GMX1777: a novel inhibitor of NAD+ biosynthesis via inhibition of nicotinamide phosphoribosyl transferase.

A81 GMX1777 (EB1627) is a soluble prodrug of the pharmacologically active compound GMX1778 (CHS828), and is currently in Phase I clinical trials. The mechanism of action of this small molecule with potent and broad antitumor activity was believed to involve NF-κB inhibition. We now report that GMX1778 functions by inhibiting nicotinamide phosphoribosyl transferase (NAMPRT), an enzyme involved in nicotinamide adenine dinucleotide (oxidized) (NAD+) biosynthesis and that NF-κB inhibition is only a consequence of NAD+ decline. To determine the primary mechanism of action of GMX1778, a combination of global metabolomics and targeted biochemical pathway profiling studies were undertaken to track intracellular physiological changes over time. Of the limited physiological changes in IM-9 cells observed after treatment with GMX1778 for 6 hours, there occurred a 60% decline of intracellular NAD+ levels, with a 91% decline noted at 13 hours. NAD+ decline is followed by a gradual decline of ATP and cell death 48 to 72 hours post treatment. Addition of nicotinamide or nicotinic acid completely rescued GMX1778-treated HeLa cells from NAD+ decline, NF-κB inhibition and cell death; thereby substantiating that lack of NAD+ is the cause of GMX1778-induced NF-κB inhibition and cytotoxicity. Modulating NAD+ levels in a similar manner did not affect the activity of an unrelated cytotoxic agent 5-FU. Metabolic experiments using labeled precursors showed that GMX1778 inhibits the generation of NAD+ from 14 C nicotinamide but not from 14 C nicotinic acid in HeLa cells and cell extracts. Further experiments using purified human enzyme demonstrated that GMX1778 inhibits NAMPRT, the rate-limiting enzyme that converts nicotinamide (NAM) to nicotinamide mononucleotide (NAMN); the apparent K i (1-3 nM) of GMX1778 for NAMPRT correlates well with the low nanomolar IC 50 of this compound in multiple human cell lines. In vitro cytotoxicity studies showed a positive correlation between the level of NAMPRT expression and sensitivity to GMX1778. In support of these results, partial siRNA knockdown of NAMPRT in HeLa cells sensitized cells to GMX1778. Together these results demonstrate that GMX1778 is a potent and specific inhibitor of NAMPRT, resulting in a rapid drop in cellular NAD+. Tumor cells have elevated NAMPRT and a high rate of NAD+ turnover due to high ADP-ribosylation activity required for DNA repair, genome stability, telomere maintenance making them more susceptible to NAMPRT inhibition than normal cells. This novel mechanism supports the clinical use of GMX1777 as an anti-cancer agent and provides a rationale for the use of GMX1777 in combination with DNA damaging agents for future trials.