the increased resistance of an M. smegmatis strain overexpressing the target and by a rapid decline in NAD(H) levels. A cocrystal structure of MtNadD with N2-8 inhibitor reveals that the binding of the inhibitor induced the formation of a new quaternary structure, a dimer-of-dimers where two copies of the inhibitor occupy symmetrical positions in the dimer interface, thus paving the way for the development of a new generation of selective MtNadD bioactive inhibitors. All these results strongly suggest that pharmacological inhibition of MtNadD is an effective strategy to combat dormant and resistant Mtb strains.

Role of Nicotinamide Adenine Dinucleotide and Related Precursors as Therapeutic Targets for Age-Related Degenerative Diseases: Rationale, Biochemistry, Pharmacokinetics, and Outcomes.

Significance: Nicotinamide adenine dinucleotide (NAD+) is an essential pyridine nucleotide that serves as an essential cofactor and substrate for a number of critical cellular processes involved in oxidative phosphorylation and ATP production, DNA repair, epigenetically modulated gene expression, intracellular calcium signaling, and immunological functions. NAD+ depletion may occur in response to either excessive DNA damage due to free radical or ultraviolet attack, resulting in significant poly(ADP-ribose) polymerase (PARP) activation and a high turnover and subsequent depletion of NAD+, and/or chronic immune activation and inflammatory cytokine production resulting in accelerated CD38 activity and decline in NAD+ levels. Recent studies have shown that enhancing NAD+ levels can profoundly reduce oxidative cell damage in catabolic tissue, including the brain. Therefore, promotion of intracellular NAD+ anabolism represents a promising therapeutic strategy for age-associated degenerative diseases in general, and is essential to the effective realization of multiple benefits of healthy sirtuin activity. The kynurenine pathway represents the de novo NAD+ synthesis pathway in mammalian cells. NAD+ can also be produced by the NAD+ salvage pathway. Recent Advances: In this review, we describe and discuss recent insights regarding the efficacy and benefits of the NAD+ precursors, nicotinamide (NAM), nicotinic acid (NA), nicotinamide riboside (NR), and nicotinamide mononucleotide (NMN), in attenuating NAD+ decline in degenerative disease states and physiological aging. Critical Issues: Results obtained in recent years have shown that NAD+ precursors can play important protective roles in several diseases. However, in some cases, these precursors may vary in their ability to enhance NAD+ synthesis via their location in the NAD+ anabolic pathway. Increased synthesis of NAD+ promotes protective cell responses, further demonstrating that NAD+ is a regulatory molecule associated with several biochemical pathways. Future Directions: In the next few years, the refinement of personalized therapy for the use of NAD+ precursors and improved detection methodologies allowing the administration of specific NAD+ precursors in the context of patients' NAD+ levels will lead to a better understanding of the therapeutic role of NAD+ precursors in human diseases.

Nicotinamide-Containing Di- and Trinucleotides as Chemical Tools for Studies of NAD-Capped RNAs.

We report the chemical synthesis of a set of nicotinamide adenine dinucleotide (NAD) cap analogues containing chemical modifications that reduce their susceptibility to NAD-RNA-degrading enzymes. These analogues can be incorporated into transcripts in a similar way as NAD. Biochemical characterization of RNAs carrying these caps with DXO, NudC, and Nudt12 enzymes led to the identification of compounds that can be instrumental in unraveling so far unaddressed biological aspects of NAD-RNAs.

The Pharmacology of CD38/NADase: An Emerging Target in Cancer and Diseases of Aging.

Recent reports indicate that intracellular NAD levels decline in tissues during chronological aging, and that therapies aimed at increasing cellular NAD levels could have beneficial effects in many age-related diseases. The protein CD38 (cluster of differentiation 38) is a multifunctional enzyme that degrades NAD and modulates cellular NAD homeostasis. At the physiological level, CD38 has been implicated in the regulation of metabolism and in the pathogenesis of multiple conditions including aging, obesity, diabetes, heart disease, asthma, and inflammation. Interestingly, many of these functions are mediated by CD38 enzymatic activity. In addition, CD38 has also been identified as a cell-surface marker in hematologic cancers such as multiple myeloma, and a cytotoxic anti-CD38 antibody has been approved by the FDA for use in this disease. Although this is a remarkable development, killing CD38-positive tumor cells with cytotoxic anti-CD38 antibodies is only one of the potential pharmacological uses of targeting CD38. The present review discusses the biology of the CD38 enzyme and the current state of development of pharmacological tools aimed at CD38, and explores how these agents may represent a novel approach for treating human conditions including cancer, metabolic disease, and diseases of aging.

Oleate ameliorates palmitate-induced reduction of NAMPT activity and NAD levels in primary human hepatocytes and hepatocarcinoma cells.

BACKGROUND: Nicotinamide phosphoribosyltransferase (NAMPT) and nicotinamide adenine dinucleotide (NAD) levels are crucial for liver function. The saturated fatty acid palmitate and the unsaturated fatty acid oleate are the main free fatty acids in adipose tissue and human diet. We asked how these fatty acids affect cell survival, NAMPT and NAD levels in HepG2 cells and primary human hepatocytes. METHODS: HepG2 cells were stimulated with palmitate (0.5mM), oleate (1mM) or a combination of both (0.5mM/1mM) as well as nicotinamide mononucleotide (NMN) (0.5 mM) or the specific NAMPT inhibitor FK866 (10nM). Cell survival was measured by WST-1 assay and Annexin V/propidium iodide staining. NAD levels were determined by NAD/NADH Assay or HPLC. Protein and mRNA levels were analysed by Western blot analyses and qPCR, respectively. NAMPT enzyme activity was measured using radiolabelled 14C-nicotinamide. Lipids were stained by Oil red O staining. RESULTS: Palmitate significantly reduced cell survival and induced apoptosis at physiological doses. NAMPT activity and NAD levels significantly declined after 48h of palmitate. In addition, NAMPT mRNA expression was enhanced which was associated with increased NAMPT release into the supernatant, while intracellular NAMPT protein levels remained stable. Oleate alone did not influence cell viability and NAMPT activity but ameliorated the negative impact of palmitate on cell survival, NAMPT activity and NAD levels, as well as the increased NAMPT mRNA expression and secretion. NMN was able to normalize intracellular NAD levels but did not ameliorate cell viability after co-stimulation with palmitate. FK866, a specific NAMPT inhibitor did not influence lipid accumulation after oleate-treatment. CONCLUSIONS: Palmitate targets NAMPT activity with a consequent cellular depletion of NAD. Oleate protects from palmitate-induced apoptosis and variation of NAMPT and NAD levels. Palmitate-induced cell stress leads to an increase of NAMPT mRNA and accumulation in the supernatant. However, the proapoptotic action of palmitate seems not to be mediated by decreased NAD levels.

NAD Synthesis Pathway Interference Is a Viable Therapeutic Strategy for Chondrosarcoma.

Nicotinamide phosphoribosyltransferase (NAMPT) and nicotinic acid phosphoribosyltransferase (NAPRT) are rate-limiting enzymes in the NAD+ synthesis pathway. Chondrosarcoma is a malignant cartilage forming bone tumor, in which mutations altering isocitrate dehydrogenase-1 and -2 (IDH1 and IDH2) activity have been identified as potential driver mutations. Vulnerability for NAD+ depletion has been reported for IDH1/2-mutant cells. Here, the potency of NAMPT inhibitors as a treatment of chondrosarcoma was explored. Eleven chondrosarcoma cell lines were treated with NAMPT inhibitors, in which the effect on cell viability, colony formation, and 3D collagen invasion was assessed. The expression level of NAMPT and NAPRT transcripts in chondrosarcoma cells was determined by qRT-PCR. Methylation of the NAPRT promoter was evaluated using a previously published dataset of genome-wide methylation. In addition, a methylation dataset was used to determine methylation of the NAPRT promoter in 20 IDH1/2-mutated cartilage tumors. Chondrosarcoma cells showed a dose-dependent decrease in cell viability, 3D collagen invasion, and colony formation upon treatment with NAMPT inhibitors, in which nearly half of the cell lines demonstrated absolute IC50s in the low nanomolar range. Increasing IC50s correlated to increasing NAPRT expression levels and decreasing NAPRT promoter methylation. No correlation between IDH1/2 mutation status and sensitivity for NAMPT inhibitors was observed. Strikingly, higher methylation of the NAPRT promoter was observed in high-grade versus low-grade chondrosarcomas. In conclusion, this study identified NAMPT as a potential target for treatment of chondrosarcoma.Implications: Chondrosarcoma patients, especially those of high histologic grade with lower expression and hypermethylation of NAPRT, may benefit from inhibition of the NAD synthesis pathway. Mol Cancer Res; 15(12); 1714-21. ©2017 AACR.

Inhibition of nicotinamide phosphoribosyltransferase and depletion of nicotinamide adenine dinucleotide contribute to arsenic trioxide suppression of oral squamous cell carcinoma.

Emerging evidence suggests that increased nicotinamide phosphoribosyltransferase (NAMPT) expression is associated with the development and prognosis of many cancers, but it remains unknown regarding its role in oral squamous cell carcinoma (OSCC). In the present study, the results from tissue microarray showed that NAMPT was overexpressed in OSCC patients and its expression level was directly correlated with differential grades of cancer. Interestingly, treatment of OSCC cells with chemotherapy agent arsenic trioxide (ATO) decreased the levels of NAMPT protein and increased cellular death in an ATO dose- and time-dependent manner. Most importantly, combination of low concentration ATO with FK866 (a NAMPT inhibitor) exerted enhanced inhibitive effect on NAMPT protein and mRNA expressions, leading to synergistic cytotoxicity on cancer cells through increasing cell apoptosis and depleting intracellular nicotinamide adenine dinucleotide levels. These findings demonstrate the crucial role of NAMPT in the prognosis of OSCC and reveal inhibition of NAMPT as a novel mechanism of ATO in suppressing cancer cell growth. Our results suggest that ATO can significantly enhance therapeutic efficacy of NAMPT inhibitor, and combined treatment may be a novel and effective therapeutic strategy for OSCC patients.

Hepatic NAD(+) deficiency as a therapeutic target for non-alcoholic fatty liver disease in ageing.

BACKGROUND AND PURPOSE: Ageing is an important risk factor of non-alcoholic fatty liver disease (NAFLD). Here, we investigated whether the deficiency of nicotinamide adenine dinucleotide (NAD(+) ), a ubiquitous coenzyme, links ageing with NAFLD. EXPERIMENTAL APPROACH: Hepatic concentrations of NAD(+) , protein levels of nicotinamide phosphoribosyltransferase (NAMPT) and several other critical enzymes regulating NAD(+) biosynthesis, were compared in middle-aged and aged mice or patients. The influences of NAD(+) decline on the steatosis and steatohepatitis were evaluated in wild-type and H247A dominant-negative, enzymically-inactive NAMPT transgenic mice (DN-NAMPT) given normal or high-fat diet (HFD). KEY RESULTS: Hepatic NAD(+) level decreased in aged mice and humans. NAMPT-controlled NAD(+) salvage, but not de novo biosynthesis pathway, was compromised in liver of elderly mice and humans. Given normal chow, middle-age DN-NAMPT mice displayed systemic NAD(+) reduction and had moderate NAFLD phenotypes, including lipid accumulation, enhanced oxidative stress, triggered inflammation and impaired insulin sensitivity in liver. All these NAFLD phenotypes, especially release of pro-inflammatory factors, Kupffer cell accumulation, monocytes infiltration, NLRP3 inflammasome pathway and hepatic fibrosis (Masson's staining and α-SMA staining), deteriorated further under HFD challenge. Oral administration of nicotinamide riboside, a natural NAD(+) precursor, completely corrected these NAFLD phenotypes induced by NAD(+) deficiency alone or HFD, whereas adenovirus-mediated SIRT1 overexpression only partially rescued these phenotypes. CONCLUSIONS AND IMPLICATIONS: These results provide the first evidence that ageing-associated NAD(+) deficiency is a critical risk factor for NAFLD, and suggest that supplementation with NAD(+) substrates may be a promising therapeutic strategy to prevent and treat NAFLD.

Inhibition of an NAD⁺ salvage pathway provides efficient and selective toxicity to human pluripotent stem cells.

The tumorigenic potential of human pluripotent stem cells (hPSCs) is a major limitation to the widespread use of hPSC derivatives in the clinic. Here, we demonstrate that the small molecule STF-31 is effective at eliminating undifferentiated hPSCs across a broad range of cell culture conditions with important advantages over previously described methods that target metabolic processes. Although STF-31 was originally described as an inhibitor of glucose transporter 1, these data support the reclassification of STF-31 as a specific NAD⁺ salvage pathway inhibitor through the inhibition of nicotinamide phosphoribosyltransferase (NAMPT). These findings demonstrate the importance of an NAD⁺ salvage pathway in hPSC biology and describe how inhibition of NAMPT can effectively eliminate hPSCs from culture. These results will advance and accelerate the development of safe, clinically relevant hPSC-derived cell-based therapies.

Redox control of glutamine utilization in cancer.

Glutamine utilization promotes enhanced growth of cancer cells. We propose a new concept map of cancer metabolism in which mitochondrial NADH and NADPH, in the presence of a dysfunctional electron transfer chain, promote reductive carboxylation from glutamine. We also discuss why nicotinamide nucleotide transhydrogenase (NNT) is required in vivo for glutamine utilization by reductive carboxylation. Moreover, NADPH, generated by both the pentose phosphate pathway and the cancer-specific serine glycolytic diversion, appears to sustain glutamine utilization for amino-acid synthesis, lipid synthesis, and for ROS quenching. The fact that the supply of NAD(+) precursors reduces tumor aggressiveness suggests experimental approaches to clarify the role of the NADH-driven redox network in cancer.

Multiphoton fluorescence lifetime imaging microscopy reveals free-to-bound NADH ratio changes associated with metabolic inhibition.

Measurement of endogenous free and bound NAD(P)H relative concentrations in living cells isa useful method for monitoring aspects of cellular metabolism, because the NADH∕NAD⁺ reduction-oxidation pair is crucial for electron transfer through the mitochondrial electron transport chain. Variations of free and bound NAD(P)H ratio are also implicated in cellular bioenergetic and biosynthetic metabolic changes accompanying cancer. This study uses two-photon fluorescence lifetime imaging microscopy (FLIM) to investigate metabolic changes in MCF10A premalignant breast cancer cells treated with a range of glycolysis inhibitors: namely, 2 deoxy-D-glucose, oxythiamine, lonidamine, and 4-(chloromethyl) benzoyl chloride, as well as the mitochondrial membrane uncoupling agent carbonyl cyanide m-chlorophenylhydrazone. Through systematic analysis of FLIM data from control and treated cancer cells, we observed that all glycolytic inhibitors apart from lonidamine had a slightly decreased metabolic rate and that the presence of serum in the culture medium generally marginally protected cells from the effect of inhibitors. Direct production of glycolytic L-lactate was also measured in both treated and control cells. The combination of these two techniques gave valuable insights into cell metabolism and indicated that FLIM was more sensitive than traditional biochemical methods, as it directly measured metabolic changes within cells as compared to quantification of lactate secreted by metabolically active cells.

Metabolic and bactericidal effects of targeted suppression of NadD and NadE enzymes in mycobacteria.

UNLABELLED: Mycobacterium tuberculosis remains a major cause of death due to the lack of treatment accessibility, HIV coinfection, and drug resistance. Development of new drugs targeting previously unexplored pathways is essential to shorten treatment time and eliminate persistent M. tuberculosis. A promising biochemical pathway which may be targeted to kill both replicating and nonreplicating M. tuberculosis is the biosynthesis of NAD(H), an essential cofactor in multiple reactions crucial for respiration, redox balance, and biosynthesis of major building blocks. NaMN adenylyltransferase (NadD) and NAD synthetase (NadE), the key enzymes of NAD biosynthesis, were selected as promising candidate drug targets for M. tuberculosis. Here we report for the first time kinetic characterization of the recombinant purified NadD enzyme, setting the stage for its structural analysis and inhibitor development. A protein knockdown approach was applied to validate bothNadD and NadE as target enzymes. Induced degradation of either target enzyme showed a strong bactericidal effect which coincided with anticipated changes in relative levels of NaMN and NaAD intermediates (substrates of NadD and NadE, respectively) and ultimate depletion of the NAD(H) pool. A metabolic catastrophe predicted as a likely result of NAD(H) deprivation of cellular metabolism was confirmed by (13)C biosynthetic labeling followed by gas chromatography-mass spectrometry (GC-MS) analysis. A sharp suppression of metabolic flux was observed in multiple NAD(P)(H)-dependent pathways, including synthesis of many amino acids (serine, proline, aromatic amino acids) and fatty acids. Overall, these results provide strong validation of the essential NAD biosynthetic enzymes, NadD and NadE, as antimycobacterial drug targets. IMPORTANCE: To address the problems of M. tuberculosis drug resistance and persistence of tuberculosis, new classes of drug targets need to be explored. The biogenesis of NAD cofactors was selected for target validation because of their indispensable role in driving hundreds of biochemical transformations. We hypothesized that the disruption of NAD production in the cell via genetic suppression of the essential enzymes (NadD and NadE) involved in the last two steps of NAD biogenesis would lead to cell death, even under dormancy conditions. In this study, we confirmed the hypothesis using a protein knockdown approach in the model system of Mycobacterium smegmatis. We showed that induced proteolytic degradation of either target enzyme leads to depletion of the NAD cofactor pool, which suppresses metabolic flux through numerous NAD(P)-dependent pathways of central metabolism of carbon and energy production. Remarkably, bactericidal effect was observed even for nondividing bacteria cultivated under carbon starvation conditions.

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

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.

Structure-activity relationship studies and biological characterization of human NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase inhibitors.

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

Extracellular NAD(+) inhibits human neutrophil apoptosis.

Regulation of neutrophil apoptosis plays a critical role in the inflammatory response. Inflammation has previously been shown to increase levels of extracellular ß-nicotinamide adenine dinucleotide (NAD(+)). The present study demonstrates that extracellular NAD(+) at concentrations found in the inflamed tissues profoundly delays spontaneous apoptosis of human neutrophils as was evidenced by inhibition of phosphatidylserine (PS) exposure, DNA fragmentation and caspase-3 activation. The effect was abrogated by NF157, an antagonist of P2Y11 receptor, and was pertussis toxin-insensitive. The NAD(+)-mediated delay of neutrophil apoptosis was reversed by 2',5'-dideoxyadenosine, an inhibitor of adenylyl cyclase, and Rp-8-Br-cAMPS, an inhibitor of type I cAMP-dependent protein kinase A (PKA). Blocking of NAD(+)-induced influx of extracellular Ca(2+) with EGTA did not abolish the pro-survival effect of NAD(+). Extracellular NAD(+) inhibited proteasome-dependent degradation of Mcl-1 upstream of caspase activation and, furthermore, suppressed Bax translocation to the mitochondria and attenuated both dissipation of mitochondrial transmembrane potential (ΔΨm) and cytochrome c release from the mitochondria into the cytosol. Finally, we found that extracellular NAD(+) inhibited spontaneous activation of caspase-9, but not caspase-8, and the pro-survival effect of extracellular NAD(+) was abrogated by the inhibitor of caspase-9, but not by the inhibitor of caspase-8. Together, these results demonstrate that extracellular NAD(+) inhibits neutrophil apoptosis via P2Y11 receptor and cAMP/PKA pathway by regulating Mcl-1 level, Bax targeting to the mitochondria and mitochondrial apoptotic pathway. Thus, extracellular NAD(+) acts as a neutrophil survival factor that can contribute to prolonged neutrophil lifespan in inflammatory response.

A secondary mode of action of polymyxins against Gram-negative bacteria involves the inhibition of NADH-quinone oxidoreductase activity.

Polymyxin B and colistin were examined for their ability to inhibit the type II NADH-quinone oxidoreductases (NDH-2) of three species of Gram-negative bacteria. Polymyxin B and colistin inhibited the NDH-2 activity in preparations from all of the isolates in a concentration-dependent manner. The mechanism of NDH-2 inhibition by polymyxin B was investigated in detail with Escherichia coli inner membrane preparations and conformed to a mixed inhibition model with respect to ubiquinone-1 and a non-competitive inhibition model with respect to NADH. These suggest that the inhibition of vital respiratory enzymes in the bacterial inner membrane represents one of the secondary modes of action for polymyxins.