GMX1777 is a prodrug of the small molecule GMX1778, currently in phase I clinical trials for the treatment of cancer. We describe findings indicating that GMX1778 is a potent and specific inhibitor of the NAD
+ biosynthesis enzyme nicotinamide phosphoribosyltransferase (NAMPT). Cancer cells have a very high rate of NAD
+ turnover, which makes NAD
+ modulation an attractive target for anticancer therapy. Selective inhibition by GMX1778 of NAMPT blocks the production of NAD
+ and results in tumor cell death. Furthermore, GMX1778 is phosphoribosylated by NAMPT, which increases its cellular retention. The cytotoxicity of GMX1778 can be bypassed with exogenous nicotinic acid (NA), which permits NAD
+ repletion via NA phosphoribosyltransferase 1 (NAPRT1). The cytotoxicity of GMX1778 in cells with NAPRT1 deficiency, however, cannot be rescued by NA. Analyses of NAPRT1 mRNA and protein levels in cell lines and primary tumor tissue indicate that high frequencies of glioblastomas, neuroblastomas, and sarcomas are deficient in NAPRT1 and not susceptible to rescue with NA. As a result, the therapeutic index of GMX1777 can be widended in the treatment animals bearing NAPRT1-deficient tumors by coadministration with NA. This provides the rationale for a novel therapeutic approach for the use of GMX1777 in the treatment of human cancers.The cyanoguanidinopyridine GMX1778 (previously known as CHS828) is the active form of the prodrug GMX1777 and has potent antitumor activity in vitro and in vivo against cell lines derived from several different tumor origins (
11). The antitumor activity of GMX1778 has been widely studied since its discovery (
1,
11,
19-
21,
24), but positive identification of the molecular target and the mechanism of action of GMX1778 has been elusive. Here, we demonstrate that GMX1778 exerts its antitumor activity via its potent and selective antagonism of NAD
+ biosynthesis. GMX1777 is currently being assessed in phase I clinical trials for treatment of patients with refractory solid tumors.The pyridine nucleotide NAD
+ plays a major role in the regulation of several essential cellular processes (
7,
22,
25,
38). In addition to being a biochemical cofactor for enzymatic redox reactions involved in cellular metabolism, including ATP production, NAD
+ is important in diverse cellular pathways responsible for calcium homeostasis (
17), gene regulation (
5), longevity (
18), genomic integrity (
33), and apoptosis (
36). Cancer cells exhibit a significant dependence on NAD
+ for support of the high levels of ATP production necessary for rapid cell proliferation. They also consume large amounts of this cofactor via reactions that utilize poly(ADP) ribosylation, including DNA repair pathways (
10,
37,
39).In eukaryotes, the biosynthesis of NAD
+ occurs via two biochemical pathways: the de novo pathway, in which NAD
+ synthesis occurs through the metabolism of
l-tryptophan via the kynurenine pathway, and the salvage pathway. The NAD
+ salvage pathway can use either nicotinamide (niacinamide) (NM) or nicotinic acid (niacin) (NA) (via the Preiss-Handler pathway) as a substrate for NAD
+ production.
Saccharomyces cerevisiae species predominantly use NA as the substrate for NAD
+ biosynthesis, through the deamidation of NM by the nicotinamidase PNC1 (
25). However, mammalian cells do not express a nicotinamidase enzyme and use NM as the preferred substrate for the NAD
+ salvage pathway. The mammalian NAD
+ biosynthesis salvage pathway using NM is composed of NA phosphoribosyltransferase (NAMPT), which is the rate-limiting and penultimate enzyme that catalyzes the phosphoribosylation of NM to produce nicotinamide mononucleotide (NMN) (
27,
29). NMN is subsequently converted to NAD
+ by NMN adenyltransferases (NMNAT). The gene encoding NAMPT was originally identified as encoding a cytokine named pre-B-cell colony-enhancing factor (PBEF1) (
30). NAMPT was also identified as a proposed circulating adipokine named visfatin (thought to be secreted by fat cells) and was suggested to function as an insulin mimetic; however, this role of NAMPT currently remains controversial (
8). In mice, NAMPT has been shown to act as a systemic NAD
+ biosynthetic enzyme that regulates insulin secretion from β cells (
28). The molecular structure of NAMPT from human (
15), rat (
16) and mouse (
35) tissue, containing either NMN or the inhibitor APO866, have been determined by X-ray crystallography. These structures revealed that NAMPT is a dimeric type II phosphoribosyltransferase.Here, we report that the anticancer compound GMX1778 is a specific inhibitor of NAMPT in vivo and in vitro and is itself a substrate for the enzyme. Phosphoribosylated GMX1778 inhibits NAMPT as potently as GMX1778 but is preferentially retained within cells. Finally, we have identified a novel anticancer strategy utilizing NA rescue of GMX1778 cytotoxicity to increase the therapeutic index of GMX1777 activity in tumors that are deficient in NA phosphoribosyltransferase 1 (NAPRT1).
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