Nicotinamide Phosphoribosyltransferase Activity Research Articles

Chemical synthesis, in vitro testing and in silico Nampt-based molecular docking of novel aniline aromatic ring-substituted 2-Aminothiazole analogues.

The heterocyclic 2-Aminothiazoles scaffolds are used in a wide range of therapeutic applications against various diseases for its antioxidant, anti-inflammatory, antimicrobial and anticancer actions. In the present study, we synthesized novel aniline aromatic ring-substituted 2-Aminothiazole derivatives. Molecular docking was performed using Glide module of the Schrödinger Suite to fit compounds JG-49, JG-62, and KBA-18 against the Nicotinamide phosphoribosyl transferase (Nampt) enzyme, an intracellular regulator of Nicotinamide adenine dinucleotide (NAD) redox cofactor involved in energy metabolism and epigenetics and are implicated in aging and metabolic diseases. The three compounds viz. JG-49, JG-62, and KBA-18 showed an increase in Nampt enzymatic activity in vitro. All three substituted derivatives of 2-Aminothiazole showed no cytotoxicity with the mouse C2C12 myoblasts cultures assessed with the MTT cell viability assay. Moreover, the wound closure of the mouse C2C12 myoblasts in vitro displayed no significant difference between the treatment groups of the 2-Aminothiazole derivatives compared with the control naïve and DMSO treated myoblasts cultures, except for the 2-Aminothiazole substituted derivatives JG-62 and KBA-18, which showed a significant increase in the wound closure compared with the control cells at different concentrations. Taken together, we demonstrated that 2-Aminothiazole substituted derivatives provide enhanced Nampt activity, wound closure, and no cytotoxic effects in vitro. Further studies will allow to improve the substitution of 2-Aminothiazole derivatives and test their potential therapeutic applications.

Inhibition of NAMPT by PAK4 Inhibitors.

The serine/threonine kinase PAK4 plays a crucial role in regulating cell proliferation, survival, migration, and invasion. Overexpression of PAK4 correlates with poor prognosis in some cancers. KPT-9274, a PAK4 inhibitor, significantly reduces the growth of triple-negative breast cancer cells and mammary tumors in mouse models, and it also inhibits the growth of several other types of cancer cells. Interestingly, although it was first identified as a PAK4 inhibitor, KPT-9274 was also found to inhibit the enzyme NAMPT (nicotinamide phosphoribosyltransferase), which is crucial for NAD (nicotinamide adenine dinucleotide) synthesis and vital for cellular energy and growth. These results made us question whether growth inhibition in response to KPT-9274 was due to PAK4 inhibition, NAMPT inhibition, or both. To address this, we tested several other PAK4 inhibitors that also inhibit cell growth, to determine whether they also inhibit NAMPT activity. Our findings confirm that multiple PAK4 inhibitors also inhibit NAMPT activity. This was assessed both in cell-free assays and in a breast cancer cell line. Molecular docking studies were also used to help us better understand the mechanism by which PAK4 inhibitors block PAK4 and NAMPT activity, and we identified specific residues on the PAK4 inhibitors that interact with NAMPT and PAK4. Our results suggest that PAK4 inhibitors may have a more complex mechanism of action than previously understood, necessitating further exploration of how they influence cancer cell growth.

NAD+ Metabolism Reprogramming Mediates Irradiation-Induced Immunosuppressive Polarization of Macrophages

Purposeradiotherapy stands as an important complementary treatment for head and neck squamous cell carcinoma (HNSCC), yet it does not invariably result in complete tumor regression. The infiltration of immunosuppressive macrophages is believed to mediate the radiotherapy resistance, which mechanism remains largely unexplored. This study aimed to elucidate the role of immunosuppressive macrophages during radiotherapy and the associated underlying mechanisms. Materials and MethodsMale C3H mice bearing syngeneic SCC-VII tumor were received irradiation (2 × 8Gy). The impact of irradiation on tumor-infiltrating macrophages were assessed. Bone marrow derived macrophages were evaluated in differentiation, proliferation, migration, and inflammatory cytokines after treatment of irradiated tumor culture medium (irCM) and irradiated tumor derived extracellular vesicles (irTEVs). A comprehensive metabolomics profiling of the irTEVs was conducted using liquid chromatography-mass spectrometry, while key metabolites were investigated the mechanism in macrophage in vitro and in vivo. ResultsRadiotherapy on SCC-VII syngeneic graft tumors increased polarization of both M1 and M2 macrophages in tumor microenvironment and drove infiltrated macrophages towards an immunosuppressive phenotype. Irradiation-induced polarization and immunosuppression of macrophages were dependent on irTEVs which delivered an increased amount of nicotinamide (NAM) to macrophages. NAM directly bound to the NF-κB transcriptional activity regulator USP7, through which NAM reduced translocation of NF-κB into the nucleus, thereby decreasing the release of cytokines IL6 and IL8. Increased enzyme activity of nicotinamide phosphoribosyl transferase (NAMPT) which is the rate-limiting enzyme of NAD+ metabolism, contributed to the irradiation-induced accumulation levels of NAM in irradiated HNSCC and irTEVs. Inhibition of NAMPT decreased NAM levels in irTEVs and increased radiotherapy sensitivity through alleviating immunosuppressive function of macrophages. ConclusionsRadiotherapy could induce NAD+ metabolic reprogramming of HNSCC cells, which regulate macrophage towards an immunosuppressive phenotype. Pharmacological targeting NAD+ metabolism might be a promising strategy for radiotherapy sensitization of HNSCC.

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 Aβ 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.

Design of NAMPTs with Superior Activity by Dual-Channel Protein Engineering Strategy.

The nicotinamide phosphoribosyltransferase (NAMPT)-catalyzed substitution reaction plays a pivotal role in the biosynthesis of nucleotide compounds. However, industrial applications are hindered by the low activity of NAMPTs. In this study, a novel dual-channel protein engineering strategy was developed to increase NAMPT activity by enhancing substrate accessibility. The best mutant (CpNAMPTY13G+Y15S+F76P) with a remarkable 5-fold increase in enzyme activity was obtained. By utilizing CpNAMPTY13G+Y15S+F76P as a biocatalyst, the accumulation of β-nicotinamide mononucleotide reached as high as 19.94 g L-1 within 3 h with an impressive substrate conversion rate of 99.8%. Further analysis revealed that the newly generated substrate channel, formed through crack propagation, facilitated substrate binding and enhanced byproduct tolerance. In addition, three NAMPTs from different sources were designed based on the dual-channel protein engineering strategy, and the corresponding dual-channel mutants with improved enzyme activity were obtained, which proved the effectiveness and practicability of the approach.

Synergistic effects of RPT1G, a first-in-class small molecule NAMPT inhibitor, with venetoclax and olaparib in hematological malignancies.

e18512 Background: MultipleBCL-2 and PARP inhibitors are currently in clinical trials, or have been approved for use, in hematological malignancies and solid tumors. Venetoclax, an inhibitor of the apoptosis regulator B-cell lymphoma-2 (BCL-2), has activity in patients with chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). PARP inhibitors, such as Olaparib, exploit synthetic lethality in homologous recombination-deficient tumors, notably in breast and ovarian cancers. However, patients treated with BCL-2 or PARP inhibitors often face relapse and refractory disease, requiring alternative strategies to counter adaptive responses. Nicotinamide Phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the nicotinamide adenine dinucleotide (NAD+) salvage pathway, is upregulated in malignancies and plays a key role in cancer metabolism (e.g., Warburg effect). NAMPT makes nicotinamide mononucleotide which is then converted to NAD+, while PARP enzymes consume NAD+, placing NAMPT directly upstream of PARP. NAMPT overexpression is associated with Venetoclax resistance in leukemia patients, highlighting the link between NAD+, mitochondrial metabolism, and BCL-2 inhibition. Consequently, NAMPT inhibition may synergize with BCL-2 or PARP inhibitors, potentially enhancing 1st-line of treatment outcomes and re-sensitizing refractory cancer cells to BCL-2 or PARP inhibition. Methods: In this study, we investigated the combination effects of Venetoclax and Olaparib with RPT1G, a novel hyperbolic NAMPT inhibitor that simultaneously inhibits and stabilizes NAMPT in a catalytically active state. RPT1G disrupts NAMPT activity without ever turning it off completely avoiding the severe on-target toxicities seen with complete NAMPT inhibitors. Several cell lines, including OCI-AML3 and MV4;11 (AML), RS4;11 (ALL), and RL (diffuse large B-cell lymphoma) were treated with RPT1G and either Venetoclax or Olaparib to determine the degree of drug interactions. In a follow up in vivo study, we treated mice in a human xenograft model using semi-resistant MV4;11 cells with RPT1G alone or in combination with Venetoclax to measure the ability of RPT1G to enhance the effects of Venetoclax. Results: RPT1G achieves significant single-agent efficacy in leukemia and lymphoma mouse xenograft models. When RTP1G is used in combination with either Venetoclax or Olaparib significant synergy is seen across a broad range of concentrations in vitro. Consistent with in vitro synergy results, in a xenograft model of human AML, mice treated with a combination of RPT1G and Venetoclax show enhanced efficacy as compared to Venetoclax alone. Conclusions: Combination of RPT1G with either Venetoclax or Olaparib synergistically enhances killing of leukemic cells and presents as an effective strategy to improve treatment outcomes for patients with diverse malignancies.

Structural insights into Xanthomonas campestris pv. campestris NAD+ biosynthesis via the NAM salvage pathway

Nicotinamide phosphoribosyltransferase (NAMPT) plays an important role in the biosynthesis of nicotinamide adenine dinucleotide (NAD+) via the nicotinamide (NAM) salvage pathway. While the structural biochemistry of eukaryote NAMPT has been well studied, the catalysis mechanism of prokaryote NAMPT at the molecular level remains largely unclear. Here, we demonstrated the NAMPT-mediated salvage pathway is functional in the Gram-negative phytopathogenic bacterium Xanthomonas campestris pv. campestris (Xcc) for the synthesis of NAD+, and the enzyme activity of NAMPT in this bacterium is significantly higher than that of human NAMPT in vitro. Our structural analyses of Xcc NAMPT, both in isolation and in complex with either the substrate NAM or the product nicotinamide mononucleotide (NMN), uncovered significant details of substrate recognition. Specifically, we revealed the presence of a NAM binding tunnel that connects the active site, and this tunnel is essential for both catalysis and inhibitor binding. We further demonstrated that NAM binding in the tunnel has a positive cooperative effect with NAM binding in the catalytic site. Additionally, we discovered that phosphorylation of the His residue at position 229 enhances the substrate binding affinity of Xcc NAMPT and is important for its catalytic activity. This work reveals the importance of NAMPT in bacterial NAD+ synthesis and provides insights into the substrate recognition and the catalytic mechanism of bacterial type II phosphoribosyltransferases.

Improvement of an enzymatic cascade synthesis of nicotinamide mononucleotide via protein engineering and reaction-process reinforcement.

Enzymatic synthesis of β-nicotinamide mononucleotide (NMN) from D-ribose has garnered widespread attention due to its cheap material, the use of mild reaction conditions, and the ability to produce highly pure products with the desired optical properties. However, the overall NMN yield of this method is impeded by the low activity of rate-limiting enzymes. The ribose-phosphate diphosphokinase (PRS) and nicotinamide phosphoribosyltransferase (NAMPT), that control the rate of the reaction, were engineered to improve the reaction efficacy. The actives of mutants PRS-H150Q and NAMPT-Y15S were 334% and 57% higher than that of their corresponding wild-type enzymes, respectively. Furthermore, by adding pyrophosphatase, the byproduct pyrophosphate which can inhibit the activity of NAMPT was degraded, leading to a 6.72% increase in NMN yield. Following with reaction-process reinforcement, a high yield of 8.10gL-1 NMN was obtained after 3h of reaction, which was 56.86-fold higher than that of the stepwise reaction synthesis (0.14gL-1 ), indicating that the in vitro enzymatic synthesis of NMN from D-ribose and niacinamide is an economical and feasible route.

RPT1G: A 1 st-in-Class Small Molecule NAMPT Inhibitor As a Novel Therapeutic for Acute Lymphocytic Leukemia

Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the rate limiting step in the nicotinamide adenine dinucleotide (NAD +) salvage pathway: the conversion of nicotinamide and 5-phosphoribosyl-1-pyrophosphate into nicotinamide mononucleotide. Because NAMPT plays a central role in cancer metabolism (e.g., Warburg effect) and is upregulated in many hematologic malignancies, it is a high-value target for oncology applications. Despite promising preclinical data showing the potential of complete NAMPT inhibitors (NAMPTi) as effective oncology agents, early clinical trials yielded limited benefit due to the small therapeutic window for the agents tested. Complete NAMPT deactivation disrupts essential biological pathways in both cancer and healthy cells, and hence could not induce efficacy without causing dose-limiting toxicities. Here we introduce a novel partial NAMPTi, RPT1G, which was optimized to allow residual activity to meet the metabolic needs of healthy cells and to overcome the challenge of on-target toxicity. RPT1G's 1 st-in-class mechanism of action (MOA) simultaneously inhibits and stabilizes NAMPT in a catalytically active state, disrupting NAMPT enzymatic activity without ever turning it off. RPT1G achieves significant efficacy in a B-cell acute lymphoblastic leukemia (B-ALL) mouse xenograft model and avoids the severe on-target toxicities seen with complete NAMPTi. To demonstrate the importance of NAMPT residual activity in preserving cellular metabolism in healthy cells, we determined the mitochondrial respiration rate for RS4;11 (B-ALL) and human peripheral blood mononuclear cells (huPBMCs) treated with RPT1G. The oxygen consumption rate (OCR) markedly decreased in treated RS4;11 cells, consistent with RPT1G-induced inhibition of cellular bioenergetics. In contrast, in huPBMC, increasing concentrations of RPT1G elicited a dose-dependent decrease of OCR, up to a concentration equal to 5-fold the IC50 in RS4;11, beyond which, further reductions in OCR were not observed. This indicates that RPT1G's influence on mitochondrial respiration had reached its maximum effect, consistent with huPBMCs being able to use their reserve capacity to maintain cell survival. To further validate the novel MOA of RPT1G in vivo, we tested the viability of >100 cancer cell lines as well as huPBMCs upon RPT1G treatment. Leukemias, specifically ALL cells, displayed high sensitivity to RPT1G. Also, the concentrations required to achieve >90% NAD + reduction in RS4;11 cells do not cause NAD + reduction, nor significant decrease in viability in huPBMCs, highlighting the specificity of RPT1G against cancer versus healthy cells. In contrast, complete inhibitors led to a decrease in cell health for both leukemia and healthy cells, which likely explains their poor tolerability in previous clinical trials. To narrow down the sensitivity profile of RPT1G, we performed multiomic analyses across >200 genomically diverse cancer cell lines using a pharmacologically related analog of RPT1G and identified TET2 mutations as prospective prognostic biomarkers. We then conducted efficacy and non-GLP tolerability proof of concept studies to assess the therapeutic index (TI) of RPT1G. The efficacy was evaluated in a human B-ALL RS4;11 xenograft mouse model at 25 mg/kg BID and 150mg/kg QD, while the tolerability was assessed at doses up to 200 mg/kg QD. Comparison of the exposure levels from both studies revealed that RPT1G has an enhanced therapeutic window, with a TI >7.9 (previous NAMPTi TI < 3). RPT1G exhibited significant efficacy at 25 mg/kg BID while maintaining an acceptable level of tolerability at 200mg/kg QD, showing no meaningful toxicological change in hematology or clinical chemistry, and no gross organ lesions, thereby providing crucial insights on dose selection for its clinical development. Our results demonstrate that the 1 st-in-class partial NAMPTi RPT1G modulates NAMPT activity and exploits the different sensitivities and metabolic demands of tumor and normal cells. This results in selective killing of leukemic cells, without on-target toxicity seen with complete NAMPTi. RPT1G emerges as an ideal clinical candidate - its favorable efficacy and tolerability target profile in preclinical models provides robust support for its utility in treating leukemias, warranting the initiation of our phase I clinical study to assess both safety and efficacy in patients.

Extracellular nicotinamide phosphoribosyltransferase: role in disease pathophysiology and as a biomarker.

Nicotinamide phosphoribosyltransferase (NAMPT) plays a central role in mammalian cell metabolism by contributing to nicotinamide adenine dinucleotide biosynthesis. However, NAMPT activity is not limited to the intracellular compartment, as once secreted, the protein accomplishes diverse functions in the extracellular space. Extracellular NAMPT (eNAMPT, also called visfatin or pre-B-cell colony enhancing factor) has been shown to possess adipocytokine, pro-inflammatory, and pro-angiogenic activities. Numerous studies have reported the association between elevated levels of circulating eNAMPT and various inflammatory and metabolic disorders such as obesity, diabetes, atherosclerosis, arthritis, inflammatory bowel disease, lung injury and cancer. In this review, we summarize the current state of knowledge on eNAMPT biology, proposed roles in disease pathogenesis, and its potential as a disease biomarker. We also briefly discuss the emerging therapeutic approaches for eNAMPT inhibition.

NAMPT and PARylation Are Involved in the Pathogenesis of Atopic Dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disease of very high prevalence, especially in childhood, with no specific treatment or cure. As its pathogenesis is complex, multifactorial and not fully understood, further research is needed to increase knowledge and develop new targeted therapies. We have recently demonstrated the critical role of NAD+ and poly (ADP-ribose) (PAR) metabolism in oxidative stress and skin inflammation. Specifically, we found that hyperactivation of PARP1 in response to DNA damage induced by reactive oxygen species, and fueled by NAMPT-derived NAD+, mediated inflammation through parthanatos cell death in zebrafish and human organotypic 3D skin models of psoriasis. Furthermore, the aberrant induction of NAMPT and PARP activity was observed in the lesional skin of psoriasis patients, supporting the role of these signaling pathways in psoriasis and pointing to NAMPT and PARP1 as potential novel therapeutic targets in treating skin inflammatory disorders. In the present work, we report, for the first time, altered NAD+ and PAR metabolism in the skin of AD patients and a strong correlation between NAMPT and PARP1 expression and the lesional status of AD. Furthermore, using a human 3D organotypic skin model of AD, we demonstrate that the pharmacological inhibition of NAMPT and PARP reduces pathology-associated biomarkers. These results help to understand the complexity of AD and reveal new potential treatments for AD patients.

Novel carbon skeletons activate human NicotinAMide Phosphoribosyl Transferase (NAMPT) enzyme in biochemical assay.

Nicotinamide adenine dinucleotide (NAD) is a central molecule in cellular metabolism that has been implicated in human health, the aging process, and an array of human diseases. NAD is well known as an electron storage molecule, cycling between NAD and the reduced NADH. In addition, NAD is cleaved into nicotinamide and Adenine diphosphate ribose by NAD-consuming enzymes such as sirtuins, PARPs and CD38. There are numerous pathways for the biosynthesis of NAD to maintain a baseline concentration and thus avoid cellular death. The NAD salvage pathway, a two-step process to regenerate NAD after cleavage, is the predominant pathway for humans. Nicotinamide PhosphribosylTransferase (NAMPT) is the rate-limiting enzyme within the salvage path. Exposure to pharmacological modulators of NAMPT has been reported to either deplete or increase NAD levels. This study used a curated set of virtual compounds coupled with biochemical assays to identify novel activators of NAMPT. Autodock Vina generated a ranking of the National Cancer Institute's Diversity Set III molecular library. The library contains a set of organic molecules with diverse functional groups and carbon skeletons that can be used to identify lead compounds. The target NAMPT surface encompassed a novel binding location that included the NAMPT dimerization plane, the openings to the two active site channels, and a portion of the known binding location for NAMPT substrate and product. Ranked molecules were evaluated in a biochemical assay using purified recombinant NAMPT enzyme. Two novel carbon skeletons were confirmed to stimulate NAMPT activity. Compound 20 (NSC9037) is a polyphenolic xanthene derivative in the fluorescein family, while compound 2 (NSC19803) is the polyphenolic myricitrin nature product. Micromolar quantities of compound 20 or compound 2 can double NAMPT's product formation. In addition, natural products that contain high concentrations of polyphenolic flavonoids, similar to myricitrin, also stimulate NAMPT activity. Confirmation of a novel binding site for these compounds will further our understanding of the cellular mechanism leading to NAD homeostasis and better human health outcomes.

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.

Nicotine rebalances NAD+ homeostasis and improves aging-related symptoms in male mice by enhancing NAMPT activity

Imbalances in NAD+ homeostasis have been linked to aging and various diseases. Nicotine, a metabolite of the NAD+ metabolic pathway, has been found to possess anti-inflammatory and neuroprotective properties, yet the underlying molecular mechanisms remained unknown. Here we find that, independent of nicotinic acetylcholine receptors, low-dose nicotine can restore the age-related decline of NAMPT activity through SIRT1 binding and subsequent deacetylation of NAMPT, thus increasing NAD+ synthesis. 18F-FDG PET imaging revealed that nicotine is also capable of efficiently inhibiting glucose hypermetabolism in aging male mice. Additionally, nicotine ameliorated cellular energy metabolism disorders and deferred age-related deterioration and cognitive decline by stimulating neurogenesis, inhibiting neuroinflammation, and protecting organs from oxidative stress and telomere shortening. Collectively, these findings provide evidence for a mechanism by which low-dose nicotine can activate NAD+ salvage pathways and improve age-related symptoms.

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

BackgroundLiver 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. MethodsWe 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. ResultWe 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)-1β. 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.

Porcine placental extract increase the cellular NAD levels in human epidermal keratinocytes

Nicotinamide adenine dinucleotide (NAD) is an essential cofactor for numerous enzymes involved in energy metabolism. Because decreasing NAD levels is a common hallmark of the aging process in various tissues and organs, maintaining NAD levels has recently been of interest for the prevention of aging and age-related diseases. Although placental extract (PE) are known to possess several anti-aging effects, the NAD-boosting activity of PE remains unknown. In this study, we found that porcine PE (PPE) significantly increased intracellular NAD levels in normal human epidermal keratinocytes (NHEKs). PPE also attenuated the NAD depletion induced by FK866, an inhibitor of nicotinamide phosphoribosyltransferase (NAMPT). Interestingly, only the fraction containing nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), and nicotinamide (NAM) restored NAD content in NHEKs in the absence of NAMPT activity. These results suggest that PPE increases intracellular NAD by providing NAD precursors such as NMN, NR, and NAM. Finally, we showed that the application of PPE to the stratum corneum of the reconstructed human epidermis significantly ameliorated FK866-induced NAD depletion, suggesting that topical PPE may be helpful for increasing skin NAD levels. This is the first study to report the novel biological activity of PE as an NAD booster in human epidermal cells.

Addressing the Enzyme-independent tumor-promoting function of NAMPT via PROTAC-mediated degradation

Aberrant overexpression of nicotinamide phosphoribosyltransferase (NAMPT) has been reported in a variety of tumor cells and is a poor prognosis factor for patient survival. It plays an important role in tumor cell proliferation, acting concurrently as an nicotinamide adenine dinucleotide (NAD+) synthase and, unexpectedly, as an extracellular signaling molecule for several tumor-promoting pathways. Although previous efforts to modulate NAMPT activity were limited to enzymatic inhibitors with low success in clinical studies, protein degradation offers the possibility to simultaneously disrupt NAMPT's enzyme activity and ligand capabilities. Here we report the development of two highly selective proteolysis-targeting chimeras (PROTACs) that promote NAMPT degradation in a cereblon-dependent manner. Both PROTAC degraders outperform a clinical candidate, FK866, in killing effect on hematological tumor cells. These results emphasize the importance and feasibility of applying PROTACs as a superior strategy for targeting proteins with multiple tumor-promoting functions like NAMPT, which is not easily achieved by conventional enzymatic inhibitors.

AMPK phosphorylates NAMPT to regulate NAD+ homeostasis under ionizing radiation.

Radiation-induced oral mucositis is the most common complication for patients who receive head/neck radiotherapy. Nicotinamide adenine dinucleotide (NAD+) is vital for DNA damage repair under ionizing radiation, through functioning as either the substrate for protein poly(ADP-ribosyl)ation at DNA break sites or the cofactor for multiple DNA repair-related enzymes, which therefore can result in a significant consumption of cellular NAD+ during DNA repair. Mammalian cells produce NAD+ mainly by recycling nicotinamide via the salvage pathway, in which the rate-limiting step is governed by nicotinamide phosphoribosyltransferase (NAMPT). However, whether NAMPT is co-opted under ionizing radiation to timely fine-tune NAD+ homeostasis remains elusive. Here we show that ionizing radiation evokes NAMPT activation within 30 min without apparent changes in its protein expression. AMPK rapidly phosphorylates NAMPT at S314 under ionizing radiation, which reinforces the enzymatic activity of NAMPT by increasing NAMPT binding with its substrate phosphoribosyl pyrophosphate (PRPP). AMPK-mediated NAMPT S314 phosphorylation substantially restores NAD+ level in the irradiated cells and facilitates DNA repair and cell viability. Our findings demonstrate a new post-translational modification-based signalling route, by which cells can rapidly orchestrate NAD+ metabolism to support DNA repair, thereby highlighting NAMPT as a potential target for the prevention of ionizing radiation-induced injuries.

Correlation of serum nicotinamide phosphoribosyl transferase with hypoxia and Framingham risk score in patients with obstructive sleep apnea-hypopnea syndrome.

Obstructive sleep apnea-hypopnea syndrome (OSAHS) is an independent risk factor for atherosclerosis (AS), but the mechanism is different from classical AS risk factors. Nicotinamide phosphoribosyltransferase (NAMPT) is involved in the pathophysiology of AS via multiple pathways, and its expression is closely related to hypoxia. The association of NAMPT with hypoxia and the risk of cardiovascular morbidity in the patients of OSAHS remains to be defined. Therefore, we carried out this study to investigate the association of NAMPT with hypoxia and the risk of early cardiovascular disease [based on the Framingham risk score (FRS)] in patients with OSAHS. A total of 82 patients diagnosed with OSAHS were enrolled in this cross-sectional survey design, along with 18 healthy controls who were age- and gender-matched. The general characteristic parameters including height and weight as well as biochemical parameters including blood glucose and lipid were collected from the subjects. The Framingham vascular risk score calculates the risk of developing vascular disease based on the above indicators. Polysomnography was performed in patients with OSAHS, and blood oxygen saturation and apnea-hypopnea index (AHI) were collected, and patients were grouped by disease extent by AHI. The serum NAMPT level of the research subjects was detected using an enzyme-linked immunosorbent assay. Spearman correlation analysis and multiple linear regression to explore the independent correlations of hypoxia on serum NAMPT activity in OSAHS patients. Serum NAMPT level in patients with OSAHS increased with the severity of the disease. Correlation analysis showed that NAMPT was significantly positively correlated with FRS in patients with OSAHS (r=0.829, P<0.05). Multiple linear regression analysis with FRS as the outcome measure showed that NAMPT activity and minimum blood oxygen saturation were independent associated with the risk of developing cardiovascular disease (β=0.03, P=0.000; β=-0.13, P=0.034). Univariate and multivariate regression analyses revealed that hypoxia was significantly associated with NAMPT levels in OSAHS patients, and the oxygen desaturation index (ODI) was independent associated with the expression of NAMPT activity (β=4.09, P=0.000). In patients with OSAHS, hypoxia is independently associated with NAMPT. NAMPT increases the risk of cardiovascular morbidity in this population may be influenced by hypoxia.

The Functional Effects of Visfatin on Human Left Internal Mammary Artery.

Visfatin may play a role in vascular dysfunction in metabolic disorders. Apart from its insulin-mimetic actions, it has divergent actions in the cardiovascular system with discordant results in the literature. Thus, we aimed to study the effects of visfatin on vascular responses of the human left internal mammary artery. Sections of redundant human left internal mammary artery were cut into 3-mm wide rings and hung in 20-mL organ baths containing physiologic salt solution and attached to an isometric force transducer connected to a computer-based data acquisition system. Removing endothelium caused an increase in pD2 values for visfatin-induced relaxation responses (10 -12 -10 -7 M) (9.06 ± 0.21 and 11.08 ± 0.92, respectively). Nicotinamide phosphoribosyltransferase inhibitor FK866 (10 µM) reversed the visfatin-induced relaxations (10 -12 -10 -7 M) ( P = 0.024). Incubations with nitric oxide synthase inhibitor nitro- l -arginine methylester and guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ) caused significant reductions in relaxation responses of visfatin ( P = 0.011 and 0.008, respectively). Visfatin incubations decreased relaxation responses to acetylcholine but not to sodium nitroprusside. Incubations with visfatin did not change contractile responses to angiotensin II, endothelin-1, noradrenalin, and phenylephrine. In this study, visfatin caused endothelium-dependent relaxations mediated by nitric oxide-cyclic guanosine monophosphate pathway and nicotinamide phosphoribosyltransferase activity. Furthermore, visfatin-induced decreases in relaxation responses were also related to endothelium-derived nitric oxide.