Nicotinamide Phosphoribosyl Transferase (NAMPT): STF-118804 is a highly specific inhibitor of NAMPT, a rate-limiting enzyme in the nicotinamide adenine dinucleotide (NAD⁺) biosynthesis salvage pathway. For MV411 cells, the IC₅₀ of STF-118804 is not explicitly given as a single value, but it inhibits cell viability with nanomolar potency (e.g., 100 nM induces significant apoptosis and NAD⁺ reduction) [1]

In vitro activity: STF-118804 reduces the viability of B-ALL cell lines containing MLL chromosomal translations, with IC50 values in the low nanomolar range. In addition, leukemic samples from five pediatric ALL patients are also sensitive to STF-118804 in the low nanomolar range. STF-118804 induces leukemia MV411 cell apoptosis without antecedent cell cycle arrest.

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Kinase Assay: The enzymatic activities of NAMPT and NMNAT are measured using in vitro kits and using the Two-Step Method per manufacturer’s instructions. Compounds, NAMPT and/or NMNAT enzymes, and their substrates are mixed and incubated at 30°C for 1 hour. Reagents for the indicator reaction (Wst-1) are then added, and absorbance is read at 450 nm every 5 min at 30°C on a Tecan Infinite M100 multimode plate reader.

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Cell Assay: Human cell lines or lineage negative cord blood cells were seeded into 96-well plates (6×105 cells per milliliter). Compounds are added in increasing concentrations, and cells are incubated at 37°C/5% CO2 for 72 hours. To detect viability, CellTiter-Blue reagent is added at 1:10 dilution, and plates are incubated for 4 hours at 37°C/5% CO2 prior to reading on a Flexstation II 384 or a Synergy H1 reader at an excitation of 555 nm and emission detection of 590 nm. Cell viability is also measured by CellTiter-Fluor. The cell-permeable fluorogenic peptide substrate GF-AFC reagent is added at 1:2 dilution, and plates are incubated for 30 min at 37°C/5% CO2 prior to reading on a Synergy H1 reader at an excitation of 380 nm and emission detection of 505 nm. Cord blood cells are enumerated on a hemocytometer, and cell viability is assessed with trypan blue exclusion dye. Inhibitory concentration (IC50) is calculated using Prism software. Primary patient samples are plated in 96-well plates and treated with increasing concentrations of STF-118804 for 48 hours at 37°C in 5% CO2. WST-1 reagent is added to the culture medium (1:10 dilution), and absorbance is measured at 450 nm using a Bio-Rad model 680 microplate reader.

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Antileukemic Cell Viability Inhibition: STF-118804 exhibits nanomolar potency against human B-acute lymphoblastic leukemia (B-ALL) cell lines. In MV411 cells, treatment with STF-118804 for 72 hours reduces viability in a dose-dependent manner (measured by resazurin-based CellTiter-Blue and live cell protease-based CellTiter-Fluor assays), with significant inhibition observed at nanomolar concentrations. Structurally related analogs of STF-118804 show reduced or no activity, confirming structure-activity relationship (SAR) specificity. For example, analogs with modifications to key chemical moieties fail to inhibit MV411 cell viability as effectively as STF-118804 [1]
- Apoptosis Induction Without Cell Cycle Arrest: STF-118804 (100 nM) induces apoptosis in MV411 cells without prior cell cycle arrest. Flow cytometry analysis (annexin V-FITC/propidium iodide staining) shows a time-dependent increase in late apoptotic (double-positive) cells, with significant accumulation by 48–72 hours. Propidium iodide staining reveals a sub-G₀ population at 48 hours (indicating apoptotic cells) but no reduction in S/G₂/M phases (unlike idarubicin, which causes cell cycle arrest). Western blot analysis confirms cleaved PARP (89 kDa, a marker of apoptosis) in MV411 and SEM leukemia cells treated with 100 nM STF-118804 for 24–36 hours [1]
- NAMPT Target Validation: Knockdown of NAMPT via shRNA increases MV411 cell sensitivity to STF-118804. MV411 cells expressing NAMPT shRNAs (four distinct sequences) show lower IC₅₀ values for STF-118804 compared to cells with empty vector or Renilla luciferase shRNA (control). qPCR confirms reduced NAMPT transcript levels in shRNA-transduced cells. Conversely, overexpression of wild-type (wt) NAMPT in HEK293T cells rescues STF-118804-mediated viability inhibition, while mutant NAMPT (inactive) does not. This confirms NAMPT as the specific target of STF-118804 [1]
- NAD⁺ Biosynthesis Inhibition: STF-118804 specifically inhibits NAMPT-mediated NAD⁺ production in vitro. In a coupled enzyme assay (NAMPT + NMNAT), STF-118804 reduces NAD⁺ formation (indirectly measured via Wst-1 formazan absorbance at 450 nm) in a dose-dependent manner. It does not inhibit NMNAT (a downstream enzyme in NAD⁺ biosynthesis) or other unrelated enzymes (ADH, diaphorase) in the assay. Nicotinic acid (10 μM, which bypasses NAMPT via the Preiss-Handler pathway) rescues MV411 cell viability from STF-118804 treatment but not from idarubicin, confirming STF-118804 acts via NAD⁺ depletion [1]

STF-118804 (25 mg/kg twice daily, s.c.) improves survival in an orthotopic xenotransplant model of high-risk acute lymphoblastic leukemia, and effectively depletes leukemia-initiating cells

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Orthotopic Xenograft Model of High-Risk ALL: Sublethally irradiated (2.5 Gy) NOD scid gamma (NSG) mice were transplanted with MV411 cells (5×10⁶) expressing firefly luciferase. Two weeks post-transplant, mice were randomized to receive subcutaneous (s.c.) STF-118804 (50 mg/kg split dose, n=7) or vehicle (n=5) for 20 days (Days 14–34). STF-118804 significantly extended survival: treated mice survived an average of 34 days longer than vehicle controls (P<0.001, log-rank test). Bioluminescent imaging showed no detectable tumor in STF-118804-treated mice by Day 36, and disease suppression persisted for at least 18 days after treatment cessation. Some mice remained relapse-free [1]
- Leukemia Stem Cell (LSC) Targeting: At Day 35, limit-dilution secondary transplantation showed a 8.3-fold reduction in LSC frequency in STF-118804-treated mice (1/11,744, 95% CI: 1/4,480–1/30,784) compared to vehicle controls (1/1,411, 95% CI: 1/600–1/3,320). Bone marrow from STF-118804-treated mice also had significantly fewer leukemia colony-forming cells (P=0.03, Student’s t-test) when plated in human methocult (which does not support murine progenitors) [1]

Coupled NAMPT-NMNAT Enzyme Assay for NAD⁺ Production: The assay uses NAMPT (catalyzes nicotinamide → nicotinamide mononucleotide, NMN) and NMNAT (catalyzes NMN + ATP → NAD⁺) in a reaction mixture. STF-118804 (at varying concentrations) is added to the mixture, which is incubated to allow NAD⁺ synthesis. NAD⁺ is indirectly detected via alcohol dehydrogenase (ADH)-mediated conversion of NAD⁺ to NADH, which reduces Wst-1 to formazan (measured by absorbance at 450 nm). The reduction in absorbance relative to vehicle controls indicates NAMPT inhibition by STF-118804. To confirm specificity, the assay is repeated with NMNAT alone (using NMN as substrate) to show STF-118804 does not inhibit NMNAT [1]
- shRNA-Mediated Target Validation Assay: An ultracomplex shRNA lentiviral library is transduced into wild-type MV411 cells. Transduced cells undergo four rounds of STF-118804 treatment or passage (untreated control). High-throughput sequencing quantifies shRNA frequency in treated vs. untreated cells. NAMPT shRNAs are significantly depleted in STF-118804-treated cells (vs. negative control shRNAs), indicating NAMPT knockdown sensitizes cells to STF-118804. Mann-Whitney U tests confirm statistical significance of NAMPT shRNA depletion across two independent replicates [1]

Leukemia Cell Viability Assay: Human B-ALL cell lines (MV411, SEM, etc.) are seeded in 96-well plates and treated with STF-118804 (0–1000 nM) or analogs for 72 hours. Cell viability is measured using two methods: (1) CellTiter-Blue (resazurin reduction to fluorescent resorufin, read via fluorometry) and (2) CellTiter-Fluor (live cell protease activity, read via fluorometry). Data from three independent triplicate experiments are used to generate dose-response curves and calculate IC₅₀ values. For idarubicin (positive control), viability is measured similarly at 25 nM [1]
- Apoptosis and Cell Cycle Analysis: MV411 cells are treated with 100 nM STF-118804 or 25 nM idarubicin for 0–72 hours. For apoptosis: cells are stained with annexin V-FITC (binds phosphatidylserine) and propidium iodide (labels dead cells), then analyzed by flow cytometry to quantify early (annexin V⁺/PI⁻) and late (annexin V⁺/PI⁺) apoptotic cells. For cell cycle: cells are fixed, permeabilized, stained with propidium iodide (binds DNA), and analyzed by flow cytometry to determine G₀/G₁, S, G₂/M, and sub-G₀ (apoptotic) populations [1]
- Western Blot for Apoptosis Markers: MV411 or SEM cells are treated with 100 nM STF-118804 (24–36 hours) or 25 nM idarubicin (24 hours). Cells are lysed, and proteins are separated by SDS-PAGE. Blots are probed with antibodies against cleaved PARP (89 kDa, apoptosis marker) and a loading control (e.g., GAPDH). Signals are detected via chemiluminescence, and bands are quantified to confirm apoptotic induction [1]
- NAMPT Rescue Assay: HEK293T cells are transfected with empty vector, wt NAMPT, or mutant NAMPT constructs. Transfected cells are treated with STF-118804 (0–1000 nM) for 72 hours. Cell viability is measured via CellTiter-Blue or CellTiter-Fluor. Rescue of viability in wt NAMPT-transfected cells (vs. empty vector or mutant NAMPT) confirms NAMPT as the target of STF-118804 [1]

20% [w/v] [2-hydroxypropyl]-γ-cyclodextrin/5% [v/v] DMSO;25 mg/kg twice daily; s.c.
Orthotopic xenograft model of ALL transplanted with MV411 cells

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Orthotopic Xenograft of MV411 ALL Cells: NSG mice are sublethally irradiated with 2.5 Gy to suppress immune function. One day later, mice are intravenously injected with 5×10⁶ MV411 cells stably expressing firefly luciferase (to enable bioluminescent imaging). Two weeks post-transplant, mice are randomized into two groups based on baseline bioluminescence (to ensure balanced tumor burden): (1) Vehicle group (n=5): subcutaneous injection of vehicle (formulation not specified) for 20 days (Days 14–34); (2) STF-118804 group (n=7): subcutaneous injection of 50 mg/kg STF-118804 (split dose, frequency not specified) for 20 days. Bioluminescent imaging is performed weekly to monitor tumor growth. Mice are euthanized when they show signs of disease (e.g., weight loss, lethargy), and survival is recorded. For secondary transplantation: bone marrow from primary treated mice (Day 35) is harvested, and limiting dilutions (1×10³–1×10⁶ cells) are injected into new NSG mice. LSC frequency is calculated via Poisson statistics based on tumor engraftment [1]
- Leukemia Colony-Forming Assay: At Day 35 post-primary transplant, bone marrow is collected from STF-118804-treated (n=8) and vehicle-treated (n=6) mice. Whole bone marrow is plated in human methocult (without cytokines, to exclude murine progenitor growth) and incubated for 14 days. Colonies are counted, and the number of leukemia colony-forming cells per mouse is compared between groups using Student’s t-test [1]

[1]. Next-generation NAMPT inhibitors identified by sequential high-throughput phenotypic chemical and functional genomic screens. Chem Biol. 2013 Nov 21;20(11):1352-63.

STF-118804 was discovered through a series of high-throughput phenotypic chemical screenings and ultra-complex shRNA screenings of small molecule libraries (to validate the target). Phenotypic screening identified STF-118804 as a cytotoxic compound against ALL cells, while shRNA screening confirmed that NAMPT was its target of action[1]
- NAMPT is a key enzyme in the NAD⁺ rescue pathway, which is essential for NAD⁺-dependent biochemical processes such as energy metabolism and DNA repair. STF-118804 induces apoptosis in ALL cells by inhibiting NAMPT and depleting intracellular NAD⁺, particularly targeting leukemia stem cells, which are often resistant to conventional chemotherapy[1]. STF-118804 is highly specific for NAMPT: it does not inhibit other enzymes (such as NMNAT) or irrelevant enzymes (ADH, dihydroflavinase) in NAD⁺ biosynthesis, and its cytotoxicity can be reversed by nicotinic acid (which bypasses NAMPT). This specificity makes it an important tool for studying NAMPT-dependent pathways in cancer [1].

C25H23N3O4S

461.53

461.14

894187-61-2

20916937

White to light yellow solid powder

1.3±0.1 g/cm3

1.609

3.08

1

6

7

33

739

0

DLFCEZOMHBPDGI-UHFFFAOYSA-N

InChI=1S/C25H23N3O4S/c1-17-5-11-22(12-6-17)33(30,31)16-23-18(2)32-25(28-23)21-9-7-20(8-10-21)24(29)27-15-19-4-3-13-26-14-19/h3-14H,15-16H2,1-2H3,(H,27,29)

4-[5-methyl-4-[(4-methylphenyl)sulfonylmethyl]-1,3-oxazol-2-yl]-N-(pyridin-3-ylmethyl)benzamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)