Anticancer Activities of Novel Nicotinamide Phosphoribosyltransferase Inhibitors in Hematological Malignancies.

Targeting cancer cells that are highly dependent on the nicotinamide adenine dinucleotide (NAD+) metabolite is a promising therapeutic strategy. Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme catalyzing NAD+ production. Despite the high efficacy of several developed NAMPT inhibitors (i.e., FK866 (APO866)) in preclinical studies, their clinical activity was proven to be limited. Here, we report the synthesis of new NAMPT Inhibitors, JJ08, FEI191 and FEI199, which exhibit a broad anticancer activity in vitro. Results show that these compounds are potent NAMPT inhibitors that deplete NAD+ and NADP(H) after 24 h of drug treatment, followed by an increase in reactive oxygen species (ROS) accumulation. The latter event leads to ATP loss and mitochondrial depolarization with induction of apoptosis and necrosis. Supplementation with exogenous NAD+ precursors or catalase (ROS scavenger) abrogates the cell death induced by the new compounds. Finally, in vivo administration of the new NAMPT inhibitors in a mouse xenograft model of human Burkitt lymphoma delays tumor growth and significantly prolongs mouse survival. The most promising results are collected with JJ08, which completely eradicates tumor growth. Collectively, our findings demonstrate the efficient anticancer activity of the new NAMPT inhibitor JJ08 and highlight a strong interest for further evaluation of this compound in hematological malignancies.

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.

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.

Genomic and tumor biological aspects of the anticancer nicotinamide phosphoribosyltransferase inhibitor FK866 in resistant human colorectal cancer cells.

Cancer cells' resistance to drugs remains an important problem affecting cancer treatment strategies. We previously studied the nicotinamide phosphoribosyltransferase (NAMPT) inhibitor FK866's resistance mechanisms in the human colorectal cancer HCT116 cells. We established an acquired FK866-resistant cell line, HCT116RFK866. In this study, we investigated gene mutations in parental HCT116 and HCT116RFK866 cells using exome sequencing technology. The results indicated cluster genes related to NAD+ biosynthesis (including NAMPT), DNA repair, and ATP-binding cassette transporters were differentially altered in these cells. Interestingly, HCT116RFK866 cells, which are resistant to other class NAMPT inhibitors, were more sensitive to the anticancer 5-fluorouracil and cisplatin and γ-ray irradiation compared to parental HCT116 cells. This higher sensitivity appears to cause a genetic change in the identified gene clusters by resistance to the NAMPT inhibitor FK866. Collectively, these novel findings provide a better understanding of anticancer candidate NAMPT inhibitors with regard to resistance mechanisms and cancer chemotherapy strategies.

Discovery of trans-3-(pyridin-3-yl)acrylamide-derived sulfamides as potent nicotinamide phosphoribosyltransferase (NAMPT) inhibitors for the potential treatment of cancer.

Nicotinamide phosphoribosyltransferase (NAMPT) has emerged as a promising target for the discovery of anticancer drugs. Based on NAMPT inhibitor FK866 that has been advanced into phase II trial, we identified a trans-3-(pyridin-3-yl)acrylamide compound 13 incorporating with a biarylsulfanilamide moiety as a new NAMPT inhibitor. Further structure-activity relationship (SAR) exploration led to additional biarylsulfanilamide-derived compounds with high in vitro NAMPT inhibitory potency and antiproliferative activity. In particular, compound 23, the most potent NAMPT inhibitor (IC50 = 5.08 nM), showed single-digit nanomolar antiproliferative activity against DU145, Hela, and H1975 cells with IC50 values of 2.90 nM, 2.34 nM, and 2.24 nM, respectively, and even subnanomolar level against K562, MCF-7, and HUH7 cells with IC50 values of 0.46 nM, 0.23 nM and 0.53 nM, respectively. Our findings provided promising lead compounds for the discovery of more potent NAMPT inhibitors as anticancer drugs.

Nicotinaldehyde, a Novel Precursor of NAD Biosynthesis, Abrogates the Anti-Cancer Activity of an NAD-Lowering Agent in Leukemia.

Targeting NAD depletion in cancer cells has emerged as an attractive therapeutic strategy for cancer treatment, based on the higher reliance of malignant vs. healthy cells on NAD to sustain their aberrant proliferation and altered metabolism. NAD depletion is exquisitely observed when NAMPT, a key enzyme for the biosynthesis of NAD, is inhibited. Growing evidence suggests that alternative NAD sources present in a tumor environment can bypass NAMPT and render its inhibition ineffective. Here, we report the identification of nicotinaldehyde as a novel precursor that can be used for NAD biosynthesis by human leukemia cells. Nicotinaldehyde supplementation replenishes the intracellular NAD level in leukemia cells treated with NAMPT inhibitor APO866 and prevents APO866-induced oxidative stress, mitochondrial dysfunction and ATP depletion. We show here that NAD biosynthesis from nicotinaldehyde depends on NAPRT and occurs via the Preiss-Handler pathway. The availability of nicotinaldehyde in a tumor environment fully blunts the antitumor activity of APO866 in vitro and in vivo. This is the first study to report the role of nicotinaldehyde in the NAD-targeted anti-cancer treatment, highlighting the importance of the tumor metabolic environment in modulating the efficacy of NAD-lowering cancer therapy.

Targeting NAD+ metabolism: dual roles in cancer treatment.

Nicotinamide adenine dinucleotide (NAD+) is indispensable for various oxidation-reduction reactions in mammalian cells, particularly during energy production. Malignant cells increase the expression levels of NAD+ biosynthesis enzymes for rapid proliferation and biomass production. Furthermore, mounting proof has indicated that NAD-degrading enzymes (NADases) play a role in creating the immunosuppressive tumor microenvironment (TME). Interestingly, both inhibiting NAD+ synthesis and targeting NADase have positive implications for cancer treatment. Here we summarize the detrimental outcomes of increased NAD+ production, the functions of NAD+ metabolic enzymes in creating an immunosuppressive TME, and discuss the progress and clinical translational potential of inhibitors for NAD+ synthesis and therapies targeting NADase.

Discovery of a novel NAMPT inhibitor that selectively targets NAPRT-deficient EMT-subtype cancer cells and alleviates chemotherapy-induced peripheral neuropathy.

Background: Exploiting synthetic lethality (SL) relationships between protein pairs has emerged as an important avenue for the development of anti-cancer drugs. Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme of the NAD+ salvage pathway, having an SL relationship with nicotinic acid phosphoribosyltransferase (NAPRT), the key enzyme in the NAD+ Preiss-Handler pathway. NAMPT inhibitor holds clinical potential not only as a promising cancer treatment but also as a means of protection against chemotherapy-induced-peripheral-neuropathy (CIPN). However, as NAD+ is essential for normal cells, the clinical use of NAMPT inhibitors is challenging. This study aimed to identify a novel NAMPT inhibitor with enhanced selective cytotoxicity against NAPRT-deficient cancer cells as well as prominent efficacy in alleviating CIPN. Methods: We began by conducting drug derivatives screening in a panel of lung cancer cell lines to select an agent with the broadest therapeutic window between the NAPRT-negative and-positive cancer cell lines. Both in vitro and In vivo comparative analyses were conducted between A4276 and other NAMPT inhibitors to evaluate the NAPRT-negative cancer cell selectivity and the underlying distinct NAMPT inhibition mechanism of A4276. Patient-derived tumor transcriptomic data and protein levels in various cancer cell lines were analyzed to confirm the correlation between NAPRT depletion and epithelial-to-mesenchymal transition (EMT)-like features in various cancer types. Finally, the efficacy of A4276 for axonal protection and CIPN remedy was examined in vitro and in vivo. Results: The biomarker-driven phenotypic screening led to a discovery of A4276 with prominent selectivity against NAPRT-negative cancer cells compared with NAPRT-positive cancer cells and normal cells. The cytotoxic effect of A4276 on NAPRT-negative cells is achieved through its direct binding to NAMPT, inhibiting its enzymatic function at an optimal and balanced level allowing NAPRT-positive cells to survive through NAPRT-dependent NAD+ synthesis. NAPRT deficiency serves as a biomarker for the response to A4276 as well as an indicator of EMT-subtype cancer in various tumor types. Notably, A4276 protects axons from Wallerian degeneration more effectively than other NAMPT inhibitors by decreasing NMN-to-NAD+ ratio. Conclusion: This study demonstrates that A4276 selectively targets NAPRT-deficient EMT-subtype cancer cells and prevents chemotherapy-induced peripheral neuropathy, highlighting its potential as a promising anti-cancer agent for use in cancer monotherapy or combination therapy with conventional chemotherapeutics.

Drug repositioning strategy for the identification of novel telomere-damaging agents: A role for NAMPT inhibitors.

Drug repositioning strategy represents a valid tool to accelerate the pharmacological development through the identification of new applications for already existing compounds. In this view, we aimed at discovering molecules able to trigger telomere-localized DNA damage and tumor cell death. By applying an automated high-content spinning-disk microscopy, we performed a screening aimed at identifying, on a library of 527 drugs, molecules able to negatively affect the expression of TRF2, a key protein in telomere maintenance. FK866, resulting from the screening as the best candidate hit, was then validated at biochemical and molecular levels and the mechanism underlying its activity in telomere deprotection was elucidated both in vitro and in vivo. The results of this study allow us to discover a novel role of FK866 in promoting, through the production of reactive oxygen species, telomere loss and deprotection, two events leading to an accumulation of DNA damage and tumor cell death. The ability of FK866 to induce telomere damage and apoptosis was also demonstrated in advanced preclinical models evidencing the antitumoral activity of FK866 in triple-negative breast cancer-a particularly aggressive breast cancer subtype still orphan of targeted therapies and characterized by high expression levels of both NAMPT and TRF2. Overall, our findings pave the way to the development of novel anticancer strategies to counteract triple-negative breast cancer, based on the use of telomere deprotecting agents, including NAMPT inhibitors, that would rapidly progress from bench to bedside.

The immunomodulatory role of IDO1-Kynurenine-NAD+ pathway in switching cold tumor microenvironment in PDAC.

Pancreatic ductal adenocarcinoma (PDAC) is the most common exocrine tumor of the pancreas characterized by late diagnosis, adverse overall 5-year survival, a higher propensity for metastatic disease, and lack of efficacy of systemic therapy options. These adverse outcomes can be partly attributed to complex tumor microenvironment (TME). Over the past decade, immunotherapy has revolutionized the management of certain cancers; thus far, the immunologically 'non-inflamed' tumor microenvironment in PDACs has proven to be challenging. Indolamine 2,3-dioxygenase 1 (IDO1) is the rate-limiting enzyme in the catabolic pathway of L-Tryptophan, an essential amino acid, that gives rise to the immunosuppressive metabolite Kynurenine. IDO1, Indolamine 2,3-dioxygenase 2 (IDO2), and Tryptophan 2,3-dioxygenase (TDO) are the key enzymes in the tryptophan catabolic pathway but we focus on the role of the predominant enzyme form IDO1 in this review. Nicotinamide phosphoribosyl transferase (iNAMPT) regulates the intracellular concentration of NAD and is upregulated in the tumor. In light of the potential role of IDO1 as a driver of hostile TME in PDAC and NAD+ as a key coenzyme in anti-tumor immune response, this review urges focus on extensive research and initiation of clinical trials using IDO1 and NAMPT inhibitors in pancreatic cancer in the future.