Nicotinamide Phosphoribosyltransferase Activity Research Articles

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.

Cardioprotective Effects of 1-(3,6-Dibromo-carbazol-9-yl)-3-Phenylamino-Propan-2-Ol in Diabetic Hearts via Nicotinamide Phosphoribosyltransferase Activation.

Diabetes is associated with increased cardiac injury and sudden death. Nicotinamide phosphoribosyltransferase (Nampt) is an essential enzyme for the NAD+ salvage pathway and is dysregulated in diabetes. Nampt activation results in rescued NADH/NAD+ ratios and provides pharmacological changes necessary for diabetic cardioprotection. Computer docking shows that 1-(3,6-Dibromo-carbazol-9-yl)-3-phenylamino-propan-2-ol (P7C3) allows for enhanced Nampt dimerization and association. To test the pharmacological application, we used male leptin receptor-deficient (db/db) mice and treated them with Nampt activator P7C3. The effects of 4-week P7C3 treatment on cardiac function were evaluated along with molecular signaling changes for phosphorylated protein kinase B (p-AKT), phosphorylated endothelial nitric oxide synthase (p-eNOS), and sirtuin 1 (SIRT1). The cardiac function evaluated by ECG and echocardiography were significantly improved after 4 weeks of P7C3 treatment. Biochemically, higher NADH/NAD+ ratios in diabetic hearts were rescued by P7C3 treatment. Moreover, activities of Nampt and SIRT1 were significantly increased in P7C3-treated diabetic hearts. P7C3 treatment significantly decreased the blood glucose in diabetic mice with 4-week treatment as noted by glucose tolerance test and fasting blood glucose measurements compared with vehicle-treated mice. P7C3 activated Nampt enzymatic activity both in vitro and in the 4-week diabetic mouse hearts, demonstrating the specificity of the small molecule. P7C3 treatment significantly enhanced the expression of cardioprotective signaling of p-AKT, p-eNOS, and Beclin 1 in diabetic hearts. Nampt activator P7C3 allows for decreased infarct size with decreased Troponin I and lactose dehydrogenase (LDH) release, which is beneficial to the heart. Overall, the present study shows that P7C3 activates Nampt and SIRT1 activity and decreases NADH/NAD+ ratio, resulting in improved biochemical signaling providing cardioprotection. SIGNIFICANCE STATEMENT: This study shows that 1-(3,6-Dibromo-carbazol-9-yl)-3-phenylamino-propan-2-ol (P7C3) is effective in treating diabetes and cardiovascular diseases. The novel small molecule is antiarrhythmic and improves the ejection fraction in diabetic hearts. The study successfully demonstrated that P7C3 decreases the infarct size in hearts during myocardial infarction and ischemia-reperfusion injury. Biochemical and cellular signaling show increased NAD+ levels, along with Nampt activity involved in upregulating protective signaling in the diabetic heart. P7C3 has high therapeutic potential for rescuing heart disease.

Discovery of small-molecule activators of nicotinamide phosphoribosyltransferase (NAMPT) and their preclinical neuroprotective activity.

The decline of nicotinamide adenine dinucleotide (NAD) occurs in a variety of human pathologies including neurodegeneration. NAD-boosting agents can provide neuroprotective benefits. Here, we report the discovery and development of a class of potent activators (NATs) of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD salvage pathway. We obtained the crystal structure of NAMPT in complex with the NAT, which defined the allosteric action of NAT near the enzyme active site. The optimization of NAT further revealed the critical role of K189 residue in boosting NAMPT activity. NATs effectively increased intracellular levels of NAD and induced subsequent metabolic and transcriptional reprogramming. Importantly, NATs exhibited strong neuroprotective efficacy in a mouse model of chemotherapy-induced peripheral neuropathy (CIPN) without any overt toxicity. These findings demonstrate the potential of NATs in the treatment of neurodegenerative diseases or conditions associated with NAD level decline.

Nampt activator P7C3 ameliorates diabetes and improves skeletal muscle function modulating cell metabolism and lipid mediators.

BackgroundNicotinamide phosphoribosyltransferase (Nampt), a key enzyme in NAD salvage pathway is decreased in metabolic diseases, and its precise role in skeletal muscle function is not known. We tested the hypothesis, Nampt activation by P7C3 (3,6‐dibromo‐α‐[(phenylamino)methyl]‐9H‐carbazol‐9‐ethanol) ameliorates diabetes and muscle function.MethodsWe assessed the functional, morphometric, biochemical, and molecular effects of P7C3 treatment in skeletal muscle of type 2 diabetic (db/db) mice. Nampt+/− mice were utilized to test the specificity of P7C3.ResultsInsulin resistance increased 1.6‐fold in diabetic mice compared with wild‐type mice and after 4 weeks treatment with P7C3 rescued diabetes (P < 0.05). In the db‐P7C3 mice fasting blood glucose levels decreased to 0.96‐fold compared with C57Bl/6J wild‐type naïve control mice. The insulin and glucose tolerance tests blood glucose levels were decreased to 0.6‐fold and 0.54‐folds, respectively, at 120 min along with an increase in insulin secretion (1.76‐fold) and pancreatic β‐cells (3.92‐fold) in db‐P7C3 mice. The fore‐limb and hind‐limb grip strengths were increased to 1.13‐fold and 1.17‐fold, respectively, together with a 14.2‐fold increase in voluntary running wheel distance in db‐P7C3 mice. P7C3 treatment resulted in a 1.4‐fold and 7.1‐fold increase in medium‐sized and larger‐sized myofibres cross‐sectional area, with a concomitant 0.5‐fold decrease in smaller‐sized myofibres of tibialis anterior (TA) muscle. The transmission electron microscopy images also displayed a 1.67‐fold increase in myofibre diameter of extensor digitorum longus muscle along with 2.9‐fold decrease in mitochondrial area in db‐P7C3 mice compared with db‐Veh mice. The number of SDH positive myofibres were increased to 1.74‐fold in db‐P7C3 TA muscles. The gastrocnemius and TA muscles displayed a decrease in slow oxidative myosin heavy chain type1 (MyHC1) myofibres expression (0.46‐fold) and immunostaining (6.4‐fold), respectively. qPCR analysis displayed a 2.9‐fold and 1.3‐fold increase in Pdk4 and Cpt1, and 0.55‐fold and 0.59‐fold decrease in Fgf21 and 16S in db‐P7C3 mice. There was also a 3.3‐fold and 1.9‐fold increase in Fabp1 and CD36 in db‐Veh mice. RNA‐seq differential gene expression volcano plot displayed 1415 genes to be up‐regulated and 1726 genes down‐regulated (P < 0.05) in db‐P7C3 mice. There was 1.02‐fold increase in serum HDL, and 0.9‐fold decrease in low‐density lipoprotein/very low‐density lipoprotein ratio in db‐P7C3 mice. Lipid profiling of gastrocnemius muscle displayed a decrease in inflammatory lipid mediators n‐6; AA (0.83‐fold), and n‐3; DHA (0.69‐fold) and EPA (0.81‐fold), and a 0.66‐fold decrease in endocannabinoid 2‐AG and 2.0‐fold increase in AEA in db‐P7C3 mice.ConclusionsOverall, we demonstrate that P7C3 activates Nampt, improves type 2 diabetes and skeletal muscle function in db/db mice.

NAMPT-targeting PROTAC promotes antitumor immunity via suppressing myeloid-derived suppressor cell expansion

Nicotinamide phosphoribosyl transferase (NAMPT) is considered as a promising target for cancer therapy given its critical engagement in cancer metabolism and inflammation. However, therapeutic benefit of NAMPT enzymatic inhibitors appears very limited, likely due to the failure to intervene non-enzymatic functions of NAMPT. Herein, we show that NAMPT dampens antitumor immunity by promoting the expansion of tumor infiltrating myeloid derived suppressive cells (MDSCs) via a mechanism independent of its enzymatic activity. Using proteolysis-targeting chimera (PROTAC) technology, PROTAC A7 is identified as a potent and selective degrader of NAMPT, which degrades intracellular NAMPT (iNAMPT) via the ubiquitin–proteasome system, and in turn decreases the secretion of extracellular NAMPT (eNAMPT), the major player of the non-enzymatic activity of NAMPT. In vivo, PROTAC A7 efficiently degrades NAMPT, inhibits tumor infiltrating MDSCs, and boosts antitumor efficacy. Of note, the anticancer activity of PROTAC A7 is superior to NAMPT enzymatic inhibitors that fail to achieve the same impact on MDSCs. Together, our findings uncover the new role of enzymatically-independent function of NAMPT in remodeling the immunosuppressive tumor microenvironment, and reports the first NAMPT PROTAC A7 that is able to block the pro-tumor function of both iNAMPT and eNAMPT, pointing out a new direction for the development of NAMPT-targeted therapies.

Metoprolol Protects Against Arginine Vasopressin-Induced Cellular Senescence in H9C2 Cardiomyocytes by Regulating the Sirt1/p53/p21 Axis

Cardiomyocyte senescence is involved in the pathological mechanism of cardiac diseases. Metoprolol is a β1 receptor blocker used for the treatment of hypertension. Recent studies show that Metoprolol can protect cardiomyocytes against ischemia injury. The present study aims to investigate the protective effects of Metoprolol against arginine vasopressin (AVP)-induced cellular senescence in cultured cardiomyocytes. The cell proliferation assay and cytotoxicity lactate dehydrogenase assay showed that the highest tolerated dosage of Metoprolol in H9C2 cardiomyocytes was optimized as 10 µM. The enzyme-linked immunosorbent assay showed that Metoprolol significantly ameliorated the elevated level of the DNA oxidation product 8-hydroxy-2 deoxyguanosine. Metoprolol also decreased the percentage of senescence-associated β-galactosidase positive cells and improved the telomerase activity under AVP exposure. Moreover, treatment with Metoprolol ameliorated the decreased intracellular nicotinamide phosphoribosyltransferase activity, nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate (NAD+/NADPH) ratio, and Sirtuin1 activity in cardiomyocytes by AVP. Finally, Metoprolol was able to downregulate the AVP-induced expression of acetylated p53 and p21. Taken together, our data reveal that Metoprolol protected the cardiomyocytes from AVP-induced senescence.

Mechanisms of Dangua Recipe in Improving Glycolipid Metabolic Disorders Based on Transcriptomics.

To explore the mechanisms of Dangua Recipe (DGR) in improving glycolipid metabolism based on transcriptomics. Sprague-Dawley rats with normal glucose level were divided into 3 groups according to a random number table, including a conventional diet group (Group A), a DGR group (Group B, high-calorie diet + 20.5 g DGR), and a high-calorie fodder model group (Group C). After 12 weeks of intervention, the liver tissue of rats was taken. Gene sequence and transcriptional analysis were performed to identify the key genes related to glycolipid metabolism reflecting DGR efficacy, and then gene or protein validation of liver tissue were performed. Nicotinamide phosphoribosyl transferase (Nampt) and phosphoenolpyruvate carboxykinase (PEPCK) proteins in liver tissues were detected by enzyme linked immunosorbent assay, fatty acid synthase (FASN) protein was detected by Western blot, and fatty acid binding protein 5 (FABP5)-mRNA was detected by quantitative real-time polymerase chain reaction. Furthermore, the functional verification was performed on the diabetic model rats by Nampt blocker (GEN-617) injected in vivo. Hemoglobin A1c (HbA1c), plasma total cholesterol and triglycerides were detected. Totally, 257 differential-dominant genes of Group A vs. Group C and 392 differential-dominant genes of Group B vs. Group C were found. Moreover, 11 Gene Ontology molecular function terms and 7 Kyoto Encyclopedia of Genes and Genomes enrichment pathways owned by both Group A vs. Group C and Group C vs. Group B were confirmed. The liver tissue target validation showed that Nampt, FASN, PEPCK protein and FABP5-mRNA had the same changes consistent with transcriptome. The in vivo functional tests showed that GEN-617 increased body weight, HbA1c, triglyceride and total cholesterol levels in the diabetic rats (P<;0.05 or P<;0.01); while all the above-mentioned levels (except triglyceride) were decreased significantly by GEN-617 combined with DGR intervention (P<;0.05 or P<;0.01). Nampt activation was one of the mechanisms about DGR regulating glycolipid metabolism.