Nicotinamide phoshophoribosyltransferase (NAMPT) plays a key role in the replenishment of the NAD pool in cells. This in turn makes this enzyme an important player in bioenergetics and in the regulation of NAD-using enzymes, such as PARPs and sirtuins. Furthermore, there is now ample evidence that NAMPT is secreted and has a role as a cytokine. An important role of either the intracellular or extracellular form of NAMPT has been shown in cancer, inflammation, and metabolic diseases. The first NAMPT inhibitors (FK866 and CHS828) have already entered clinical trials, and a surge in interest in the synthesis of novel molecules has occurred. The present review summarizes the recent progress in this field.

Nicotinamide adenine dinucleotide (NAD) is a cofactor of many enzymatic reactions as well as being a substrate for a number of NAD-consuming enzymes (e.g., PARPS, sirtuins, etc). NAD can be synthesized de novo starting from tryptophan, nicotinamide, nicotinic acid, or nicotinamide riboside from the diet. On the other hand, the nicotinamide that is liberated by NAD-consuming enzymes can be salvaged to reform NAD. In this former instance, nicotinamide phosphoribosyltransferase (NAMPT) is the bottleneck enzyme. In the many cells in which the salvage pathway is predominant, NAMPT, therefore, represents an important controller of intracellular NAD concentrations, and as a consequence of energy metabolism. It is, therefore, not surprising that NAMPT is over expressed by tumoral cells, which take advantage from this to sustain growth rate and tumor progression. This has led to the initiation of numerous medicinal chemistry programs to develop NAMPT inhibitors in the context of oncology. More recently, however, it has been shown that NAMPT inhibitors do not solely target the tumor but also have an effect on the immune system. To add complexity, this enzyme can also be secreted by cells, and in the extracellular space it acts as a cytokine mainly through the activation of Toll like Receptor 4 (TLR4), although it has not been clarified yet if this is the only receptor responsible for its actions. While specific small molecules have been developed only against the intracellular form of NAMPT, growing evidences sustain the possibility to target the extracellular form. In this contribution, the most recent evidences on the medicinal chemistry of NAMPT will be reviewed, together with the key elements that sustain the hypothesis of NAMPT targeting and the drawbacks so far encountered.

Recent evidence has shown that NAD(P) plays a variety of roles in cell-signaling processes. Surprisingly, the presence of NAD(P) utilizing ectoenzymes suggests that NAD(P) is present extracellularly. Indeed, nanomolar concentrations of NAD have been found in plasma and other body fluids. Although very high concentrations of NAD have been shown to enter cells, it is not known whether lower, more physiological concentrations are able to be taken up. Here we show that two mammalian cell types are able to transport low NAD concentrations effectively. Furthermore, extracellular application of NAD was able to counteract FK866-induced cell death and restore intracellular NAD(P) levels. We propose that NAD uptake may play a role in physiological NAD homeostasis.Pyridine nucleotides, although often considered as simple redox cofactors in the cell, are in fact multifunctional molecules involved in a wide range of cellular processes (1). It is becoming clear that NAD(P) is involved in more pharmacologically attractive cellular processes such as cell signaling, transcriptional regulation, and post-translational protein modification (2, 3). For example, NAD(P) has been shown to be the precursor of molecules involved in calcium signaling (e.g. cADPR, 2 NAADP, and ADPR), to be involved in the regulation of epigenetic changes via sirtuins and to be a substrate for both monoand poly-ADP-ribosylation (3-5).The general perception is that cellular NAD is synthesized either de novo from tryptophan or via one of two possible recycling pathways: from nicotinic acid or nicotinamide (together referred to as vitamin PP, or niacin) (6). Recently, a third biosynthesis pathway has been described, which uses nicotinamide riboside as a precursor (7).The presence of membrane-bound ectoenzymes that use NAD(P) (8) has led to the investigation and description of several mechanisms for the export of NAD across the plasma membrane, including transport through connexins and stimulus-induced exocytotic release (9 -11). Surprisingly, though, the possibility that low NAD concentrations can be imported across the membrane to directly replenish the cellular NAD(P) pools bypassing biosynthetic pathways has not been conclusively addressed. However, circumstantial evidence suggests that this may occur: (i) High concentrations of extracellularly applied NAD(H) have been shown to increase intracellular NAD levels (10, 12-14): (ii) extracellular NAD counteracts PARP-induced intracellular NAD depletion (12, 15); (iii) CD38 knock-out mice, which are impaired in their ability to degrade extracellular NAD, display higher endogenous SIRT1 activity (16); (iv) uptake processes have been shown for the Ca 2ϩ -mobilizing NAD(P) metabolites cADPR and NAADP in a variety of diverse mammalian cell types (17-21); (v) NAD and at least one of the enzymes involved in biosynthesis are present extracellularly (13,22,23); and (vi) Wallerian degeneration can be slowed by the addition of extracellular NAD (24).Here we show that various mammalian cell types are able to transport pico...

The inhibition of NAD synthesis or salvage pathways has been proposed as a novel target for antitumoral drugs. Two molecules with this mechanism of action are at present undergoing clinical trials. In searching for similar novel molecules, we exploited copper-catalyzed [3 + 2] cycloaddition between azides and alkynes (click chemistry) to synthesize 185 novel analogues. The most promising compound displays an IC(50) for cytotoxicity in vitro of 3.8 +/- 0.3 nM and an IC(50) for NAD depletion of 3.0 +/- 0.4 nM. Herein, we strengthen previous data suggesting that this class of compounds induces autophagic cell death. In addition to characterizing this compound and providing a rationale via molecular docking, we reinforce the excellent potential of click chemistry for rapidly generating structure-activity relationships and for drug screening.

log EC 50 ) 1.02 ((0.18)