Conversion Of Nicotinamide Research Articles

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.

Biosynthesis of a Therapeutically Important Nicotinamide Mononucleotide through a Phosphoribosyl Pyrophosphate Synthetase 1 and 2 Engineered Strain of Escherichia coli.

Nicotinamide mononucleotide (NMN), a precursor of NAD+, can be synthesized by the conversion of nicotinamide with the help of nicotinamide phosphoribosyl transferase (NAMPT) via the salvage pathway. NMN has recently gained great attention as an excellent therapeutic option due to its long-term effective pharmacological activities. In this study, we constructed a recombinant strain of Escherichia coli by inserting NAMPT and phosphoribosyl pyrophosphate synthetase 1 (PRPS1) and PRPS2 (from Homo sapiens) genes to investigate the effect of PRPS1 and PRPS2 on NMN synthesis. The metabolically engineered strain of E. coli BL21 (DE3) exhibited 1.57 mM NMN production in the presence of Mg2+ and phosphates in batch fermentation studies. For further improvement in NMN production levels, effects of different variables were studied using a response surface methodology approach. A significant increment was achieved with a maximum of 2.31 mM NMN production when supplemented with 1% ribose, 1 mM Mg2+ and phosphate, and 0.5% nicotinamide in the presence of a lactose (1%) inducer. Additionally, insertion of the PRPS1 and PRPS2 genes in the phosphoribosyl pyrophosphate synthesis pathway and individual gene expression studies facilitated a higher NMN production at the intracellular level than the reported studies. The strain exhibited intracellular production of NMN from cheap substrates such as glucose, lactose, and nicotinamide. Hence, the overall optimized process can be further scaled up for the economical production of NMN using a recombinant strain of E. coli BL21 (DE3), which is the future perspective of the current study.

P7C3 Neuroprotective Chemicals Function by Activating the Rate-Limiting Enzyme in NAD Salvage

The P7C3 class of aminopropyl carbazole chemicals fosters the survival of neurons in a variety of rodent models of neurodegeneration or nerve cell injury. To uncover its mechanism of action, an active derivative of P7C3 was modified to contain both a benzophenone for photocrosslinking and an alkyne for CLICK chemistry. This derivative was found to bind nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme involved in the conversion of nicotinamide into nicotinamide adenine dinucleotide (NAD). Administration of active P7C3 chemicals to cells treated with doxorubicin, which induces NAD depletion, led to a rebound in intracellular levels of NAD and concomitant protection from doxorubicin-mediated toxicity. Active P7C3 variants likewise enhanced the activity of the purified NAMPT enzyme, providing further evidence that they act by increasing NAD levels through its NAMPT-mediated salvage.

Nutritional Aspect of Tryptophan Metabolism

Mammals, including humans, can synthesize the vitamin nicotinamide from tryptophan in the liver. The resultant nicotinamide is distributed to non-hepatic tissues. We have studied the effects of changes in tryptophan–nicotinamide metabolism on niacin nutritional status. The liver plays a critical role in nicotinamide supply. Animal studies showed that the tryptophan–nicotinamide pathway is affected by physiological conditions, the presence of disease, nutrients, hormones, and chemicals. Human studies have shown that 1 mg of nicotinamide is produced from 67 mg of tryptophan intake, and that the conversion ratio of tryptophan to nicotinamide is enhanced from mid to late pregnancy. These findings have contributed to the determination of dietary reference intakes for niacin recommended in the Dietary Reference Intakes for Japanese 2010. Our findings suggest that the conversion of nicotinamide from tryptophan is important in maintaining niacin nutrition.

Nicotinamide phosphoribosyltransferase/sirtuin 1 pathway is involved in human immunodeficiency virus type 1 Tat‐mediated long terminal repeat transactivation

Tat is a multifunctional transactivator encoded by human immunodeficiency virus type 1 (HIV-1). Tat transactivating activity is controlled by nicotinamide adenine nucleotide(+) (NAD(+))-dependent deacetylase sirtuin 1 (SIRT1). Nicotinamide phosphoribosyltransferase (Nampt) is a rate-limiting enzyme in the conversion of nicotinamide into NAD(+), which is crucial for SIRT1 activation. Thus, the effect of Nampt on Tat-regulated SIRT activity was studied in Hela-CD4-beta-gal (MAGI) cells. We demonstrated that Tat caused NAD(+) depletion and inhibited Nampt mRNA and protein expression in MAGI cells. Resveratrol reversed Tat-induced NAD(+) depletion and inhibition of Nampt mRNA and protein expression. Further investigation revealed that Tat-induced inhibition of SIRT1 activity was potentiated in Nampt-knockdown by Nampt siRNA compared to treatment with Tat alone. Nampt siRNA potentiated Tat-induced HIV-1 transactivation in MAGI cells. Altogether, these results indicate that Nampt is critical in the regulation of Tat-induced inhibition of SIRT1 activity and long terminal repeat (LTR) transactivation. Nampt/SIRT1 pathway could be a novel therapeutic tool for the treatment of HIV-1 infection.

Age-Related Alterations of B-Group Vitamin Contents in Urine, Blood and Liver from Rats

To investigate how aging alters B-group vitamin metabolism, rats were fed with niacin-free 20% casein diet from 3 to 80 wk old, and the urinary excretions of the B group vitamins were periodically measured. The blood and liver B-group vitamin levels in 80-wk-old rats were also compared with those in 8-wk-old rats. The urinary excretion of thiamin, riboflavin, vitamin B6 metabolite 4-pyridoxic acid, pantothenic acid, folic acid and biotin were not altered during 540 d. The urinary vitamin B12 increased by 8-fold at 29 wk old, and further increased at 80 wk old. Conversion of nicotinamide from tryptophan gradually decreased to 60% from 29 to 48 wk old. Plasma PLP, vitamin B12 and folate levels in 80-wk-old rats were lower than those in 8-wk-old rats, consistent with lower liver vitamin B6 and folate levels in aged rats. Plasma and liver biotin levels in aged rats were higher than those in young rats. Other B-group vitamins such as vitamin B1, vitamin B2, niacin and pantothenic acid levels in blood and liver from aged rats were same as those from young rats. Alteration of vitamin B6 metabolism in particular is similar to the observations in elderly humans reported previously. Our findings suggest that aged rats can be useful models to investigate aging-related B-group vitamin metabolism.

Determination of Nicotiamide by Photochemical Fluorimetryxs

Abstract An on-line photochemical spectrofluorimetric method for the determination of nicotinamide is proposed. It is based on the conversion of nicotinamide in an acidic medium into an intensively fluorescent compound. The calibration graph was linear up to 10.0 μg ml−1 nicotinamide (r = 0.999), the limit of detection was 0.60 ng ml−1 nicotinamide and the relative standard deviations were 1.5% (n = 6), 2.1% (n = 6) and 2.1% (n = 6) for 50 ng ml−1, 0.70 μg ml−1 and 1.0 μg ml−1 nicotinamide, respectively. The kinetic behaviors of the reaction and the effects of some experimental conditions were investigated. Its application to assay of vitamin B complex tablets was found to be satisfactory.

Importance of nicotinamide as an NAD precursor in the human erythrocyte

The effect of variation in the concentration of inorganic phosphate and of the pyridine precursors nicotinamide (NAm) and nicotinic acid (NA) on pyridine nucleotide synthesis was studied using intact human erythrocytes. A wide range of incubation times was employed. The results showed that under physiological conditions the rate of synthesis of NAD from NAm exceeded that from NA twofold, while the reverse situation pertained at higher and unphysiological substrate levels. The two pathways had different regulation points. For NAm the rate-limiting factor was the initial step, namely its conversion into the mononucleotide, while for NA it lay at the second step, conversion of NA mononucleotide (NAMN) to its adenine dinucleotide. At physiological substrate levels the uptake of NA and conversion to NAMN were rapid, while the uptake and conversion of NAm were time dependent. This process was stimulated significantly by inorganic phosphate only for NAm. These results indicate that while NA is the predominant precursor of human erythrocyte NAD at high (unphysiological) substrate and phosphate levels, NAm is more efficient as an NAD precursor under physiological conditions, suggesting an important and hitherto unrecognized role for nicotinamide in NAD synthesis in vivo.

Determination of Nicotinic Acid and Nicotinamide in Meats by High Performance Liquid Chromatography and Conversion of Nicotinamide in Meats during Storage

A method for determination of nicotinic acid (NA) and nicotinamide (NAA) in meat by high performance liquid chromatography (HPLC) was developed.NA and NAA were extracted from meat with methanol and the extract was cleaned up with a Sep-pak alumina N cartridge. NAA was eluted with methanol and NA was eluted with 0.1M sodium hydrogencarbonate from the cartridge. NA and NAA in each eluate were separated on a column packed with LiChrosorb RP-18 by using methanol-acetate buffer (pH 5) containing 0.1M sodium acetate and 0.01M tetrabutylammonium hydroxide (2:10v/v%), as a mobile phase, with detection at 261nm. Recoveries of NA and NAA from ground meats spiked at levels of 0.5 and 2.0mg/100g were more than 92%. NA was detected in 24 of 155 samples of commercial ground meat at the level of 1.1-45.7mg/100g and NAA was detected in all of the samples at the level of 1.1-5.9mg/100g.The effect of storage time on content of NA an NAA in meat at -18, 4, 10 and 25°C was examined. At 10°C, NAA content began to decrease on the 2nd day and NAA was no longer detected on the 5th day, whereas NA began to be detected on the 2nd day and its content subsequently rapidly increased. The total amount of NA and NAA in one sample was approximately constant during storage.Based on these results, NAA in meat appears to be converted to NA during sotrage.