human NNMT (IC50 = 1.8 µM); monkey NNMT (IC50 = 2.8 µM); mouse NNMT (IC50 = 5.0 µM)

JBSNF-000088 (6-carboxamide nicotinamide) has IC50 values of 1.6 μM and 6.3 μM against U2OS or secreted 3T3L1 cells, respectively [1].

JBSNF-000088 (6-carboxamide nicotinamide) (50 mg/kg; 4 weeks powder efficacy) demonstrated statistically significant % weight loss at day 21 and resulted in significant reductions in diabetic blood glucose [1] JBSNF -000088 (50 mg/kg; borderline gavage; twice daily for 4 weeks) resulted in significant improvements in endpoint tolerance and normalization of endpoint tolerance on day 28 [1]. JBSNF-000088 (1 mg/kg; intravenous topology; duration 4 hours) resulted in a low weave clearance of 21 mL/min·kg and 0.7 L/kg over three repeated cycles, with a very short post-intravenous half-life (0.5 )[1 JBSNF-000088 (10 mg/kg; intragastric gavage; 4 hours duration) resulted in a Cmax of 3568 ng/mL and a Tmax value of 0.5 hours, indicating rapid intraluminal absorption and destruction, with a half-life of 0.4 hours by intragastric gavage .

Fluorescence based NNMT enzymatic assay[1]
NNMT activity was measured with the fluorescence enzymatic assay. MNA formed in the NNMT reaction reacts with acetophenone in the presence of KOH and formic acid and forms a fluorescent product 2, 7- naphthyridine. JBSNF-000088 was screened using human, mouse and monkey NNMT enzymes. Different concentrations of inhibitors were preincubated along with the enzyme for 30 minutes at room temperature. Reaction was initiated by addition of SAM and nicotinamide mixture (7 µM, 20 µM, 8 µM SAM and 6 µM, 20 µM, 9 µM nicotinamide for human, mouse and monkey NNMT assays respectively) for 60 minutes at 37 °C. The final assay reaction mixture contained a buffer of 100 mM Tris Hcl pH 7.5, 0.04% BSA, 2 mM dithiothreitol and 1% DMSO. At the end of the incubation, the reaction was stopped by the addition of ethanol: acetophenone mix (75% ethanol: 25% acetophenone) and 5 M potassium hydroxide prepared with 50% ethanol. The reaction was incubated for 15 minutes following which 100 µL of 60% formic acid was added. The reaction was incubated for another 60 minutes at room temperature. The fluorescent product 2, 7- naphthyridine was measured using a Tecan reader with excitation at 375 nm and emission at 430 nm. The IC50 values were determined by fitting the inhibition curves (percent inhibition versus inhibitor concentration) using a four parametric sigmoidal dose response using GraphPad Prism software.

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Human NNMT enzymatic assay using LC-MS/MS detection[1]
MNA formed in the human NNMT reaction was measured using a fit-for-purpose LC-MS/MS method. Different concentrations of the inhibitors were preincubated along with 5ng/well human NNMT enzyme for 30 minutes at room temperature. Reaction was initiated by addition of SAM and nicotinamide mixture at 7 µM, 20 µM respectively and incubated for 60 minutes at 37 °C. The final assay reaction mixture contained a buffer of 100 mM Tris Hcl pH 7.5, 0.04% BSA, 2 mM dithiothreitol and 1% DMSO. 100 µL of acetonitrile containing internal standard d4-MNA (20ng/mL) was added to the wells and incubated for 10 minutes at room temperature. 70 µL of autoclaved water was added to the wells and mixed gently. The plate was centrifuged at 5000 g for 10 minutes at room temperature. 150 µL of supernatant was transferred into 96 well plate and analyzed by LC-MS/MS. The IC50 values were determined by fitting the inhibition curves (percent inhibition versus inhibitor concentration) using a four parametric sigmoidal dose response graph pad prism.

Cell Based U2OS Assay[1]
Human bone osteosarcoma (U2OS) cell line was procured from ATCC and maintained in DMEM F-12 growth media containing 10% heat inactivated fetal bovine serum and penstrep (filter sterilized) at 37 °C with 5% CO2. Cells were counted and 10 K cells per well were seeded into a 96 well cell culture plate followed by incubation for 24 h at 37 °C, 5% CO2, and 95% humidity. Cell culture media was replaced with 100 µl of medium/ inhibitor mixture at different concentrations and incubated for 24 hours at 37 °C, 5% CO2, and 95% humidity. Medium/compound mixture was removed and washed twice followed by addition of 100 µL Acetonitrile containing internal standard d4-MNA (20ng/mL) to the wells. The plate was centrifuged at 5000 g for 10 minutes. 150 µL of supernatant was transferred into 96 well plate (Costar 3364) and analyzed by LC-MS/MS. The IC50 values were determined by fitting the inhibition curves (percent inhibition versus inhibitor concentration) using a four parametric sigmoidal dose response graph pad prism.

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Cell Based 3T3L1 Assay[1]
3T3-L1, a cell line derived from mouse 3T3 cells was procured from ATCC and maintained in DMEM high glucose growth media containing 10% heat inactivated fetal bovine serum and penstrep at 37 °C with 5% CO2. Cells were counted and 5 K cells per well were seeded into a 96 well cell culture plate and incubated at 37 °C, 5% CO2, 95% humidity until it reached 100% confluency. Post 24 h of confluency the medium was replaced with differentiation induction media containing (500 μM, IBMX + 1 μM DEXA + 1 μg/ml insulin) and the medium was changed on alternate days. Cells were differentiated for up to 14 days: After three days, the induction medium was replaced by DMEM supplemented with 10% FBS and 1 µg/ml insulin. From day 5, cells were cultured in regular growth medium.
Post differentiation, cells were incubated with compound for 24 h at 37 °C, 5% CO2, and 95% humidity. Cells were washed with DPBS and 100 µl acetonitrile containing internal standard d4-MNA (20ng/mL final concentration) was added to the wells. 100% Acetonitrile was used to extract MNA from the cells. The plate was incubated for 20 minutes at room temperature and 100 µL of autoclaved water was added and mixed gently. The plate was centrifuged at 5000 g for 10 minutes. Supernatant was transferred into 96 well plate (Costar 3364) and submitted for LCMS/MS. The IC50 values were determined by fitting the inhibition curves (percent inhibition versus inhibitor concentration) using a four parametric sigmoidal dose response graph pad prism.

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Cytotoxicity assay[1]
Human liver cancer (HepG2) cell line was procured from ATCC and maintained in DMEM Glut Max growth media containing 10% HI FBS and 1% Pen-Strep (filter sterilized) in 37 °C incubator with 5% CO2. Cells were detached with 0.25% trypsin when they were 80% confluent in a cell culture flask. Cells were counted and 50 K cells per well were seeded into a 96 well opaque-walled multi well plate followed by Incubation overnight at 37 °C, 5% CO2, 95% humidity. Cell culture media was replaced with 100 µL of medium/ inhibitor mixture (Containing 0.5% DMSO) along with controls and incubated for 48 or 72 h at 37 °C, 5% CO2, 95% humidity. Max control samples (complete reaction with 0.5% DMSO) and min control samples (complete reaction with known inhibitor) Cell Titer-Glo® Reagent was added to all the wells containing media in 1:1 ratio (e.g., add 100 µL of reagent to 100 µL of medium containing cells for a 96-well plate). The contents were mixed for 2 minutes on an orbital shaker to induce cell lysis. Then the plate was incubated at room temperature for 10 minutes to stabilize luminescent signal. Luminescence was recorded in Victor or Top Count Luminescence counter. The cell viability over DMSO control was calculated.

Animal/Disease Models: High-fat diet (HFD)-induced obese mice [1]
Doses: 50 mg/kg
Route of Administration: Oral route for 4 weeks; the blocking bioavailability was found to be approximately 40% [1]. po (oral gavage) administration twice (two times) daily for four weeks
Experimental Results: Demonstrated significant weight loss (%) and resulted in a significant reduction in postprandial blood glucose by the oral route on day 21. On day 28, there was a statistically significant improvement in oral glucose tolerance, which was normalized by po (oral gavage).

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Animal/Disease Models: C57BL/6 mice[1]
Doses: 1 mg/kg (intravenous (iv) (iv)administration); 10 mg/kg (po (oral gavage)) (pharmacokinetic/PK/PK study)
Route of Administration: intravenous (iv) (iv)administration and po (oral gavage) ; 4 hour
Experimental Results: resulting in a low plasma clearance of 21 mL/min·kg, a steady-state volume of distribution of 0.7 L/kg, and a very short plasma half-life of 0.5 hrs (hrs (hours)) after intravenous (iv) (iv)injection. The results demonstrated that the Cmax was 3568 ng/mL, and the Tmax value was 0.5 hrs (hrs (hours)), indicating ra

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Efficacy studies[1]
Lean control + Vehicle and HFD + Vehicle Control groups (G1 and G2) mice were administered with vehicle. Dose formulations of HFD + JBSNF-000088, 50 mg kg−1, po, bid were administered at dose volume of 5 mL kg−1 body weight to G3 mice. Similar pattern was followed for ob/ob mice and db/db mice studies. The dose volume for individual animals was calculated based on the most recently recorded body weight during the study period. Throughout the study period, all animals were observed for mortality/morbidity. Cage side observations of animals for visible clinical signs, was carried out once daily throughout the study period. Individual animal body weights were recorded twice weekly during the study period.

The ob/ob mice were 12 weeks of age at the start of the study and db/db mice were 8 weeks of age at the start of the study. DIO mice were 20 weeks of age at the start of the study. In each study, non-diabetic lean mice comprised the control group which received vehicle (0.5% w/v HEC and 0.5% v/v Tween 80) and obese or diabetic mice were randomly assigned to two groups based on body weight and unfasted glucose which received either vehicle (0.5% w/v HEC and 0.5% v/v Tween 80) or JBSNF-000088, 50 mg kg−1, po, bid for 30 days. Individual animal body weights, food and water consumption were recorded twice weekly during the study period. Each group consisted of 10 animals in DIO, ob/ob and db/db efficacy studies. Animals were housed as n = 5 per cage and individual animal body weight, food and water consumption were recorded twice weekly for the duration of the study. Food consumption was expressed as cumulative energy intake. Fed blood glucose and insulin were measured on day 7, 14 and day 21 post treatment.

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OGTT[1]
Effect of JBSNF-000088 on glucose tolerance was assessed in an oral glucose tolerance test (OGTT) on day 28 of treatment, in DIO mice, ob/ob mice and on day 26 of treatment for db/db mice. Animals from DIO and ob/ob study were fasted for 4 h followed by an oral administration of glucose (2 g kg−1) while animals from db/db study were fasted for 16 h followed by an oral administration of glucose (1 g kg−1). One hour prior to glucose administration, mice were dosed orally with vehicle or JBSNF-000088, 50 mg kg−1.

Blood glucose measurements from tail snips were performed at −60 (prior to drug administration), 0 (prior to glucose administration), and 15, 30, 60, 120 and 180 minutes after glucose administration. Blood for plasma insulin measurement was collected at 0 and 15 minutes. HOMA-IR was calculated according to the formula; [HOMA-IR = (Fasting plasma insulin, ngml−1 × Fasting Blood Glucose, mmol l−1) / 22.5]. Animals were re-fed after the last time point of blood glucose and dosing was continued until termination on day 30.

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Study termination[1]
On day 30 of treatment, animals were fasted for 4 h and sacrificed by CO2 asphyxiation. One h prior to sacrifice, mice were dosed orally with vehicle or JBSNF-000088, 50 mg kg−1. Blood and tissue samples (liver, subcutaneous fat, renal fat, epididymal fat and mesentery fat) were collected from each animal. Plasma and tissue samples were stored at −80 °C until analysis.

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DIO studies using WT and NNMT KO animals[1]
NNMT fl/fl mice on a C57BL/6 background were generated using VelociGene® technology31. A loxP locus was placed 1.6 kb upstream of the transcription starting site encompassing the gene promoter. A loxP-Frt-Hyg-Frt cassette was inserted 212 bp downstream of NNMT exon 1. The floxed coordinates were chr9:48,412,881–48,414,698. NNMT fl/fl mice were crossed with ZP3-cre mice (C57BL/6, Jackson Laboratories) to generate whole-body NNMT knockout mice. The DIO mouse study was conducted in accordance to the German Animal Protection Law, as well as according to international animal welfare legislation and rules. Female wild-type and NNMT knockout animals were put on high-fat diet (Ssniff HFD adjusted TD.97366) for 18 weeks and then treated with either
(50 mg/kg bid by oral gavage) or vehicle for four weeks (n = 9–10 per group). Throughout the study, Mice were housed in an environmentally controlled room at 23 °C on a 12 h:12 h light dark cycle (light on at 06:00 AM), and food and water was offered ad libitum. An oral glucose tolerance test was performed on day 25 of treatment: Animals were fasted overnight. 60 minutes before an oral glucose bolus (2 g kg−1), JBSNF-000088 (50 mg kg−1) or vehicle (0.5% HEC + 0.5% Tween80) was administered by oral gavage. For blood glucose analysis, blood was collected from the tail of conscious mice at time points 15, 30, 60 and 120 minutes after the glucose bolus.

[1]. A small molecule inhibitor of Nicotinamide N-methyltransferase for the treatment of metabolic disorders. Sci Rep. 2018 Feb 26;8(1):3660.

Nicotinamide N-methyltransferase (NNMT) is a cytosolic enzyme that catalyzes the transfer of a methyl group from the co-factor S-adenosyl-L-methionine (SAM) onto the substrate, nicotinamide (NA) to form 1-methyl-nicotinamide (MNA). Higher NNMT expression and MNA concentrations have been associated with obesity and type-2 diabetes. Here we report a small molecule analog of NA, JBSNF-000088, that inhibits NNMT activity, reduces MNA levels and drives insulin sensitization, glucose modulation and body weight reduction in animal models of metabolic disease. In mice with high fat diet (HFD)-induced obesity, JBSNF-000088 treatment caused a reduction in body weight, improved insulin sensitivity and normalized glucose tolerance to the level of lean control mice. These effects were not seen in NNMT knockout mice on HFD, confirming specificity of JBSNF-000088. The compound also improved glucose handling in ob/ob and db/db mice albeit to a lesser extent and in the absence of weight loss. Co-crystal structure analysis revealed the presence of the N-methylated product of JBSNF-000088 bound to the NNMT protein. The N-methylated product was also detected in the plasma of mice treated with JBSNF-000088. Hence, JBSNF-000088 may act as a slow-turnover substrate analog, driving the observed metabolic benefits.[1]

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Our attempts to determine the binding mode of JBSNF-000088 using mouse and human NNMT proteins by X-ray crystallography led to the finding that JBSNF-000088 is methylated and the methylated form of JBSNF-000088 is bound to the active substrate binding site in both proteins with the co-factor SAM turning into the demethylated form (SAH). This supports the hypothesis that JBSNF-000088 may act as a substrate analog, competing with the natural substrate NA and inhibiting the binding of NA to the active site of the enzyme. Once bound to the active site, transfer of methyl group from SAM can lead to the formation of N-methylated JBSNF-000088. Interestingly, the N-methylated product was also found in the circulating plasma of animals dosed with the un-methylated small molecule (JBSNF-000088), though only to a small extent. The latter may be explained by JBSNF-000088 being a potent binder but poor substrate for NNMT with resulting slow turnover. However, in the NNMT co-crystal, due to the high enzyme concentration, formation of the N-methylated compound was clearly detectable. The N-methylated product of JBSNF-000088 itself is a poor inhibitor of NNMT, with an IC50 value of > 30 µM (20% inhibition at 30 µM). Therefore, JBSNF-000088 may act as a competitive substrate analog that is very slowly converted to its N-methylated product. To our knowledge, this is the first proof-of-concept study using a small molecule modulator of NNMT in animal models of metabolic disease to demonstrate pharmacological benefits. Our study opens up the possibility of developing small molecule modulators of NNMT to test in patients with metabolic disorders.[1]

C7H8N2O2

152.15

152.059

C, 55.26; H, 5.30; N, 18.41; O, 21.03

7150-23-4

7150-23-4

250810

White to light yellow solid powder

1.213g/cm3

301.6ºC at 760mmHg

136.2ºC

1.55

0.889

1

3

2

11

149

0

O(C([H])([H])[H])C1C([H])=C([H])C(C(N([H])[H])=O)=C([H])N=1

KXDSMFBEVSJYRF-UHFFFAOYSA-N

InChI=1S/C7H8N2O2/c1-11-6-3-2-5(4-9-6)7(8)10/h2-4H,1H3,(H2,8,10)

6-methoxypyridine-3-carboxamide

JBSNF-000088; 6-Methoxynicotinamide; 6-METHOXYNICOTINAMIDE; 7150-23-4; 6-methoxypyridine-3-carboxamide; CHEMBL4206972; 3-Pyridinecarboxamide,6-methoxy-; NSC70628; MFCD00229166; JBSNF 000088; JBSNF000088

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)

DMSO: ~30 mg/mL (~197.2 mM)
Ethanol: ~7 mg/mL (~46 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (13.67 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 20.8 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (13.67 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (13.67 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 2.94 mg/mL (19.32 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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