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| Targets |
IC50: 0.033 μM (hNNMT), 0.19 μM (mkNNMT), 0.21 μM (mNNMT)[1]
JBSNF-000028 hydrochloride targets nicotinamide N-methyltransferase (NNMT), a cytosolic methyltransferase that uses S-adenosylmethionine (SAM) to methylate nicotinamide, producing 1-methylnicotinamide (MNA). By inhibiting NNMT, JBSNF-000028 reduces the consumption of SAM and the production of MNA and SAH, thereby modulating cellular methylation potential and metabolic pathways, including NAD+ metabolism, insulin sensitivity, and lipid homeostasis. Species-specific IC50 values are: 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 hydrochloride for 24 hours suppresses NNMT activity with an EC50 of 2.5 μM[1]. HepG2 cells are not susceptible to JBSNF-000028 hydrochloride (10–100 μM; 72 h) cytotoxicity[1]. At the nicotinamide pocket, JBSNF-000028 hydrochloride binds beneath a hairpin structural motif and stacks between Tyr-204 (from the hairpin) and Leu-164 (from the central domain)[1]. Hydrochloride of JBSNF-000028 is inert against a wide range of targets associated with safety and metabolism[1].
In cell-free assays, JBSNF-000028 hydrochloride inhibits recombinant NNMT with high potency: IC50 = 0.033 uM for human NNMT (hNNMT), 0.19 uM for monkey NNMT (mkNNMT), and 0.21 uM for mouse NNMT (mNNMT). The compound shows selectivity for NNMT over other methyltransferases and metabolizing enzymes. The inhibition is reversible and competitive with respect to the nicotinamide substrate. The hydrochloride salt is stable and improves solubility compared to the free base. |
| ln Vivo |
In mice suffering from diet-induced obesity (DIO), JBSNF-000028 hydrochloride (50 mg/kg; po; twice daily for 27 days) enhances the management of glucose and lipids[1]. NNMT knockout mice with diet-induced obesity respond better to JBSNF-000028 hydrochloride (50 mg/kg; po; twice daily for 27 days) daily for 4 weeks.
In cell-based assays using primary human adipocytes or hepatocytes, JBSNF-000028 hydrochloride (0.1-10 uM) reduces the production of 1-methylnicotinamide (MNA), the product of NNMT catalysis, which can be measured in cell culture supernatants or lysates by LC-MS/MS. Inhibition of NNMT alters gene expression profiles, including upregulation of genes involved in insulin sensitivity (e.g., GLUT4, IRS1) and downregulation of lipogenic and inflammatory genes (e.g., FAS, SREBP1c, TNF-alpha). The compound also increases cellular NAD+ levels. |
| Enzyme Assay |
JBSNF-000028 hydrochloride is evaluated in a cell-free NNMT enzyme inhibition assay as described for the TFA salt. Recombinant human, monkey, or mouse NNMT is incubated with nicotinamide (10-100 uM), SAM (10 uM), and varying concentrations (0.0001-100 uM) of JBSNF-000028 hydrochloride in assay buffer (50 mM Tris-HCl, pH 7.5, 1 mM DTT) at 37degC for 30-60 minutes. The reaction is stopped by heating or acidification, and MNA is quantified by LC-MS/MS. The IC50 is calculated from dose-response curves using nonlinear regression analysis.
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| Cell Assay |
Primary human adipocytes are differentiated from preadipocytes and maintained in adipocyte differentiation medium for 7-14 days. Cells are treated with JBSNF-000028 hydrochloride (0.1-10 uM) for 24-48 hours. Culture supernatants are collected for MNA measurement by LC-MS/MS. Cells are lysed for RNA extraction, and gene expression is analyzed by qRT-PCR. To assess insulin sensitivity, cells are treated with insulin (100 nM) for 10-15 minutes, and AKT phosphorylation (p-AKT) is measured by Western blot. Cellular NAD+ levels are measured using a NAD/NADH assay kit.
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| Animal Protocol |
JBSNF-000028 hydrochloride is evaluated in mouse models of diet-induced obesity (DIO). Male C57BL/6J mice are fed a high-fat diet (60% kcal from fat) for 12-16 weeks to induce obesity and insulin resistance. Mice are dosed orally with JBSNF-000028 hydrochloride (50 mg/kg; twice daily) for 27 days. Glucose tolerance is assessed by oral glucose tolerance test (OGTT; 2 g/kg glucose). Blood samples are collected at 0, 15, 30, 60, and 120 minutes for glucose measurement. Insulin levels at t=0 and t=30 minutes are measured by ELISA. Liver and adipose tissue are harvested for histology (H&E, Oil Red O) and gene expression analysis (qRT-PCR).
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| ADME/Pharmacokinetics |
JBSNF-000028 hydrochloride is an orally active compound. For in vivo studies, it is typically formulated in 0.5% methylcellulose (MC) or 10% DMSO/40% PEG300/5% Tween 80/45% saline. The compound is administered via oral gavage at doses of 10-50 mg/kg, twice daily (BID). Following oral administration in rodents, peak plasma concentrations (Cmax) are achieved within 1-2 hours (Tmax). The terminal half-life is likely 2-4 hours. The compound is metabolized by liver enzymes, and elimination is primarily via the hepatobiliary route. The hydrochloride salt improves aqueous solubility for in vivo formulation.
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| Toxicity/Toxicokinetics |
In preclinical studies, JBSNF-000028 hydrochloride is well-tolerated at doses up to 50 mg/kg BID for 4 weeks in mice. No significant body weight loss, behavioral changes, or mortality were observed. Clinical chemistry parameters (ALT, AST, BUN, creatinine) remained within normal ranges. The compound is not classified as a hazardous substance or mixture according to MSDS. Standard laboratory safety precautions (gloves, lab coat, safety glasses) should be followed when handling the powder. Not for human use.
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| References |
[1]. Ruf S, et al. Novel tricyclic small molecule inhibitors of Nicotinamide N-methyltransferase for the treatment of metabolic disorders. Sci Rep. 2022 Sep 14;12(1):15440.
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| Additional Infomation |
JBSNF-000028 hydrochloride is a research compound not approved for clinical use. It is a potent, orally bioavailable NNMT inhibitor that has been shown to improve glucose and lipid handling in diet-induced obesity models. NNMT is a metabolic enzyme whose expression is upregulated in obesity and type 2 diabetes. JBSNF-000028 represents a promising lead for the development of therapeutics targeting metabolic diseases, including type 2 diabetes, obesity, and nonalcoholic fatty liver disease (NAFLD). The hydrochloride salt is stable and suitable for in vivo studies.
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| Molecular Formula |
C11H14CLN3
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| Molecular Weight |
223.70
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| Related CAS # |
JBSNF-000028 TFA;JBSNF-000028 free base
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| Appearance |
Off-white to light yellow 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 :~16.67 mg/mL (~74.52 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 | 4.4703 mL | 22.3514 mL | 44.7027 mL | |
| 5 mM | 0.8941 mL | 4.4703 mL | 8.9405 mL | |
| 10 mM | 0.4470 mL | 2.2351 mL | 4.4703 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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