The Fluorinated NAD Precursors Enhance FK866 Cytotoxicity by Activating SARM1 in Glioblastoma Cells.

Glioblastoma, a formidable brain tumor characterized by dysregulated NAD metabolism, poses a significant therapeutic challenge. The NAMPT inhibitor FK866, which induces NAD depletion, has shown promise in controlling tumor proliferation and modifying the tumor microenvironment. However, the clinical efficacy of FK866 as a single drug therapy for glioma is limited. In this study, we aim to disrupt NAD metabolism using fluorinated NAD precursors and explore their synergistic effect with FK866 in inducing cytotoxicity in glioblastoma cells. The synthesized analogue of nicotinamide riboside (NR), ara-F nicotinamide riboside (F-NR), inhibits nicotinamide ribose kinase (NRK) activity in vitro, reduces cellular NAD levels, and enhances FK866's cytotoxicity in U251 glioblastoma cells, indicating a collaborative impact on cell death. Metabolic analyses reveal that F-NR undergoes conversion to fluorinated nicotinamide mononucleotide (F-NMN) and other metabolites, highlighting the intact NAD metabolic pathway in glioma cells. The activation of SARM1 by F-NMN, a potent NAD-consuming enzyme, is supported by the synergistic effect of CZ-48, a cell-permeable SARM1 activator. Temporal analysis underscores the sequential nature of events, establishing NAD depletion as a precursor to ATP depletion and eventual massive cell death. This study not only elucidates the molecular intricacies of glioblastoma cell death but also proposes a promising strategy to enhance FK866 efficacy through fluorinated NAD precursors, offering potential avenues for innovative therapeutic interventions in the challenging landscape of glioblastoma treatment.

Effect of alternating nicotinamide phosphoribosyltransferase expression levels on mitophagy in Alzheimer's disease mouse models.

AD is the abbreviation for Alzheimer's Disease, which is a neurodegenerative disorder that features progressive dysfunction in cognition. Previous research has reported that mitophagy impairment and mitochondrial dysfunction have been crucial factors in the AD's pathogenesis. More recently, literature has emerged which offers findings suggesting that the nicotinamide adenine dinucleotide (short for NAD+) augmentation eliminates the defective mitochondria and restores mitophagy. Meanwhile, as an enzyme which is rate-limiting, the Nicotinamide phosphoribosyltransferase, or NAMPT, is part of the salvage pathway of NAD+ synthesis. Therefore, the aim of the research project has been to produce proof for how the NAMPT-NAD +-silent information-regulated transcription factors1/3 (short for SIRT1/3) axis function in mediating mitophagy in APP/PS1 mice aged six months. The results revealed that the NAMPT-NAD+-SIRT1/3 axis in the APP/PS1 mice's hippocampus was considerably declined. Surprisingly, P7C3 (an NAMPT activator) noticeably promoted the NAD+-SIRT1/3 axis, improved mitochondrial structure and function, enhanced mitophagy activity along with the ability of learning and memory. While FK866 (an NAMPT inhibitor) reversed the decreased NAD+-SIRT1/3 axis, and even exacerbated Aß plaque deposition level in the APP/PS1 mice's hippocampus. The findings observed in this study indicate two main points: avoiding downregulation of the NAMPT activity can prevent AD-related mitophagy impairment; on the other hand, NAMPT characterizes a potential therapeutic intervention regarding AD pathogenesis.

Distinct Capabilities in NAD Metabolism Mediate Resistance to NAMPT Inhibition in Glioblastoma.

Glioblastoma (GBM) cells require high levels of nicotinamide adenine dinucleotide (NAD) to fuel metabolic reactions, regulate their cell cycle and support DNA repair in response to chemotherapy and radiation. Inhibition of a key enzyme in NAD biosynthesis, NAMPT, has demonstrated significant anti-neoplastic activity. Here, we sought to characterise NAD biosynthetic pathways in GBM to determine resistance mechanisms to NAD inhibitors. GBM cells were treated with the NAMPT inhibitor FK866 with and without NAD precursors, and were analysed by qPCR, Western blot and proliferation assays (monolayer and spheroid). We also measured changes in the cell cycle, apoptosis, NAD/NADH levels and energy production. We performed orthoptic xenograft experiments in athymic nude mice to test the efficacy of FK866 in combination with temozolomide (TMZ). We show that the expression of key genes involved in NAD biosynthesis is highly variable across GBM tumours. FK866 inhibits proliferation, reduces NAD levels and limits oxidative metabolism, leading to G2/M cell cycle arrest; however, this can be reversed by supplementation with specific NAD precursors. Furthermore, FK866 potentiates the effects of radiation and TMZ in vitro and in vivo. NAMPT inhibitors should be considered for the treatment of GBM, with patients stratified based on their expression of key enzymes in other NAD biosynthetic pathways.

The Effect of Visfatin on the Functioning of the Porcine Pituitary Gland: An In Vitro Study.

Visfatin (VIS), also known as nicotinamide phosphoribosyltransferase (NAMPT), is the rate-limiting enzyme in the biosynthesis of nicotinamide adenine dinucleotide (NAD+). Recently, VIS has been also recognized as an adipokine. Our previous study revealed that VIS is produced in the anterior and posterior lobes of the porcine pituitary. Moreover, the expression and secretion of VIS are dependent on the phase of the estrous cycle and/or the stage of early pregnancy. Based on this, we hypothesized that VIS may regulate porcine pituitary function. This study was conducted on anterior pituitary (AP) glands harvested from pigs during specific phases of the estrous cycle. We have shown the modulatory effect of VIS in vitro on LH and FSH secretion by porcine AP cells (determined by ELISA). VIS was also found to stimulate cell proliferation (determined by Alamar Blue) without affecting apoptosis in these cells (determined using flow cytometry technique). Moreover, it was indicated that VIS may act in porcine AP cells through the INSR, AKT/PI3K, MAPK/ERK1/2, and AMPK signaling pathways (determined by ELISA or Western Blot). This observation was further supported by the finding that simultaneous treatment of cells with VIS and inhibitors of these pathways abolished the observed VIS impact on LH and FSH secretion (determined by ELISA). In addition, our research indicated that VIS affected the mentioned processes in a manner that was dependent on the dose of VIS and/or the phase of the estrous cycle. Thus, these findings suggest that VIS may regulate the functioning of the porcine pituitary gland during the estrous cycle.

Design of PLGA nanoparticles for sustained release of hydroxyl-FK866 by microfluidics.

The use of nanoparticle (NP) delivery systems in cancer treatment has received significant interest, however use of such systems in delivery of cytotoxic chemotherapy agents can be limited by low encapsulation efficiency and burst release of the cytotoxin, as well issues with throughput and reproducibility during the fabrication of drug-loaded NPs. In this study, we used a hydrodynamic flow-focusing microfluidic system to successfully produce poly(lactic-co-glycolic acid) (PLGA) NPs. The physico-chemical properties of PLGA NPs were controlled by changing the manufacturing parameters, such as flow rate ratio, total flow rate, PLGA and surfactant concentration. The NAMPT inhibitor-polymer conjugate, hydroxyl-FK866-PLGA, was synthesized and used to fabricate hydroxyl-FK866-PLGA NPs for the formulation of localized delivery systems able to release low doses of cytotoxins and enhance the efficacy of NAMPT inhibitors. Hydroxyl-FK866-PLGA NPs were prepared with optimized fabrication parameters, having average Z-size of 128 ± 8 nm (PDI < 0.2), ζ-potential of -14.8 ± 5.3 mV and high encapsulation efficiency (98.6 ± 5.8 %). The pH-dependent release of hydroxyl-FK866 was monitored over time in conditions mimicking the normal (pH 7.4) and inflamed/tumor (pH 6.4) microenvironments, observing a sustained release pattern (over two months) without any initial burst release. Finally, toxicity of hydroxyl-FK866-PLGA NPs were tested in selected human cell lines, the human leukemia monocytic cell line (THP-1), and the human triple negative breast cancer cell line (MDA-MB-231). Our work suggests that microfluidic systems are a promising technology for a rapid and efficient manufacturing of PLGA-based NPs for the controlled release of cytotoxins. Moreover, the use of drug-polymer conjugates is an effective approach for the manufacturing of polymeric NPs enabling high encapsulation efficiency and a prolonged and sustained pH-dependent drug release.

Discovery of potent NAMPT-Targeting PROTACs using FK866 as the warhead.

Nicotinamide phosphoribosyltransferase (NAMPT) has emerged as a promising target for cancer therapy due to its strong correlation with nicotinamide adenine dinucleotide (NAD+) metabolism and tumorigenesis. Proteolysis targeting chimeras (PROTACs) provided an attractive strategy for developing NAMPT-targeting NAD+-depleting cancer drugs. Herein, a series of von Hippel-Lindau (VHL)-recruiting NAMPT-targeting PROTACs were designed using NAMPT inhibitor FK866 as the warhead. Among them, compound C5 degraded NAMPT (DC50 = 31.7 nM) in a VHL- and proteasome-dependent manner. Moreover, compound C5 effectively inhibited the proliferation of A2780 cells (IC50 = 30.6 nM) and significantly reduced the general cytotoxicity of FK866 to normal cells.

NAMPT inhibition relieves intestinal inflammation by regulating macrophage activation in experimental necrotizing enterocolitis.

Nicotinamide phosphoribosyl transferase (NAMPT) is associated with various NAD+ -consuming enzymatic reactions. The precise role in intestinal mucosal immunity in necrotizing enterocolitis (NEC) is not well defined. Here, we examined whether NAMPT inhibition by the highly specific inhibitor FK866 could alleviate intestinal inflammation during the pathogenesis of NEC. In the present study, we showed that NAMPT expression was upregulated in the human terminal ileum of human infants with NEC. FK866 administration attenuated M1 macrophage polarization and relieved the symptoms of experimental NEC pups. FK866 inhibited intercellular NAD+ levels, macrophage M1 polarization, and the expression of NAD+ -dependent enzymes, such as poly (ADP ribose) polymerase 1 (PARP1) and Sirt6. Consistently, the capacity of macrophages to phagocytose zymosan particles, as well as antibacterial activity, were impaired by FK866, whereas NMN supplementation to restore NAD+ levels reversed the changes in phagocytosis and antibacterial activity. In conclusion, FK866 reduced intestinal macrophage infiltration and skewed macrophage polarization, which is implicated in intestinal mucosal immunity, thereby promoting the survival of NEC pups.

Mitochondrial rewiring drives metabolic adaptation to NAD(H) shortage in triple negative breast cancer cells.

Nicotinamide phosphoribosyltransferase (NAMPT) is a key metabolic enzyme in NAD+ synthesis pathways and is found upregulated in several tumors, depicting NAD(H) lowering agents, like the NAMPT inhibitor FK866, as an appealing approach for anticancer therapy. Like other small molecules, FK866 triggers chemoresistance, observed in several cancer cellular models, which can prevent its clinical application. The molecular mechanisms sustaining the acquired of resistance to FK866 were studied in a model of triple negative breast cancer (MDA-MB-231 parental - PAR), exposed to increasing concentrations of the small molecule (MDA-MB-231 resistant - RES). RES cells are not sensitive to verapamil or cyclosporin A, excluding a potential role of increased efflux pumps activity as a mechanism of resistance. Similarly, the silencing of the enzyme Nicotinamide Riboside Kinase 1 (NMRK1) in RES cells does not increase FK866 toxicity, excluding this pathway as a compensatory mechanism of NAD+ production. Instead, Seahorse metabolic analysis revealed an increased mitochondrial spare respiratory capacity in RES cells. These cells presented a higher mitochondrial mass compared to the FK866-sensitive counterparts, as well as an increased consumption of pyruvate and succinate for energy production. Interestingly, co-treatment of PAR cells with FK866 and the mitochondrial pyruvate carrier (MPC) inhibitors UK5099 or rosiglitazone, as well as with the transient silencing of MPC2 but not of MPC1, induces a FK866-resistant phenotype. Taken together, these results unravel novel mechanisms of cell plasticity to counteract FK866 toxicity, that, besides the previously described LDHA dependency, rely on mitochondrial rewiring at functional and energetic levels.

The NAMPT Inhibitor FK866 in Combination with Cisplatin Reduces Cholangiocarcinoma Cells Growth.

It is well established that Cholangiocarcioma (CCA) drug resistance plays a crucial role in the spread and survival of cancer cells. The major enzyme in the nicotinamide-adenine dinucleotide (NAD+)-mediated pathways, nicotinamide phosphoribosyltransferase (NAMPT), is essential for cancer cell survival and metastasis. Previous research has shown that the targeted NAMPT inhibitor FK866 reduces cancer cell viability and triggers cancer cell death; however, whether FK866 affects CCA cell survival has not been addressed before. We show herein that NAMPT is expressed in CCA cells, and FK866 suppresses the capacity of CCA cells to grow in a dose-dependent manner. Furthermore, by preventing NAMPT activity, FK866 significantly reduced the amount of NAD+ and adenosine 5'-triphosphate (ATP) in HuCCT1, KMCH, and EGI cells. The present study's findings further show that FK866 causes changes in mitochondrial metabolism in CCA cells. Additionally, FK866 enhances the anticancer effects of cisplatin in vitro. Taken together, the results of the current study suggest that the NAMPT/NAD+ pathway may be a possible therapeutic target for CCA, and FK866 may be a useful medication targeting CCA in combination with cisplatin.

Visfatin induces ovarian cancer resistance to anoikis by regulating mitochondrial activity.

PURPOSE: Ovarian cancer is characterized by recurrent peritoneal and distant metastasis. To survive in a non-adherent state, floating ovarian cancer spheroids develop mechanisms to resist anoikis. Moreover, ascitic fluid from ovarian cancer patients contains high levels of visfatin with anti-apoptotic properties. However, the mechanism by which visfatin induces anoikis resistance in ovarian cancer spheroids remains unknown. Here, we aimed to assess wheather visfatin which possess anti-apoptotic properties can induce resistance of anoikis in ovarian cancer spheroids. METHODS: Visfatin synthesis were examined using a commercial human visfatin ELISA Kit. Spheroid were exposed to visfatin and cell viability and caspase 3/7 activity were measured using CellTiter-Glo 3D cell viability assay and Caspase-Glo® 3/7 Assay System. mRNA and protein expression were analyzed by Real-time PCR and Western Blot analysis, respectively. Analysis of mitochondrial activity was estimated by JC-1 staining. RESULTS: First, our results suggested higher expression and secretion of visfatin by epithelial than by granulosa ovarian cells, and in non-cancer tissues versus cancer tissues. Interestingly, visfatin increased the proliferation/apoptosis ratio in ovarian cancer spheroids. Specifically, both the intrinsic and extrinsic pathways of anoikis were regulated by visfatin. Moreover, the effect of the visfatin inhibitor (FK866) was opposite to that of visfatin. Furthermore, both NAMPT and FK866 affected mitochondrial activity in ovarian cancer cells. CONCLUSION: In conclusion, visfatin acts as an anti-apoptotic factor by regulating mitochondrial activity, leading to anoikis resistance in ovarian cancer spheroids. The finding suggest visfatin as a potential novel therapeutic target for the treatment of ovarian carcinoma with peritoneal dissemination.

NAMPT inhibition reduces macrophage inflammation through the NAD+/PARP1 pathway to attenuate liver ischemia-reperfusion injury.

BACKGROUND: Liver ischemia-reperfusion injury (IRI) is a major complication in the perioperative period and often leads to liver failure and even systemic inflammation. Previous studies have suggested that the inflammatory response participated in the liver damage during liver IRI. Nicotinamide phosphoribosyl transferase (NAMPT) is required for the maintenance of cellular nicotinamide adenine dinucleotide (NAD+) levels, catalyzing the rate-limiting step in the NAD + salvage pathway. NAMPT is strongly upregulated during inflammation and constitutes an important mechanistic link between inflammatory, metabolic, and transcriptional pathways. The aim of our study was to investigate the role of NAMPT in liver IRI. METHODS: We investigated the effect of pharmacological inhibition of NAMPT with FK866 in models of liver IRI. Liver damage was assessed by HE staining, serum ALT/AST, and TUNEL staining. To examine the mechanism, primary hepatocytes, liver macrophages and RAW264.7 cells were treated with or without NAMPT inhibitors before hypoxia-reoxygenation. Liver macrophages and RAW 264.7 cells activation in vitro was evaluated by western blotting, flow cytometry, and ELISA. RESULT: We found that NAMPT was upregulated in liver IRI. Treatment with the NAMPT inhibitor FK866 ameliorated liver IRI and suppressed inflammation in mice. Although NAMPT plays an important role both in hepatocytes and liver macrophages, we focused on the impact of NAMPT on liver macrophages. The mechanism revealed that FK866 potently inhibited NAMPT activity, as demonstrated by reduced liver NAD+ and intracellular NAD+, resulting in reduced abundance and activity of NAD + -dependent enzymes, including poly (ADP-ribose) polymerase 1 (PARP1), thus inhibiting macrophage M1 polarization by reducing CD86, iNOS, TNF-α, and interleukin (IL)-1ß. Taken together, our data suggested that NAMPT can regulate macrophage polarization through NAD+/PARP1 to ameliorate liver injury, and that FK866-mediated NAMPT blockade may be a therapeutic approach in liver IRI.

Visfatin is a multifaceted molecule that exerts regulation effects on inflammation and apoptosis in RAW264.7 cells and mice immune organs.

Visfatin, a multifunctional adipocytokine, is particularly important in the regulation of apoptosis and inflammation through an unidentified mechanism. Clarifying the control mechanisms of visfatin on inflammation and apoptosis in RAW264.7 cells and mice immunological organs was the goal of the current investigation. In order to create a pathophysiological model, the RAW264.7 cells were stimulated with 200 ng/mL visfatin and 20 µg/mL lipopolysaccharide (LPS), either separately or combined. The effects of exogenous visfatin on inflammation and apoptosis in RAW264.7 cells were investigated by flow cytometry assay, RNA-seq analysis and fluorescence quantitative PCR. According to the findings, exogenous visfatin exhibits dual effects on inflammation by modulating the expression of IL-1α, TNFRSF1B, and LIF as well as taking part in various signaling pathways, including the MAPK and Rap1 signaling pathways. By controlling the expression levels of Bcl2l1, Bcl2a1a, and Fas and primarily participating in the PI3K/AKT signaling pathway and Hippo signaling pathway, exogenous visfatin can inhibit apoptosis in RAW264.7 cells. The visfatin inhibitor FK866 was used to further confirm the effects of visfatin on inflammation and apoptosis in mice immune organs. Subsequently, mice spleen and thymus were collected. It is interesting to note that in LPS-treated mice, suppression of endogenous visfatin might worsen the immune system's inflammatory response and even result in rapid mortality. Additionally, endogenous visfatin promotes the apoptosis in mice immune organs by regulating the expression levels of Bcl2l1, Fas, Caspase 3, Bcl2a1a, and Bax. Together, these results imply that visfatin is a multifaceted molecule that regulates inflammation and apoptosis in RAW264.7 cells and mice immunological organs by taking part in a variety of biological processes and regulating the amounts of associated cytokines expression. Our findings offer additional understandings of how visfatin affects apoptosis and inflammation.

Integrating Pharmacogenomics Data-Driven Computational Drug Prediction with Single-Cell RNAseq to Demonstrate the Efficacy of a NAMPT Inhibitor against Aggressive, Taxane-Resistant, and Stem-like Cells in Lethal Prostate Cancer.

Metastatic prostate cancer/PCa is the second leading cause of cancer deaths in US men. Most early-stage PCa are dependent on overexpression of the androgen receptor (AR) and, therefore, androgen deprivation therapies/ADT-sensitive. However, eventual resistance to standard medical castration (AR-inhibitors) and secondary chemotherapies (taxanes) is nearly universal. Further, the presence of cancer stem-like cells (EMT/epithelial-to-mesenchymal transdifferentiation) and neuroendocrine PCa (NEPC) subtypes significantly contribute to aggressive/lethal/advanced variants of PCa (AVPC). In this study, we introduced a pharmacogenomics data-driven optimization-regularization-based computational prediction algorithm ("secDrugs") to predict novel drugs against lethal PCa. Integrating secDrug with single-cell RNA-sequencing/scRNAseq as a 'Double-Hit' drug screening tool, we demonstrated that single-cells representing drug-resistant and stem-cell-like cells showed high expression of the NAMPT pathway genes, indicating potential efficacy of the secDrug FK866 which targets NAMPT. Next, using several cell-based assays, we showed substantial impact of FK866 on clinically advanced PCa as a single agent and in combination with taxanes or AR-inhibitors. Bulk-RNAseq and scRNAseq revealed that, in addition to NAMPT inhibition, FK866 regulates tumor metastasis, cell migration, invasion, DNA repair machinery, redox homeostasis, autophagy, as well as cancer stemness-related genes, HES1 and CD44. Further, we combined a microfluidic chip-based cell migration assay with a traditional cell migration/'scratch' assay and demonstrated that FK866 reduces cancer cell invasion and motility, indicating abrogation of metastasis. Finally, using PCa patient datasets, we showed that FK866 is potentially capable of reversing the expression of several genes associated with biochemical recurrence, including IFITM3 and LTB4R. Thus, using FK866 as a proof-of-concept candidate for drug repurposing, we introduced a novel, universally applicable preclinical drug development pipeline to circumvent subclonal aggressiveness, drug resistance, and stemness in lethal PCa.

Inhibition of visfatin alleviates sepsis-induced intestinal damage by inhibiting Hippo signaling pathway.

BACKGROUND: The aim of this study is to investigate role of Visfatin, one of the pro-inflammatory adipokines, in sepsis-induced intestinal injury and to clarify the potential mechanism. METHODS: C57BL/6 mice underwent cecal ligation and puncture (CLP) surgery to establish sepsis model in vivo. Intestinal epithelial cells were stimulated with LPS to mimic sepsis-induced intestinal injury in vitro. FK866 (the inhibitor of Visfatin) with or without XMU-MP-1 (the inhibitor of Hippo signaling) was applied for treatment. The expression levels of Visfatin, NF-κB and Hippo signaling pathways-related proteins were detected by western blot or immunohistochemistry. The intestinal cell apoptosis and intestinal injury were investigated by TUNEL staining and H&E staining, respectively. ELISA was used to determine the production of inflammatory cytokines. RESULTS: The expression of Visfatin increased in CLP mice. FK866 reduced intestinal pathological injury, inflammatory cytokines production, and intestinal cell apoptosis in sepsis mice. Meanwhile, FK866 affected NF-κB and Hippo signaling pathways. Additionally, the effects of FK866 on inflammatory response, apoptosis, Hippo signaling and NF-κB signaling were partly abolished by XMU-MP-1, the inhibitor of Hippo signaling. In vitro experiments also revealed that FK866 exhibited a protective role against LPS-induced inflammatory response and apoptosis in intestinal cells, as well as regulating NF-κB and Hippo signaling, whereas addition of XMU-MP-1 weakened the protective effects of FK866. CONCLUSION: In short, this study demonstrated that inhibition of Visfatin might alleviate sepsis-induced intestinal injury through Hippo signaling pathway, supporting a further research on Visfatin as a therapeutic target.

Quantitative Analysis of Daporinad (FK866) and Its In Vitro and In Vivo Metabolite Identification Using Liquid Chromatography-Quadrupole-Time-of-Flight Mass Spectrometry.

Daporinad (FK866) is one of the highly specific inhibitors of nicotinamide phosphoribosyl transferase (NAMPT) and known to have its unique mechanism of action that induces the tumor cell apoptosis. In this study, a simple and sensitive liquid chromatography-quadrupole-time-of-flight-mass spectrometric (LC-qTOF-MS) assay has been developed for the evaluation of drug metabolism and pharmacokinetics (DMPK) properties of Daporinad in mice. A simple protein precipitation method using acetonitrile (ACN) was used for the sample preparation and the pre-treated samples were separated by a C18 column. The calibration curve was evaluated in the range of 1.02~2220 ng/mL and the quadratic regression (weighted 1/concentration2) was used for the best fit of the curve with a correlation coefficient ≥ 0.99. The qualification run met the acceptance criteria of ±25% accuracy and precision values for QC samples. The dilution integrity was verified for 5, 10 and 30-fold dilution and the accuracy and precision of the dilution QC samples were also satisfactory within ±25% of the nominal values. The stability results indicated that Daporinad was stable for the following conditions: short-term (4 h), long-term (2 weeks), freeze/thaw (three cycles). This qualified method was successfully applied to intravenous (IV) pharmacokinetic (PK) studies of Daporinad in mice at doses of 5, 10 and 30 mg/kg. As a result, it showed a linear PK tendency in the dose range from 5 to 10 mg/kg, but a non-linear PK tendency in the dose of 30 mg/kg. In addition, in vitro and in vivo metabolite identification (Met ID) studies were conducted to understand the PK properties of Daporinad and the results showed that a total of 25 metabolites were identified as ten different types of metabolism in our experimental conditions. In conclusion, the LC-qTOF-MS assay was successfully developed for the quantification of Daporinad in mouse plasma as well as for its in vitro and in vivo metabolite identification.

NAD+ depletion enhances reovirus-induced oncolysis in multiple myeloma.

Cancer cell energy metabolism plays an important role in dictating the efficacy of oncolysis by oncolytic viruses. To understand the role of multiple myeloma metabolism in reovirus oncolysis, we performed semi-targeted mass spectrometry-based metabolomics on 12 multiple myeloma cell lines and revealed a negative correlation between NAD+ levels and susceptibility to oncolysis. Likewise, a negative correlation was observed between the activity of the rate-limiting NAD+ synthesis enzyme NAMPT and oncolysis. Indeed, depletion of NAD+ levels by pharmacological inhibition of NAMPT using FK866 sensitized several myeloma cell lines to reovirus-induced killing. The myelomas that were most sensitive to this combination therapy expressed a functional p53 and had a metabolic and transcriptomic profile favoring mitochondrial metabolism over glycolysis, with the highest synergistic effect in KMS12 cells. Mechanistically, U-13C-labeled glucose flux, extracellular flux analysis, multiplex proteomics, and cell death assays revealed that the reovirus + FK866 combination caused mitochondrial dysfunction and energy depletion, leading to enhanced autophagic cell death in KMS12 cells. Finally, the combination of reovirus and NAD+ depletion achieved greater antitumor effects in KMS12 tumors in vivo and patient-derived CD138+ multiple myeloma cells. These findings identify NAD+ depletion as a potential combinatorial strategy to enhance the efficacy of oncolytic virus-based therapies in multiple myeloma.

Synthesis and Evaluation of Reactive Oxygen Species Sensitive Prodrugs of a NAMPT Inhibitor FK866.

NAMPT is an attractive target in cancer therapy and numerous NAMPT inhibitors have been developed. However, the clinical activities of NAMPT inhibitors have displayed disappointing results in clinical trials for their dose-limiting toxicities. In this study, reactive oxygen species (ROS)-responsive prodrugs of a NAMPT inhibitor FK866 were designed and synthesized. A short synthesis method was developed to shield the activity of FK866 through a quaternary ammonium connection. Two prodrugs, with boronic acid as a responsive group to ROS, were prepared and one of the prodrugs 122-066 also contained a fluorescence carrier. Both of the prodrugs released the active compound by the treatment of H2O2,, and the biological evaluation showed that they exhibited a higher potency in cells with high levels of ROS. Moreover, prodrug 122-066 had the ability to release FK866 and simultaneously induce the fluorescence activation under the stimulation of H2O2. This method has the potential to improve the therapeutic window of NAMPT inhibitors.

Optical Redox Imaging of Treatment Responses to Nampt Inhibition and Combination Therapy in Triple-Negative Breast Cancer Cells.

We evaluated the utility of optical redox imaging (ORI) to identify the therapeutic response of triple-negative breast cancers (TNBC) under various drug treatments. Cultured HCC1806 and MDA-MB-231 cells treated with FK866 (nicotinamide phosphoribosyltransferase (Nampt) inhibitor), FX11 (lactate dehydrogenase A inhibitor), paclitaxel, and their combinations were subjected to ORI, followed by imaging fluorescently labeled reactive oxygen species (ROS). Cell growth inhibition was measured by a cell viability assay. We found that both cell lines experienced significant NADH decrease and redox ratio (Fp/(NADH+Fp)) increase due to FK866 treatment; however, HCC1806 was much more responsive than MDA-MB-231. We further studied HCC1806 with the main findings: (i) nicotinamide riboside (NR) partially restored NADH in FK866-treated cells; (ii) FX11 induced an over 3-fold NADH increase in FK866 or FK866+NR pretreated cells; (iii) FK866 combined with paclitaxel caused synergistic increases in both Fp and the redox ratio; (iv) FK866 sensitized cells to paclitaxel treatments, which agrees with the redox changes detected by ORI; (v) Fp and the redox ratio positively correlated with cell growth inhibition; and (vi) Fp and NADH positively correlated with ROS level. Our study supports the utility of ORI for detecting the treatment responses of TNBC to Nampt inhibition and the sensitization effects on standard chemotherapeutics.

Nicotinamide phosphoribosyltransferase inhibitor ameliorates mouse aging-induced cognitive impairment.

Nicotinamide phosphoribosyltransferase (NAMPT) is the key enzyme for the synthesis of nicotinamide adenine dinucleotide (NAD) in the salvaging pathway. Though NAMPT inhibitors such as FK866 were originally developed as anti-cancer drugs, they also display neuroprotective effects. Here we show that the administration of FK866 at 0.5 mg/kg (ip, qod) for four weeks, i.e., ∼1% of the dose used for the treatment of cancer, significantly alleviates the aging-induced impairment of cognition and locomotor activity. Mechanistically, FK866 enhanced autophagy, reduced protein aggregation, and inhibited neuroinflammation indicated by decreasing TNFα, IL-6, GFAP, and Iba1 levels in the aged mouse brain. Though FK866 did not affect the total NAD and nicotinamide mononucleotide (NMN) levels in the mouse brain at the dose we used, FK866 increased nicotinamide (NAM) level in the young mouse brain and decreased NAM level in the aged mouse brain. On the other hand, FK866 did not affect the serum glucose, cholesterol, and triglyceride of young and aged mice and exhibited no effects on the various indices of young mice. Thus, the NAMPT inhibitor can be repurpose to counteract the cognitive impairment upon aging. We also envision that NAMPT inhibitor can be used for the treatment of age-related neurodegenerative diseases.

Inhibition of nicotinamide phosphoribosyltransferase protects against acute pancreatitis via modulating macrophage polarization and its related metabolites.

BACKGROUND & OBJECTIVES: Acute pancreatitis is a common inflammatory disorder of the exocrine pancreas with no specific therapy. Intracellular nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in nicotinamide adenine dinucleotide (NAD) salvage pathway, is involved in many inflammatory disorders. In this study, we investigated the role of NAMPT in experimental acute pancreatitis. METHODS: Acute pancreatitis was induced in mice using three disparate models: (1) caerulein hyperstimulation, (2) ethanol plus palmitoleic acid, and (3) retrograde biliopancreatic ductal infusion of sodium taurocholate. The NAMPT inhibitor FK866 and NAMPT downstream product nicotinamide mononucleotide (NMN) was administered. Serum and pancreas were collected and analyzed biochemically and histologically. Bone marrow derived macrophages were isolated, cultured with cytokines or pancreatic acini, then analyzed by quantitative PCR and non-targeted metabolomics. RESULTS: The levels of pancreatic NAMPT and NAD were down-regulated upon acute pancreatitis. NAMPT inhibitor FK866 suppressed M1 macrophage polarization while NMN boosted it. In co-culture of macrophages with acinar cells, inhibition of NAMPT prevented M1-like macrophage differentiation induced by injured pancreatic acini. The injured pancreatic acinar milieu induced a unique metabolic signature linked to macrophage polarization, and inhibition of NAMPT reversed these metabolites changes. Furthermore, NMN supplementation aggravated caerulein hyperstimulation pancreatitis and alcoholic pancreatitis, and inhibition of NAMPT protected against caerulein hyperstimulation, alcoholic and biliary acute pancreatitis and reducing pancreatic macrophage infiltration in vivo. CONCLUSIONS: NAMPT inhibition protects against acute pancreatitis via preventing M1 macrophage polarization and restoring the metabolites related to macrophage polarization and that NAMPT could be a promising therapeutic target for acute pancreatitis.