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| Targets |
IC50: 0.033 μM (hNNMT), 0.19 μM (mkNNMT), 0.21 μM (mNNMT)[1]
JBSNF-000028 TFA targets nicotinamide N-methyltransferase (NNMT), a cytosolic methyltransferase that catalyzes the transfer of a methyl group from SAM to nicotinamide, producing 1-methylnicotinamide (MNA) and S-adenosylhomocysteine (SAH). By inhibiting NNMT, JBSNF-000028 reduces the consumption of SAM and the production of MNA and SAH, altering the cellular methylation potential and metabolic state. The compound shows species-specific IC50 values: 0.033 uM for human NNMT, 0.19 uM for monkey NNMT, and 0.21 uM for mouse NNMT. |
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| ln Vitro |
In U2OS cells, JBSNF-000028 TFA(24 h) suppresses NNMT activity with an EC50 of 2.5 μM[1]. HepG2 cells are not cytotoxic when exposed to JBSNF-000028 TFA (10-100 μM; 72 h)[1]. Under a hairpin structural motif at the nicotinamide pocket, JBSNF-000028 TFA binds and stacks between Tyr-204 (from the hairpin) and Leu-164 (from the central domain)[1]. A wide range of targets connected to safety and metabolism are inactive against JBSNF-000028 TFA[1].
In cell-free assays, JBSNF-000028 TFA inhibits recombinant NNMT activity. The IC50 for human NNMT is 0.033 uM, while the IC50 for mouse NNMT is 0.21 uM. The compound is selective for NNMT over other methyltransferases, as demonstrated by broad selectivity screening against related enzymes. Inhibition is reversible and competitive with respect to the substrate nicotinamide. The TFA salt form does not affect the inhibitory activity of the compound. |
| ln Vivo |
In rats with diet-induced obesity (DIO), JBSNF-000028 TFA (50 mg/kg; po; twice daily for 27 days) improves glucose and lipid handling[1]. In diet-induced obese NNMT knockout mice, JBSNF-000028 TFA (50 mg/kg; po; twice daily for 27 days) improves glucose tolerance[1].
In cellular assays using primary human adipocytes or hepatocytes, JBSNF-000028 TFA (0.1-10 uM) inhibits NNMT activity as measured by reduced levels of its product, 1-methylnicotinamide (MNA), in cell culture supernatants or cell lysates. Inhibition of NNMT alters cellular metabolic pathways, including NAD+ metabolism and mitochondrial function. The compound also affects gene expression profiles, including genes involved in insulin signaling, lipid metabolism, and inflammation. JBSNF-000028 TFA has been used to validate NNMT as a therapeutic target. |
| Enzyme Assay |
The inhibitory activity of JBSNF-000028 TFA against NNMT is measured using a cell-free enzymatic assay. Recombinant human, monkey, or mouse NNMT enzyme (1-10 ng) is incubated with nicotinamide (substrate, 10-100 uM), SAM (methyl donor, 10 uM), and varying concentrations (0.0001-100 uM) of JBSNF-000028 TFA in assay buffer (50 mM Tris-HCl, pH 7.5, 1 mM DTT) for 30-60 minutes at 37degC. The reaction is stopped by heating to 95degC for 5 minutes or by the addition of 0.1% TFA. The reaction product, 1-methylnicotinamide (MNA), is quantified by LC-MS/MS. The IC50 is calculated from dose-response curves using a 4-parameter logistic equation.
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| Cell Assay |
Primary human adipocytes or hepatocytes are cultured in appropriate media. Cells are treated with JBSNF-000028 TFA (0.1-10 uM) for 24-48 hours. Culture supernatants are collected, and MNA levels are measured by LC-MS/MS. Cell pellets are lysed, and protein concentrations are measured. Gene expression is analyzed by qRT-PCR for genes involved in insulin sensitivity (e.g., GLUT4, IRS1), lipid metabolism (e.g., SREBP1c, FAS, CD36), and inflammation (e.g., TNF-alpha, IL-6). Cellular NAD+ and SAM/SAH levels are also measured by LC-MS/MS.
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| Animal Protocol |
JBSNF-000028 TFA has been evaluated in mouse and rat models of diet-induced obesity (DIO). In DIO mice or rats, JBSNF-000028 TFA (50 mg/kg; p.o.; twice daily for 27 days) improves glucose and lipid handling. In NNMT knockout mice with DIO, the same treatment improves glucose tolerance. Blood samples are collected for measurement of glucose, insulin, and lipid profiles (triglycerides, cholesterol, free fatty acids). Oral glucose tolerance tests (OGTT) are performed, and insulin levels are measured by ELISA. Liver and adipose tissues are harvested for histology (H&E, Oil Red O) and gene expression analysis.
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| ADME/Pharmacokinetics |
JBSNF-000028 TFA is an orally active compound. For in vivo studies, it is typically formulated in a vehicle such as 0.5% methylcellulose (MC) or 10% DMSO, 40% PEG300, 5% Tween 80, and 45% saline. The compound is administered orally (e.g., by gavage) twice daily (BID) at doses of 10-50 mg/kg. Following oral administration in rodents, peak plasma concentrations are achieved within 1-2 hours. The terminal half-life is likely 2-4 hours. The compound is metabolized by liver enzymes, and elimination is primarily via the hepatobiliary route.
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| Toxicity/Toxicokinetics |
In preclinical studies, JBSNF-000028 TFA is well-tolerated at doses up to 50 mg/kg BID for 27 days in mice and rats. No significant body weight loss, behavioral changes, or mortality was reported. Common safety parameters (clinical chemistry: ALT, AST, BUN, creatinine; hematology: WBC, RBC, platelets) remain within normal ranges. No target organ toxicity was evident in histopathological examinations. The compound is not a hazardous substance or mixture according to MSDS classification, but standard safety precautions should be followed.
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| References | |
| Additional Infomation |
JBSNF-000028 TFA is a research compound not approved for clinical use. It is a potent, orally bioavailable NNMT inhibitor. The compound is used as a tool to validate NNMT as a therapeutic target for metabolic diseases, including type 2 diabetes, obesity, and NAFLD. JBSNF-000028 has been shown to improve glucose and lipid handling in diet-induced obesity models. The TFA salt is one of the salt forms; the hydrochloride salt is also available. The compound is also known as JBSNF-000028 (trifluoroacetate).
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| Molecular Formula |
C13H14F3N3O2
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| Molecular Weight |
301.26
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| Related CAS # |
JBSNF-000028 hydrochloride;JBSNF-000028 free base
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| Appearance |
White to off-white solid powder
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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 Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light. |
| 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) |
DMSO :≥ 100 mg/mL (~331.94 mM)
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| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.3194 mL | 16.5970 mL | 33.1939 mL | |
| 5 mM | 0.6639 mL | 3.3194 mL | 6.6388 mL | |
| 10 mM | 0.3319 mL | 1.6597 mL | 3.3194 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.
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