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GNE-617 is a novel, potent and specific inhibitor NAMPT (nicotinamide phosphoribosyltransferase) that inhibits the biochemical activity of NAMPT with an IC50 of 5 nM and exhibits efficacy in xenograft models of cancer. NAMPT is a pleiotropic protein with intra- and extra-cellular functions as an enzyme, cytokine, growth factor, and hormone. NAMPT is of interest in oncology, because it catalyzes the rate-limiting step in the salvage pathway to generate nicotinamide adenine dinucleotide (NAD), which is considered a universal energy- and signal-carrying molecule involved in cellular energy metabolism and many homeostatic functions. The activity of GNE-617 hydrochloride is evaluated on a panel 53 non-small cell lung cancer (NSCLC) cell lines in the presence or absence of 10 μM nicotinic acid. GNE-617 inhibits NAMPT IC50 of 18.9 nM in A549 cell.The majority of cell lines exhibit a steep dose response to GNE-617 when evaluated by decrease in ATP or total nucleic acid, and the cytotoxicity is completely rescued by simultaneous addition of nicotinic acid.
| Targets |
Nicotinamide Phosphoribosyltransferase (NAMPT)
- IC50 ~1.2 nM (determined by recombinant human NAMPT enzyme activity assay using [¹⁴C]-nicotinamide as substrate);
- EC50 ~4.8 nM (for inhibiting NAD+ synthesis in human retinal pigment epithelial (hRPE) cells, measured by cyclic enzyme assay)[2]
[2] |
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| ln Vitro |
Tested against 53 non-small cell lung cancer (NSCLC) cell lines, the efficacy of GNE-617 hydrochloride was assessed with and without 10 μM Nicotinic Acid. With an IC50 of 18.9 nM in A549 cells, GNE-617 inhibits NAMPT. When GNE-617 was added concurrently with nicotinic acid, the majority of cell lines exhibited a steep dose response as measured by decreases in ATP or total nucleic acids. This prevented any cytotoxicity. IC50 values were found for most tested cell lines to be below 100 nM, and for about half of them to be below 10 nM [1].
1. Inhibition of NAMPT enzyme activity: Recombinant human NAMPT enzyme assays showed GNE-617 dose-dependently inhibited NAMPT-mediated conversion of nicotinamide to nicotinamide mononucleotide (NMN). At 1 nM, inhibition rate was ~45%; at 5 nM, ~90%; IC50 was calculated as ~1.2 nM. No significant inhibition of other NAD+ synthesis enzymes (e.g., NAPRT1, PRPP synthetase) at concentrations up to 100 nM[2] 2. Reduction of intracellular NAD+ levels: - In hRPE cells: GNE-617 (0.1-100 nM) dose-dependently reduced intracellular NAD+ levels. 1 nM reduced NAD+ by ~30%; 10 nM by ~75%; 100 nM by >90%, with EC50 ~4.8 nM. NAD+ levels began to decline at 6 hours post-treatment and reached a minimum at 24 hours[2] - In primary mouse retinal cells (mixed photoreceptors and Müller cells): 10 nM GNE-617 reduced NAD+ by ~65% at 24 hours; 50 nM by ~85%[2] 3. Retinal cell toxicity: - hRPE cells: GNE-617 (1-100 nM) reduced cell viability (MTT assay) in a time- and dose-dependent manner. 10 nM reduced viability by ~20% at 24 hours, ~50% at 48 hours; 50 nM reduced viability by ~40% at 24 hours, ~80% at 48 hours. TUNEL staining showed 50 nM GNE-617 increased apoptotic cells by ~6-fold at 48 hours[2] - Primary mouse photoreceptor cells: 20 nM GNE-617 reduced rhodopsin expression (marker of photoreceptor integrity) by ~50% at 48 hours (Western blot), and increased caspase-3 activation by ~4-fold[2] [2] |
| ln Vivo |
When rats were given equal doses and dosage durations, GNE-617 (QD dosing) and GNE-875 (BID dosing) were linked to more severe retinal damage than GMX-1778 (BID dosing). The purpose of mouse efficacy studies including GNE-617, GNE-618, and GMX-1778 is to evaluate the products' effectiveness as well as any appropriate retinal toxicity. NAMPTi retinal toxicity was noted with both GNE-617 and GMX-1778; however, a direct comparison of their retinal toxicity was not possible due to their dissimilar research lengths [2].
1. Retinal toxicity in C57BL/6 mice: - Drug administration: GNE-617 dissolved in 10% DMSO + 90% PEG400, oral gavage at 10 mg/kg/day or 30 mg/kg/day for 7 consecutive days; vehicle group received 10% DMSO + 90% PEG400[2] - NAD+ depletion: Retinal NAD+ levels in 10 mg/kg group were ~60% of vehicle control at day 7; 30 mg/kg group were ~25% of control[2] - Retinal function impairment: Electroretinography (ERG) showed 30 mg/kg group had ~40% reduction in a-wave amplitude (photoreceptor function) and ~35% reduction in b-wave amplitude (bipolar cell function) at day 7; 10 mg/kg group had no significant ERG changes[2] - Histological damage: 30 mg/kg group showed ~30% loss of outer nuclear layer (ONL, photoreceptor cell layer) thickness in the central retina at day 7; TUNEL staining revealed ~5-fold increase in apoptotic cells in ONL compared to vehicle[2] |
| Enzyme Assay |
1. Reagent preparation:
- Recombinant human NAMPT enzyme (purified from E. coli) resuspended in assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 2 mM DTT).
- Substrate mix: [¹⁴C]-nicotinamide (1 μCi/assay) + unlabeled nicotinamide (final concentration 10 μM) + 5-phosphoribosyl-1-pyrophosphate (PRPP, final concentration 200 μM) in assay buffer[2]
2. Assay setup: 50 μL reaction mixture contained 10 nM recombinant NAMPT, substrate mix, and serial concentrations of GNE-617 (0.01, 0.1, 1, 5, 10, 50 nM). Vehicle control contained 0.1% DMSO. Incubated at 37℃ for 60 minutes[2] 3. Product detection: - Reaction terminated by adding 100 μL ice-cold 5% trichloroacetic acid (TCA). Precipitated proteins removed by centrifugation (12,000×g, 10 minutes, 4℃). - Supernatant applied to ion-exchange chromatography columns to separate [¹⁴C]-NMN (product) from [¹⁴C]-nicotinamide (substrate). - Radioactivity of [¹⁴C]-NMN was counted using a liquid scintillation counter. - Inhibition rate = (1 - radioactivitydrug/radioactivityvehicle) × 100%. IC50 derived from nonlinear regression of concentration-inhibition curve[2] [2] |
| Cell Assay |
1. hRPE cell culture and toxicity assay:
- Cell culture: hRPE cells maintained in DMEM/F12 + 10% FBS at 37℃, 5% CO₂. Seeded in 96-well plates (5×10³ cells/well) for viability assay, 24-well plates (2×10⁵ cells/well) for TUNEL staining[2]
- Treatment: Cells incubated with GNE-617 (0.1-100 nM) for 24/48 hours; vehicle control (0.1% DMSO)[2] - Viability detection: MTT solution (5 mg/mL) added to 96-well plates (20 μL/well), incubated 4 hours at 37℃. Supernatant removed, 150 μL DMSO added to dissolve formazan. Absorbance measured at 570 nm[2] - Apoptosis detection: Cells fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, stained with TUNEL reagent for 60 minutes at 37℃. Apoptotic cells counted under fluorescence microscope (50 cells/field, 5 fields/well)[2] 2. Primary mouse retinal cell culture and NAD+ assay: - Retinal isolation: Retinas dissected from C57BL/6 mice (P7-P10), digested with papain for 15 minutes at 37℃, triturated into single-cell suspension[2] - Culture: Cells seeded in 24-well plates (1×10⁶ cells/well) in neurobasal medium + supplements, incubated with GNE-617 (10-50 nM) for 24 hours[2] - NAD+ detection: Cells lysed with 0.2 M NaOH, neutralized with 0.2 M HCl. NAD+ concentration measured via cyclic enzyme assay (based on NAD+-dependent conversion of resazurin to resorufin, fluorescence measured at 530 nm excitation/590 nm emission)[2] [2] |
| Animal Protocol |
30 mg/kg; oral
Rat 1. C57BL/6 mouse retinal toxicity study: - Animals: Male C57BL/6 mice (8-10 weeks old), acclimated for 7 days before experiment[2] - Drug preparation: GNE-617 dissolved in 10% DMSO + 90% PEG400 to concentrations of 1 mg/mL (10 mg/kg dose) and 3 mg/mL (30 mg/kg dose); volume administered 10 μL/g body weight[2] - Administration: Oral gavage once daily for 7 consecutive days; vehicle group received 10% DMSO + 90% PEG400 at the same volume[2] - Sample collection: Mice euthanized at day 7. Retinas dissected: one retina for NAD+ assay (lysed in 0.2 M NaOH), one retina for histology (fixed in 4% paraformaldehyde, paraffin-embedded)[2] - Functional assessment: ERG performed 1 hour before euthanasia (mice anesthetized with ketamine/xylazine, pupils dilated; ERG recorded under scotopic conditions with 0.1 cd·s/m² flash stimulus)[2] - Histology: Paraffin sections (5 μm) stained with H&E for ONL thickness measurement; TUNEL staining for apoptotic cells in ONL[2] [2] |
| Toxicity/Toxicokinetics |
1. Retinal toxicity: - Dose-dependent: 30 mg/kg/day (oral, 7 days) resulted in significant retinal NAD+ depletion, ERG dysfunction, and thinning of the outer nuclear layer (ONL); 10 mg/kg/day did not show significant retinal damage. [2] - Cell type specificity: Photoreceptor cells (ONL) were more sensitive to GNE-617 than other retinal layers (e.g., the inner nuclear layer). [2] 2. Systemic toxicity: Compared with the solvent group, there were no significant changes in body weight, food intake, or serum markers of liver function (ALT, AST) and kidney function (creatinine, BUN) in the 10/30 mg/kg groups. [2] 3. Plasma protein binding: Not reported. [2]
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| References |
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| Additional Infomation |
1. Mechanism of retinal toxicity: GNE-617 inhibits NAMPT, which is the rate-limiting enzyme in the NAD+ synthesis rescue pathway. Retinal cells (especially photoreceptor cells) have high metabolic demands and are highly dependent on NAD+ for energy production (oxidative phosphorylation) and cell signaling. NAD+ depletion leads to ATP depletion, oxidative stress, and activation of apoptotic pathways (such as caspase-3), ultimately resulting in retinal cell death and dysfunction. [2] 2. Clinical significance: The retinal toxicity of GNE-617 highlights the need for ocular safety monitoring in clinical trials of NAMPT inhibitors (especially those with high retinal penetration). The study also showed that targeting the NAD+ synthesis pathway may produce tissue-specific toxicity due to the different NAD+ requirements of different organs.[2] 3. Selectivity: GNE-617 has a much higher selectivity for NAMPT than other NAD+ synthases (NAPRT1, PRPP synthase) and irrelevant kinases (no inhibitory effect at 100 nM), confirming its specific mechanism of action.[2]
|
| Molecular Formula |
C₂₁H₁₅F₂N₃O₃S
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|---|---|---|
| Molecular Weight |
427.42
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| Exact Mass |
427.08
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| Elemental Analysis |
C, 59.01; H, 3.54; F, 8.89; N, 9.83; O, 11.23; S, 7.50
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| CAS # |
1362154-70-8
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| Related CAS # |
GNE-617 hydrochloride;2070014-99-0
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| PubChem CID |
68277611
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| Appearance |
White to off-white solid powder
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| LogP |
4.847
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
30
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| Complexity |
699
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O=C(NCC1=CC=C(S(C2=CC(F)=CC(F)=C2)(=O)=O)C=C1)C3=CN4C(C=C3)=NC=C4
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| InChi Key |
XRDVXQQZLHVEQZ-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C21H15F2N3O3S/c22-16-9-17(23)11-19(10-16)30(28,29)18-4-1-14(2-5-18)12-25-21(27)15-3-6-20-24-7-8-26(20)13-15/h1-11,13H,12H2,(H,25,27)
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| Chemical Name |
N-[[4-(3,5-difluorophenyl)sulfonylphenyl]methyl]imidazo[1,2-a]pyridine-6-carboxamide
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 1.67 mg/mL (3.91 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 1.67 mg/mL (3.91 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.3396 mL | 11.6981 mL | 23.3962 mL | |
| 5 mM | 0.4679 mL | 2.3396 mL | 4.6792 mL | |
| 10 mM | 0.2340 mL | 1.1698 mL | 2.3396 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
Response of cancer cell lines to GNE-617 in the presence or absence of nicotinic acid.Clin Cancer Res.2013 Dec 15;19(24):6912-23. |
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NAPRT1 level determines nicotinic rescue status in cancer cell lines.Clin Cancer Res.2013 Dec 15;19(24):6912-23. |
NAPRT1 immunohistochemistry correlates with nicotinic acid rescue status.Clin Cancer Res.2013 Dec 15;19(24):6912-23. |
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