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The small molecule GMX1778 is a potent inhibitor of NAD+ biosynthesis: strategy for enhanced therapy in nicotinic acid phosphoribosyltransferase 1-deficient tumors - PubMed

The small molecule GMX1778 is a potent inhibitor of NAD+ biosynthesis: strategy for enhanced therapy in nicotinic acid phosphoribosyltransferase 1-deficient tumors

Mark Watson et al. Mol Cell Biol. 2009 Nov.

Abstract

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.

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Figures

FIG. 1.
FIG. 1.

Characterization of GMX1778 cytotoxicity. (A) Kinetics of NAD+ depletion, ATP depletion, and cell lysis. Cellular NAD+ levels, measured by LC/MS, and ATP depletion data are from one experiment, while the cell lysis data are from an independent experiment. Data are presented as means ± standard deviations. (B) Clonogenicity, annexin V reactivity, and cell lysis (propidium iodide [PI] permeability) of IM-9 cells after 72 h of exposure to GMX1778 (25 nM). Clonogenicity was assessed in soft agar by counting colonies, and data are presented as percentages (± standard errors of the means) of colonies compared to levels of DMSO-treated cells. Annexin V reactivity and PI permeability were assessed by flow cytometric analyses of 10,000 events. (C) Caspase 3 activity and cleavage in IM-9 cells treated with GMX1778. Caspase 3 activity was measured in 25 μg of cell extract. Inset, Western blot of the accumulation of cleaved caspase 3 in 25 μg of extracts from cells treated with GMX1778. The panels are from a single Western blot, with intervening lanes removed.

FIG. 2.
FIG. 2.

Characterization of GMX1778 cytotoxicity. (A) HeLa cells are protected from GMX1778 cytotoxicity by NA. HeLa cells were exposed to GMX1778 in the presence or absence of 10 μM NA. Viability was measured after 72 h and is presented as means ± standard deviations. The inset shows cellular NAD+ levels from HeLa cells treated with 100 nM GMX1778. Cellular NAD+ levels were measured by LC/MS and are presented as means ± standard deviations. (B) NA (10 μM) rescue of GMX1778 cytotoxicity also rescues TNF-α-induced NF-κB activity. HeLa cells were exposed to GMX1778 in the presence or absence of NA. NFκB transcriptional activity was measured by luminescence generated from expression of a reporter gene and is presented as means ± standard deviations. (C) NA rescues GMX1778-mediated inhibition of IκBα phosphorylation. IκBα phosphorylation was induced by a 5-min stimulation with TNF-α following treatment of cells with bortezomib (1 h) and 100 nM GMX1778 (30 h) with or without 10 μM NA. (D) GMX1778 cytotoxicity is rescued by NMN. HeLa cells were exposed to GMX1778 in the presence or absence of 100 μM NMN. Viability was measured after 72 h and is presented as means ± standard deviations. The inset shows cellular NAD+ levels from HeLa cells treated with 100 nM GMX1778. Cellular NAD+ levels were measured by LC/MS and are presented as means ± standard deviations. (E) Yeast nicotinamidase (PNC1) expression rescues mammalian cells from GMX1778 cytotoxicity. HeLa cells were transiently transfected with vector carrying FLAG-PNC1 or empty vector and challenged by exposure to GMX1778. Viability after 72 h is presented as means ± standard deviations. Left inset, Western blot of 25 μg of cell extract from cells transfected with either vector or PNC1 as indicated and probed with anti-FLAG. Right inset, cellular NAD+ levels from transfected cells treated with 100 nM GMX1778. Cellular NAD+ levels were measured by LC/MS and are presented as means ± standard deviations.

FIG. 3.
FIG. 3.

The salvage NAD+ biosynthetic pathway from nicotinamide is inhibited by GMX1778. (A) NAD+ biosynthetic pathways in mammalian cells are the de novo pathways which synthesize NAD+ from tryptophan and quinolinic acid (QA) and the salvage pathways where NAD+ is generated from either NA or NM that is taken up by cells. NA is converted to NAD+ through a three-step enzymatic process involving NAPRT1, NMNAT, and NAD+ synthetase in sequence. NAD+ synthesis from NM is a two-step process involving NAMPT and NMNAT. The ribose portion of NAD+ is utilized or broken down by multiple enzymes, including poly(ADP-ribose) polymerase (PARP) and sirtuin proteins, to regenerate NM, which is in turn recycled back to NAD+. (B and C) Intact HeLa cells were treated with GMX1778 (20 nM) for 2 h and then with [14C]NM (1 μM) (B) or [14C]NA (100 nM) (C) for an additional 6 h. Cell extracts were as indicated. Metabolic products were separated by TLC and visualized by autoradiography or phosphorimaging. Media, [14C]NM added to cell culture media; t = 0, cultured cells harvested immediately following the addition of [14C]NM or [14C]NA; asterisk, unidentified NM metabolite; NAAD+, NA adenine dinucleotide. The three columns in panel B are from one autoradiogram with intervening lanes removed. The two columns in panel C are from one phosphorimager image with an intervening lane removed. (D) Effect of GMX1778 on recombinant NAMPT and NMNAT1. Fluorescence coupled-enzyme assay for the measurement of NAD+ production after 180 min. For NMNAT1 activity, NMNAT1 and NMN concentrations were 3 nM and 125 μM, respectively. For NAMPT activity, NAMPT and NM concentrations were 2 μM and 50 μM, respectively. Data are presented as means ± standard errors of the means. (E) Fluorescence polarization competition assay. Disruption of GMX1778-Alexa Fluor (20 nM) and NAMPT (200 nM) complex by GMX1778 or NM. Data are presented as means ± standard errors of the means.

FIG. 4.
FIG. 4.

Modulation of NAMPT expression alters the sensitivity of tumor cell lines to GMX1778. (A) Cytotoxicity of GMX1778 in HeLa cells transfected with either control or NAMPT siRNA. Cell viability was measured after 72 h of GMX1778 treatment. The inset shows the results of Western blot analysis of 25 μg of cell extract from HeLa cells transfected with either control or NAMPT siRNA. The blot was probed with anti-NAMPT and anti-GAPDH as indicated. (B) Cellular NAD+ levels from HeLa cells transfected with NAMPT siRNA. Cellular NAD+ levels were measured by LC/MS and are presented as means ± standard deviations. (C) GMX1778 sensitivity of HEK-293-TR cells transfected with vector, NAMPT, or NAMPT(G217R). Cells were grown in the presence of doxycycline (to induce transfected NAMPT protein expression). Viability after 72 h is presented as means ± standard deviations. (D) Cellular NAD+ levels from HeLa cells transfected with expression constructs for NAMPT and NAMPT(G217R) and treated with indicated concentrations of GMX1778. Cellular NAD+ levels were measured by LC/MS and are presented as means ± standard deviations. (E) Western blot analysis of FLAG-NAMPT protein expression in 25 μg of doxycycline-induced cell extract of cells from the experiment whose results are presented in panel C. (F) TLC analysis of the [14C]NM metabolic process resulting in [14C]NAD+ in extracts from HeLa cells transfected with vector, NAMPT, or NAMPT(G217R) and treated with 1 μM [14C]NM in the presence or absence of 50 nM GMX1778. Data from an experiment examining the [14C]NM metabolic process resulting in [14C]NAD in extracts from cells grown in the absence of doxycycline to repress protein expression (−dox) or in the presence of doxycycline to induce protein expression (+dox) are shown. These data are from an experiment that was conducted independently of the experiment whose results are presented in panel C.

FIG. 5.
FIG. 5.

The G217R mutation in NAMPT results in resistance to GMX1778 inhibition. (A) Docked structure of GMX17778 (space-filling model) in the active site of NAMPT (surface) superimposed with arginine (cylinders) at position 217. GMX1778 and the side chain of arginine 217 are colored according to atom type. The NAMPT structure is based on the X-ray crystal structure 2GVJ coordinates in the Protein Data Bank. (B) Inhibition of phosphoribosyltransferase activity of recombinant NAMPT and NAMPT(G217R) in vitro. The data are presented as means ± standard errors of the means. (C) SCID mouse xenograft assays using HCT-116R tumor cells. Mice carrying tumors were treated with a 24-h iv infusion of 150 mg/kg GMX1777 or vehicle. As a positive control for antitumor activity, mice carrying tumors were treated with iv infusions of 5-FU (5-fluororacil).

FIG. 6.
FIG. 6.

Inverse correlation of NAMPT expression with GMX1778 cytotoxicity in tumor cell lines. (A) Relative expression of NAMPT mRNA with 72-h GMX1778 cytotoxicity IC50 values for 25 tumor cell lines. The Pearson correlation coefficient (r) and the P value are indicated. (B) Western blot analysis of 25 μg of cell extracts from SCLC and NSCLC cell lines for NAMPT and GAPDH protein expression. The corresponding 72-h GMX1778 IC50 values are indicated below the lanes for the respective cell lines.

FIG. 7.
FIG. 7.

GMX1778 is a substrate for phosphoribosylation by NAMPT. (A) TLC of the in vitro phosphoribosylation of [14C]GMX1778 by recombinant NAMPT. The concentrations of NAMPT and [14C]NM were 1 μM and 50 μM, respectively. (B) TLC of products from the reactions performed with recombinant NAMPT (2.5 μM), [14C]NM (500 nM), and [14C]GMX1778 (100 nM) in the presence or absence of ATP (2 mM) and PRPP (100 μM). (C) Phosphoribosyl-[14C]GMX1778 was incubated with calf intestinal alkaline phosphatase (CIAP) or buffer as indicated, and the products were separated by TLC and exposed to a phosphorimaging plate. Lanes 2 and 4 represent the same material as lanes 1 and 3, respectively, but were spiked with phosphoribosyl-[14C]GMX1778 to identify the migration pattern. (D) Phosphoribosyl-[14C]GMX1778 is a potent inhibitor of recombinant NAMPT. Phosphoribosyl-[14C]GMX1778 was produced by using recombinant NAMPT to convert [14C]GMX1778. Data represent the results of a fluorescence coupled-enzyme assay of NAD+ production after 180 min. The concentrations of NAMPT and NM were 1 μM and 50 μM, respectively. (E) Cellular retention of phosphoribosyl-[14C]GMX1778 in HeLa cells exposed to [14C]GMX1778. HeLa cells were exposed to 100 nM [14C]GMX1778 for 1 h (−APO866) and were then either washed (lane 1) or washed and incubated at 37°C for 1 h in media (lane 2) before extracts were prepared and resolved using TLC. HeLa cells were exposed to 100 nM [14C]GMX1778 and 500 nM APO866 for 1 h (+ APO866) and were then either washed (lane 3) or washed and incubated at 37°C for 1 h in medium (lane 4) before extracts were prepared and resolved by TLC. Std., phosphoribosyl-[14C]GMX1778 standard.

FIG. 8.
FIG. 8.

NAPRT1 status of human tumor cell lines. (A) Frequency of rescue from GMX1778 cytotoxicity (30 nM) by 10 μM NA or 10 μM NAMN in a panel of human tumor cell lines. Cytotoxicity was measured at 72 h. (B) NAPRT1 mRNA levels measured by qRT-PCR and normalized to GAPDH mRNA expression. Statistical significance was determined by Student's t test with Welch's correction; P values are indicated. (C) Western blot of NAPRT1 protein expression in cytosolic extracts (30 μg) from human tumor cell lines that were rescued or not rescued from GMX1778 cytotoxicity by NA as indicated.

FIG. 9.
FIG. 9.

NAPRT1 mRNA and protein expression in primary human tumor tissues. (A) Comparison of NAPRT1 mRNA levels in normal tissue to levels in malignant tissue of the same origin. Levels of NAPRT1 mRNA from normal, carcinoma, brain, normal brain, glioblastoma, oligodendroglioma, and neuroblastoma frozen tissue samples were measured by qRT-PCR. Values were normalized to ribosomal protein-large P0 mRNA levels; solid bars indicate the means. Statistical significance was determined by Student's t test with Welch's correction; P values are indicated. (B) Immunohistochemical detection of NAPRT1 protein in lung, glioblastoma, and neuroblastoma tumor tissue sections. Upper panel, sections (5 μm) were stained with a rabbit polyclonal NAPRT1 antibody. Lower panel, sequential sections were stained with hematoxylin and eosin (H&E) to reveal cellular structures. Scale bars, 100 μm.

FIG. 10.
FIG. 10.

Antitumor activity of combination treatment with GMX1777 and NA in NAPRT1-deficient tumors. (A) SCID mice carrying NAPRT1-deficient HT1080 tumors were treated with a 24-h iv infusion of GMX1777 (150 mg/kg of body weight) followed by a 4-h iv infusion of NA (120 mg/kg) or left without further treatment (Vehicle IV). (B) SCID mice carrying HT1080 tumors were treated with a 24-h iv infusion of GMX1777 (650 mg/kg) followed by a 4-h iv infusion of NA (120 mg/kg) or left without further treatment (Vehicle IV). One mouse out of eight died from the GMX1777 treatment in the absence of NA. (C) SCID mice carrying NAPRT1-proficient HCT-116 tumors were treated with a 24-h iv infusion of GMX1777 (150 mg/kg) followed by a 4-h iv infusion of NA (120 mg/kg) or left without further treatment (Vehicle IV). (D) NA protects SCID mice from a toxic dose of GMX1777. Two groups (five mice each) were treated with a 24-h iv infusion of GMX1777 (750 mg/kg). One group was subsequently treated with a 4-h iv infusion of NA (120 mg/kg), and survival rates were assessed.

FIG. 11.
FIG. 11.

Model for the inhibition of NAD+ biosynthesis by GMX1778 in tumor cells. See text for details. NAAD+, NA adenine dinucleotide; QA, quinolinic acid.

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