Synthesis and structure-activity relationship of new nicotinamide phosphoribosyltransferase inhibitors with antitumor activity on solid and haematological cancer.

Cancer cells are highly dependent on Nicotinamide phosphoribosyltransferase (NAMPT) activity for proliferation, therefore NAMPT represents an interesting target for the development of anti-cancer drugs. Several compounds, such as FK866 and CHS828, were identified as potent NAMPT inhibitors with strong anti-cancer activity, although none of them reached the late stages of clinical trials. We present herein the preparation of three libraries of new inhibitors containing (pyridin-3-yl)triazole, (pyridin-3-yl)thiourea and (pyridin-3/4-yl)cyanoguanidine as cap/connecting unit and a furyl group at the tail position of the compound. Antiproliferative activity in vitro was evaluated on a panel of solid and haematological cancer cell lines and most of the synthesized compounds showed nanomolar or sub-nanomolar cytotoxic activity in MiaPaCa-2 (pancreatic cancer), ML2 (acute myeloid leukemia), JRKT (acute lymphobalistic leukemia), NMLW (Burkitt lymphoma), RPMI8226 (multiple myeloma) and NB4 (acute myeloid leukemia), with lower IC50 values than those reported for FK866. Notably, compounds 35a, 39a and 47 showed cytotoxic activity against ML2 with IC50 = 18, 46 and 49 pM, and IC50 towards MiaPaCa-2 of 0.005, 0.455 and 2.81 nM, respectively. Moreover, their role on the NAD+ synthetic pathway was demonstrated by the NAMPT inhibition assay. Finally, the intracellular NAD+ depletion was confirmed in vitro to induced ROS accumulation that cause a time-dependent mitochondrial membrane depolarization, leading to ATP loss and cell death.

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.

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.

Identification of NAPRT Inhibitors with Anti-Cancer Properties by In Silico Drug Discovery.

Depriving cancer cells of sufficient NAD levels, mainly through interfering with their NAD-producing capacity, has been conceived as a promising anti-cancer strategy. Numerous inhibitors of the NAD-producing enzyme, nicotinamide phosphoribosyltransferase (NAMPT), have been developed over the past two decades. However, their limited anti-cancer activity in clinical trials raised the possibility that cancer cells may also exploit alternative NAD-producing enzymes. Recent studies show the relevance of nicotinic acid phosphoribosyltransferase (NAPRT), the rate-limiting enzyme of the Preiss-Handler NAD-production pathway for a large group of human cancers. We demonstrated that the NAPRT inhibitor 2-hydroxynicotinic acid (2-HNA) cooperates with the NAMPT inhibitor FK866 in killing NAPRT-proficient cancer cells that were otherwise insensitive to FK866 alone. Despite this emerging relevance of NAPRT as a potential target in cancer therapy, very few NAPRT inhibitors exist. Starting from a high-throughput virtual screening approach, we were able to identify and annotate two additional chemical scaffolds that function as NAPRT inhibitors. These compounds show comparable anti-cancer activity to 2-HNA and improved predicted aqueous solubility, in addition to demonstrating favorable drug-like profiles.

Identification of new FK866 analogues with potent anticancer activity against pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal diseases for which chemotherapy has not been very successful yet. FK866 ((E)-N-(4-(1-benzoylpiperidin-4-yl)butyl)-3-(pyridin-3-yl)acrylamide) is a well-known NAMPT (nicotinamide phosphoribosyltransferase) inhibitor with anti-cancer activities, but it failed in phase II clinical trials. We found that FK866 shows anti-proliferative activity in three PDAC cell lines, as well as in Jurkat T-cell leukemia cells. More than 50 FK866 analogues were synthesized that introduce substituents on the phenyl ring of the piperidine benzamide group of FK866 and exchange its buta-1,4-diyl tether for 1-oxyprop-3-yl, (E)-but-2-en-1,4-diyl and 2- and 3-carbon tethers. The pyridin-3-yl moiety of FK866 was exchanged for chlorinated and fluorinated analogues and for pyrazin-2-yl and pyridazin-4-yl groups. Several compounds showed low nanomolar or sub-nanomolar cell growth inhibitory activity. Our best cell anti-proliferative compounds were the 2,4,6-trimethoxybenzamide analogue of FK866 ((E)-N-(4-(1-(2,4,6-trimethoxybenzoyl)piperidin-4-yl)butyl)-3-(pyridin-3-yl)acrylamide) (9), the 2,6-dimethoxybenzamide (8) and 2-methoxybenzamide (4), which exhibited an IC50 of 0.16 nM, 0.004 nM and 0.08 nM toward PDAC cells, respectively.

Gut microbiota severely hampers the efficacy of NAD-lowering therapy in leukemia.

Most cancer cells have high need for nicotinamide adenine dinucleotide (NAD+) to sustain their survival. This led to the development of inhibitors of nicotinamide (NAM) phosphoribosyltransferase (NAMPT), the rate-limiting NAD+ biosynthesis enzyme from NAM. Such inhibitors kill cancer cells in preclinical studies but failed in clinical ones. To identify parameters that could negatively affect the therapeutic efficacy of NAMPT inhibitors and propose therapeutic strategies to circumvent such failure, we performed metabolomics analyses in tumor environment and explored the effect of the interaction between microbiota and cancer cells. Here we show that tumor environment enriched in vitamin B3 (NAM) or nicotinic acid (NA) significantly lowers the anti-tumor efficacy of APO866, a prototypic NAMPT inhibitor. Additionally, bacteria (from the gut, or in the medium) can convert NAM into NA and thus fuel an alternative NAD synthesis pathway through NA. This leads to the rescue from NAD depletion, prevents reactive oxygen species production, preserves mitochondrial integrity, blunts ATP depletion, and protects cancer cells from death.Our data in an in vivo preclinical model reveal that antibiotic therapy down-modulating gut microbiota can restore the anti-cancer efficacy of APO866. Alternatively, NAphosphoribosyltransferase inhibition may restore anti-cancer activity of NAMPT inhibitors in the presence of gut microbiota and of NAM in the diet.

Reactive oxygen/nitrogen species contribute substantially to the antileukemia effect of APO866, a NAD lowering agent.

APO866 is a small molecule drug that specifically inhibits nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme involved in nicotinamide adenine dinucleotide (NAD) biosynthesis from the natural precursor nicotinamide. Although, the antitumor activity of APO866 on various types of cancer models has been reported, information regarding mechanisms by which APO866 exerts its cytotoxic effects is not well defined. Here we show that APO866 induces a strong, time-dependent increase in highly reactive ROS, nitric oxide, cytosolic/mitochondrial superoxide anions and hydrogen peroxide. We provide evidence that APO866-mediated ROS production is modulated by PARP1 and triggers cell death through mitochondria depolarization and ATP loss. Genetic or pharmacologic inhibition of PARP1 prevented hydrogen peroxide accumulation, caspase activation, mitochondria depolarization, ATP loss and abrogates APO866-induced cell death, suggesting that the integrity of PARP1 status is required for cell death. Conversely, PARP1 activating drugs enhanced the anti-leukemia activity of APO866 Collectively, our studies show that APO866 induces ROS/RNS productions, which mediate its anti-leukemia effect. These results support testing new combinatorial strategies to enhance the antitumor activities of APO866.

The NAD-Booster Nicotinamide Riboside Potently Stimulates Hematopoiesis through Increased Mitochondrial Clearance.

It has been recently shown that increased oxidative phosphorylation, as reflected by increased mitochondrial activity, together with impairment of the mitochondrial stress response, can severely compromise hematopoietic stem cell (HSC) regeneration. Here we show that the NAD+-boosting agent nicotinamide riboside (NR) reduces mitochondrial activity within HSCs through increased mitochondrial clearance, leading to increased asymmetric HSC divisions. NR dietary supplementation results in a significantly enlarged pool of progenitors, without concurrent HSC exhaustion, improves survival by 80%, and accelerates blood recovery after murine lethal irradiation and limiting-HSC transplantation. In immune-deficient mice, NR increased the production of human leucocytes from hCD34+ progenitors. Our work demonstrates for the first time a positive effect of NAD+-boosting strategies on the most primitive blood stem cells, establishing a link between HSC mitochondrial stress, mitophagy, and stem-cell fate decision, and unveiling the potential of NR to improve recovery of patients suffering from hematological failure including post chemo- and radiotherapy.

Imatinib Uptake into Cells is Not Mediated by Organic Cation Transporters OCT1, OCT2, or OCT3, But is Influenced by Extracellular pH.

BACKGROUND: Cancer cells undergo genetic and environmental changes that can alter cellular disposition of drugs, notably by alterations of transmembrane drug transporters expression. Whether the influx organic cation transporter 1 (OCT1) encoded by the gene SLC221A1 is implicated in the cellular uptake of imatinib is still controversial. Besides, imatinib ionization state may be modulated by the hypoxic acidic surrounding extracellular microenvironment. OBJECTIVE: To determine the functional contribution of OCTs and extracellular pH on imatinib cellular disposition. METHODS: We measured imatinib uptake in two different models of selective OCTs drug transporter expression (transfected Xenopus laevis oocytes and OCT-expressing HEK293 human cells), incubated at pH 7.4 and 6, using specific mass spectrometry analysis. RESULTS: Imatinib cellular uptake occurred independently of OCT1- OCT2- or OCT3-mediated drug transport at pH 7.4. Uptake of the OCTs substrate tetraethylammonium in oocytes remained intact at pH 6, while the accumulation of imatinib in oocytes was 10-fold lower than at pH 7.4, irrespectively of OCTs expressions. In OCT1- and OCT2-HEK cells at pH 6, imatinib accumulation was reduced by 2- 3-fold regardless of OCTs expressions. Since 99.5% of imatinib at pH6 is under the cationic form, the reduced cellular accumulation of imatinib at such pH may be explained by the lower amount of uncharged imatinib remaining for passive diffusion across cellular membrane. CONCLUSION: Imatinib is not a substrate of OCTs 1-3 while the environmental pH modulates cellular disposition of imatinib. The observation that a slightly acidic extracellular pH influences imatinib cellular accumulation is important, considering the low extracellular pH reported in the hematopoietic leukemia/ cancer cell microenvironment.

Experimental Drainage Device to Reduce Lymphoedema in a Rat Model.

OBJECTIVE: Despite recent advances in pharmacological research and microsurgery, lymphoedema remains an incurable disease that deeply affects quality of life. There is an urgent need for innovative approaches to restore continuous lymph flow in affected tissues. To this end, the efficacy of a subcutaneously implanted draining device in reducing lymphoedema volume in a rat hindlimb lymphoedema model was tested. METHODS: A rat model of chronic lymphoedema was developed by surgical removal of popliteal and inguinal lymph nodes, followed by irradiation. The model was characterised by monitoring limb volume via tape measure, skin water content via dielectric constant measurement, and lymphatic drainage via lymphofluoroscopy. After lymphoedema establishment in 16 Wistar rats, a device made of fenestrated tubing equipped with a miniaturised pumping system, was implanted subcutaneously in the affected limb to restore continuous recirculation of interstitial fluid. RESULTS: Lymphofluoroscopy imaging showed impaired lymphatic drainage following lymphadenectomy and irradiation. Affected limb volume and skin water content increased significantly compared with the untreated limb, with a median (interquartile range) of 3.85 (0.38) cm3 versus 3.03 (0.43) cm3 for volume (n = 16, p = .001) and 26.6 (9.1) versus 16.6 (3.7) cm3 for skin dielectric constant (n = 16, p = .001). Treatment of lymphoedema with the implanted drainage device showed that 5 weeks post-implant excess volume was significantly reduced by 51 ± 18% compared with the pre-implant situation (n = 9 sham group, n = 7 pump group). CONCLUSION: Lymphoedema volume in the rat model was significantly reduced by restoring continuous drainage of excess fluid using a novel subcutaneously implanted device, opening the way to the development of an artificial lymphatic vessel.