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Purity: = 98.95%
Denifanstat (TVB-2640; FASN-IN-2; ASC-40; TVB2640) is a novel, potent and orally bioactive fatty acid synthase (FASN) inhibitor with potential anticancer activity and may also be used for the Treatment of Nonalcoholic Steatohepatitis. TVB-2640 binds to and blocks FASN, which prevents the synthesis of palmitate needed for tumor cell growth and survival. This leads to a reduction in cell signaling, an induction of tumor cell apoptosis and the inhibition of cell proliferation in susceptible tumor cells. FASN, an enzyme responsible for the de novo synthesis of palmitic acid, is overexpressed in tumor cells and plays a key role in tumor metabolism, lipid signaling, tumor cell survival and drug resistance; tumor cells are dependent on increased fatty acid production for their enhanced metabolic needs and rapid growth. TVB-2640 significantly reduced liver fat and improved biochemical, inflammatory, and fibrotic biomarkers after 12 weeks, in a dose-dependent manner in patients with nonalcoholic steatohepatitis. ClinicalTrials.gov, Number NCT03938246.
| Targets |
Fatty Acid Synthase (FASN)(IC50 = 0.052 μM)
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|---|---|
| ln Vitro |
Denifanstat (compound 152) is a strong inhibitor of FASN [2]. Palmitoylation of the SARS-CoV2 spike protein is inhibited by fatty acid synthase (FASN) [1].
Fatty acid synthase (FASN) is an attractive therapeutic target in non-alcoholic steatohepatitis (NASH) because it drives de novo lipogenesis and mediates pro-inflammatory and fibrogenic signaling. We therefore tested pharmacological inhibition of FASN in human cell culture and in three diet induced mouse models of NASH. Three related FASN inhibitors were used; TVB-3664, TVB-3166 and clinical stage TVB-2640 (denifanstat). In human primary liver microtissues, FASN inhibiton (FASNi) decreased triglyceride (TG) content, consistent with direct anti-steatotic activity. In human hepatic stellate cells, FASNi reduced markers of fibrosis including collagen1α (COL1α1) and α-smooth muscle actin (αSMA). In CD4+ T cells exposed to NASH-related cytokines, FASNi decreased production of Th17 cells, and reduced IL-1β release in LPS-stimulated PBMCs[3]. |
| ln Vivo |
In mice with diet induced NASH l, FASNi prevented development of hepatic steatosis and fibrosis, and reduced circulating IL-1β. In mice with established diet-induced NASH, FASNi reduced NAFLD activity score, fibrosis score, ALT and TG levels. In the CCl4-induced FAT-NASH mouse model, FASN inhibition decreased hepatic fibrosis and fibrosis markers, and development of hepatocellular carcinoma (HCC) tumors by 85%. These results demonstrate that FASN inhibition attenuates inflammatory and fibrotic drivers of NASH by direct inhibition of immune and stellate cells, beyond decreasing fat accumulation in hepatocytes. FASN inhibition therefore provides an opportunity to target three key hallmarks of NASH[3].
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| Enzyme Assay |
Biochemical and cellular potency assays[3]
FASN enzymatic activity was determined using a fluorescence-based thiol quantification assay as described by Chung et al.37. The assay monitors the release of Coenzyme A (CoA), a byproduct of FASN activity, which reacts with the profluorescent dye CPM (7-Diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin) (Sigma C1484) and makes it fluorescent. For FASN biochemical assays, human FASN protein was extracted from SKBr3 cells. Palmitate synthesis assays were performed as previously described25, by cell incubation in media containing 13C-acetate and measurement of conversion to palmitate by LC–MS analysis. The mean of 2 to 15 independent assays are shown. Activity assay[4] The activities of the KR domain of the hFASN with and without Denifanstat were determined by monitoring NADPH oxidation using a CLARIOstar spectrophotometer (software version: 5.20 R5) at 334 nm in the presence of trans-1-decalone. Reaction was performed at 37 °C in 96 well microplates in a final volume of 150 µl. The assay mixture contained 50 mM sodium phosphate at pH 7.4, 1 mM DTT, 0.04% (v/v) Tween-20, 400 µM NADPH, 5 mM trans-1-decalone and 100 nM purified protein. For protein inhibition, 10 µM Denifanstat was used. The DH domain activity of the wild-type, L1097A, and H878A hFASN was measured spectrophotometrically through the decrease in absorbance caused by NADPH oxidation at 334 nm. The reactions were carried out at 37 °C in 96 well microplates in a final volume of 150 µl. The reactions mixture contained 50 mM sodium phosphate at pH 7.4, 1 mM TCEP, 1 mM EDTA, 40 µM NADPH and 800 µM DL-beta-Hydroxybutyryl CoA and 100 nM purified protein. A calibration curve was taken for 5 min before adding the substrates. The error of the graph was plotted from the two biological replications with technical duplicate conditions in each purified protein preparation unless otherwise stated. More specifically, two protein preparations from two separate transfections were used (i.e., biological duplicate). For each biological replicate, two independent assays were run on the same 96-well plate (i.e., technical duplicate). Each curve is normalized against its first point of measurement after the addition of the substrates. Each assay replicate is plotted individually in Supplementary Fig. 10. All assay data are provided as excel files containing raw and normalized absorbance values as well as calculated NADPH concentrations at each time point. |
| Cell Assay |
LMT[3]
LMTs were created using cryopreserved primary human cells and cultured in 96 well Akura plates using established protocols, as previously described27 Total TG content was measured with the TG-Glo assay. LX-2[3] The procedures were followed as previously described29. Briefly, in 6-well culture plate 150,000 LX-2 cells were seeded per well and serum starved overnight. Two sets of culture plates were run in parallel. Both sets of LX-2 cells were induced with either vehicle (DMSO) or indicated dosing of TVB-3664. One set of LX-2 cells were harvested after 48 h of TVB-3664 treatment before harvest. In other set of cells both vehicle and TVB-3664 were replaced by normal DMEM culture media with 10% FBS and maintained for additional 48 h before harvest. For RNA expression, total RNA was extracted from harvested LX-2 cells, cDNA was synthesized and expression of fibrogenic genes was quantified by qPCR. Unpaired T tests were used to compare the mean results per group appropriate since these were independent groups. View More
Immunocytochemistry of HSCs[3] PBMC and CD4+ T cells[3] Healthy human donor blood collected in sodium citrate tubes was purchased from The Stanford Blood Center, Palo Alto, CA. Appropriate informed consent and IRB approval was obtained under licenses in place at Stanford Blood Center (https://stanfordbloodcenter.org/research-updates/donating-for-research/). All samples were de-identified. All methods were performed in accordance with the Declaration of Helsinki. Freshly collected PBMC were isolated from by Ficoll Paque Plus density gradient separation. Naïve CD4+ T cells were enriched with Dynabeads™ human CD4+ T Cells Isolation Kits. Naïve human CD4+ T cells were then differentiated by plating into 48-well tissue culture plates (Costar) with 1 μg/mL anti-CD3 with anti-CD28 antibody coated beads. Differentiation to generate human Th17 naïve human CD4+ T cell cultures were placed in medium containing the following additives for 4 days: anti-IL-2 (30 ng/mL, anti-IL-4 antibody (20 ng/mL), anti-IFNγ antibody (50 ng/mL) from R&D Systems, recombinant human IL-6 (10 ng/mL), human TGFβ (1 ng/mL) (recombinant human IL-1β (10 ng/mL) recombinant human IL-23 (10 ng/mL) from BD Biosciences. The resulting Th17 cells were cultured in IMDM GlutaMAX medium supplemented with 10% heat-inactivated FCS with 500 units of penicillin–streptomycin. IL-1β from primary human PBMC supernatant was measured by ELISA according to manufacture’s instructions. Flow cytometry [3] Monoclonal antibodies specific to the following antigens (and labeled with the indicated fluorescent markers) were used: Foxp3 FITC (236A/E7; 1:100), IL-17A PE-Cy7 (eBio64DEC17; 1:100), CD4 PerCP (SK3; 1:200) For analysis of surface markers, cells were stained in PBS containing 0.25% BSA and 0.02% azide. Dead cells were excluded by LIVE/DEAD Fixable Dead Cell Stain Kit. For intracellular cytokine staining cells were treated with Brefeldin A (5 mg/mL) for 2 h and stained using the Fixation/Permeabilization Kit according to the manufacturer’s instructions. Quantitation of fluorescent cells and their staining intensity were acquired on a FACSCalibur, and data were analyzed with CELLQuest software. |
| Animal Protocol |
Preventative NASH diet-induced model[3]
5-week old male C57BL/6J mice were used. CARE is a USDA certified (Certificate Number 84-R-0081) and OLAW accredited facility. The study design and animal usage were reviewed and approved by the CARE Research IACUC for compliance with regulations prior to study initiation (IACUC number 1628). Mice were fed a diet designed to imitate a WD, containing 40 kcal% fat (vegetable shortening), 20 kcal% fructose, and 2% cholesterol, or normal standard commercial rodent diet. Liver samples were processed and histology performed at IDEXX BioResearch. Masson’s Trichrome stain was used to assess the amount of fibrous connective tissue in the liver, and Oil Red O and adipophilin stained sections were used to characterize histologic features associated with steatosis. A qualified pathologist performed histologic evaluation using the following scoring system on H&E stained sections: 0 = normal; 1 = minimal changes; 2 = mild changes; 3 = moderate changes; 4 = marked changes and 5 = severe changes. Cytokine/chemokine assays were performed by Sagimet Biosciences using terminal bleed samples, by ELISA according to manufacturers instructions. Therapeutic NASH diet-induced model[3] 5-week old male C57BL/6J mice were used. All animal experiments were conducted in accordance with Gubra’s bioethical guidelines, fully compliant to internationally accepted principles for the care and use of laboratory animals. The experiments were covered by licenses for Jacob Jelsing (2013-15-2934-00784 and 2015-15-0201-00518) issued by the Danish committee for animal research. Mice were fed a diet designed to imitate a WD, containing 40% high trans-fat with high sugar (20% fructose, 9% sucrose) and 2% cholesterol (Research Diets, Inc., #D09100310), for 44 weeks prior to treatment with TVB-3664, and during treatment. Blood samples were collected in heparinized tubes and plasma separated and stored at -80 °C until analysis. TG, TC, ALT, AST were measured using commercial kits on the Cobas TM C-501 autoanalyzer according to the manufacturer’s instructions. Liver IHC used standardized procedures. NAS scoring used the criteria outlined in Kleiner et al.28. Fibrosis was scored on the basis of 0 (none), 1 (perisinusoidal or periportal), 2 (perisinusoidal and portal/periportal), or 3 (bridging fibrosis). Statistical analysis used one-way ANOVA with Dunnett’s multiple comparison test to compare treated to vehicle. For change in histology score per animal, Fisher’s exact test followed by adjustment for multiple correction using the Bonferroni method. View More
Fibrosis and tumor (FAT)-NASH CCl4 model[3] |
| References |
[1]. Minhyoung Lee, et al. Fatty Acid Synthase inhibition prevents palmitoylation of SARS-CoV2 Spike Protein and improves survival of mice infected with murine hepatitis virus. BioRxiv, December 21, 2020.
[2]. Johan D., et al. Heterocyclic modulators of lipid synthesis. WO2012122391A1. [3]. FASN inhibition targets multiple drivers of NASH by reducing steatosis, inflammation and fibrosis in preclinical models. Sci Rep. 2022 Sep 19;12(1):15661. doi: 10.1038/s41598-022-19459-z. [4]. Atomic model for core modifying region of human fatty acid synthase in complex with Denifanstat. Nat Commun. 2023 Jun 12;14(1):3460. doi: 10.1038/s41467-023-39266-y. |
| Additional Infomation |
Fatty acid synthase (FASN) is an attractive therapeutic target in non-alcoholic steatohepatitis (NASH) because it drives de novo lipogenesis and mediates pro-inflammatory and fibrogenic signaling. We therefore tested pharmacological inhibition of FASN in human cell culture and in three diet induced mouse models of NASH. Three related FASN inhibitors were used; TVB-3664, TVB-3166 and clinical stage TVB-2640 (denifanstat). In human primary liver microtissues, FASN inhibiton (FASNi) decreased triglyceride (TG) content, consistent with direct anti-steatotic activity. In human hepatic stellate cells, FASNi reduced markers of fibrosis including collagen1α (COL1α1) and α-smooth muscle actin (αSMA). In CD4+ T cells exposed to NASH-related cytokines, FASNi decreased production of Th17 cells, and reduced IL-1β release in LPS-stimulated PBMCs. In mice with diet induced NASH l, FASNi prevented development of hepatic steatosis and fibrosis, and reduced circulating IL-1β. In mice with established diet-induced NASH, FASNi reduced NAFLD activity score, fibrosis score, ALT and TG levels. In the CCl4-induced FAT-NASH mouse model, FASN inhibition decreased hepatic fibrosis and fibrosis markers, and development of hepatocellular carcinoma (HCC) tumors by 85%. These results demonstrate that FASN inhibition attenuates inflammatory and fibrotic drivers of NASH by direct inhibition of immune and stellate cells, beyond decreasing fat accumulation in hepatocytes. FASN inhibition therefore provides an opportunity to target three key hallmarks of NASH.
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| Molecular Formula |
C27H29N5O
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|---|---|
| Molecular Weight |
439.563
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| Exact Mass |
439.2372
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| Elemental Analysis |
C, 73.78; H, 6.65; N, 15.93; O, 3.64
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| CAS # |
1399177-37-7
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| Related CAS # |
1399177-37-7;
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| PubChem CID |
66548316
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| Appearance |
Typically exists as solids (or liquids in special cases) at room temperature
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| LogP |
5.3
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| tPSA |
85.7Ų
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| SMILES |
N#CC1=CC=C(C2CCN(C(C3=CC(C4=NN=C(C)N4)=C(C5CCC5)C=C3C)=O)CC2)C=C1
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| InChi Key |
BBGOSBDSLYHMRA-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C27H29N5O/c1-17-14-24(22-4-3-5-22)25(26-29-18(2)30-31-26)15-23(17)27(33)32-12-10-21(11-13-32)20-8-6-19(16-28)7-9-20/h6-9,14-15,21-22H,3-5,10-13H2,1-2H3,(H,29,30,31)
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| Chemical Name |
4-(1-(4-Cyclobutyl-2-methyl-5-(5-methyl-4H-1,2,4-triazol-3- yl)benzoyl)piperidin-4-yl)benzonitrile
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| Synonyms |
TVB 2640; Denifanstat; FASN-IN-2; ASC-40; TVB-2640; ASC40; TVB2640
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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: This product requires protection from light (avoid light exposure) during transportation and storage. |
| 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 (~227.51 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.69 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 25.0 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: ≥ 2.5 mg/mL (5.69 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), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (5.69 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2750 mL | 11.3750 mL | 22.7500 mL | |
| 5 mM | 0.4550 mL | 2.2750 mL | 4.5500 mL | |
| 10 mM | 0.2275 mL | 1.1375 mL | 2.2750 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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