FASN[1][2].
TVB-3664 targets fatty acid synthase (FASN). It is a potent and selective inhibitor that binds to and blocks the enzymatic activity of FASN. This prevents the synthesis of palmitate, a key fatty acid required for membrane formation, cell signaling, and protein palmitoylation. The compound's high oral bioavailability and potency make it a promising candidate for therapeutic development.

In the CaCo2, HT29, and LIM2405 cell lines, TVB-3664 (0-1 μM, 7 days) demonstrates anti-tumor action [1]. The viability of several tumor cell lines from hematological and solid malignancies is decreased by TVB-3664 [1].
In vitro, TVB-3664 has been shown to be a potent inhibitor of FASN. It induces FASN inhibition, leading to significantly reduced tubulin palmitoylation and mRNA expression. The compound's activity is characterized by its low IC50 values for palmitate synthesis in both human and mouse cells.

Tumor volume and weight significantly decreased after 4 weeks of oral gavage treatment with TVB-3664 (3 mg/kg for Pt 2614 and Pt 2449PT) or 6 mg/kg for Pt 2402 and Pt 2449LM). For the Pt 2614 PDX model, the average tumor weight decreased by 30%, 37.5%, and 51.5%, respectively [1].
In vivo, TVB-3664 is being evaluated for its anti-cancer activity. As a highly bioavailable FASN inhibitor, it is expected to effectively inhibit tumor growth in various animal models. The compound's ability to inhibit microtubule protein palmitoylation may contribute to its anti-proliferative effects.

TMA analysis[1]
Immunoreactivity score of FASN expression was analyzed in matched normal colon mucosa and tumor tissues from patients diagnosed with Stage I-IV CRC who had surgery at UK Chandler Medical Center (TMA ID BH15991A, n = 57 normal and 56 tumor tissues). Scoring was carried out blindly by a pathologist. Immunoreactivity score was determined by multiplication of the values for staining intensity (0, no staining; 1, weak; 2, moderate; 3, strong staining) and the values for percentage of positive tumor cells (0, no positive cells; 1, 0–10%; 2, 11–50%; 3, 51–100% positive).

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Nuclear magnetic resonance analysis[1]
NMR spectra were obtained on an Agilent DD2 14.1 T spectrometer equipped with a 3-mm inverse triple resonance HCN cold probe. The 1D proton spectra were recorded at 288 k with an acquisition time of 2 s and a 4 s relaxation delay, during which the residual HOD resonance was irradiated. 1D 1H[13C]HSQC spectra were recorded with an acquisition time of 0.25 s and a 1.75 s relaxation delay. Internal DSS-d6 was used as a chemical shift reference and for absolute quantification. MNOVA was used for NMR spectral processing. After phasing and baseline correction, metabolites were quantified using the peak fitting routines in MNOVA, and assigned using in-house databases. Isotopomers were quantified as absolute and fractional enrichments as previously described Assigned metabolites were normalized to mg protein determined by BCA analysis.
In vitro enzyme or receptor binding (non-cell) assays for TVB-3664 involve measuring its direct inhibition of FASN enzymatic activity. The assay uses a purified recombinant FASN enzyme and a fluorescent or radiolabeled substrate to measure the rate of fatty acid synthesis. The compound is incubated with the enzyme and substrate, and the decrease in product formation is measured to determine the IC50.

Cell Proliferation Assay[1]
Cell Types: CaCo2, HT29 and LIM2405 cell lines.
Tested Concentrations: 0-1 μM.
Incubation Duration: 7 days.
Experimental Results: demonstrated anti-tumor activity.
In vitro cell-based assays for TVB-3664 are performed using cancer cell lines that rely on de novo lipogenesis. Cells are treated with the compound, and palmitate synthesis is measured by incorporating radiolabeled acetate or glucose into lipids. The compound's effects on tubulin palmitoylation are assessed by Western blotting. Cell viability and apoptosis are also measured.

Animal/Disease Models: Colorectal cancer (CRC) PDX models in NOD-SCID-IL2rg-/- (NSG) mice using specimens collected from patients who had undergone surgery for resection of primary CRC or CRC metastasis[1].
Doses: 3 mg/kg (Pt 2614 and Pt 2449PT) or 6 mg/kg (Pt 2402 and Pt 2449LM).
Route of Administration: po (oral gavage) daily for 4 weeks.
Experimental Results: Led to a significant reduction in tumor volume and tumor weight in Pt 2614, Pt 2449PT, and Pt 2402 PDX models, with an average reduction in tumor weight of 30%, 37.5% and 51.5%, respectively.

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Tissues were obtained from consented patients with Stage II-IV CRC who had undergone surgery at UK Medical Center. 6–8-week-old NSG mice (NOD.Cg-Prkdc Il2rg /SzJ) from The Jackson Laboratory were used for PDX models. All procedures were performed using protocols approved by the UK Animal Care and Use Committee. Briefly, CRC tissues (2–5 mm) obtained from CRC patients of both sexes were implanted subcutaneously into their flanks in a small pocket surgically created under the skin. Established tumors were designated as generation 0 (G0). Tumor tissues from G0 were minced and mixed with Matrigel to ensure homogeneous distribution of tissues among mice and allow implantation of an equal volume of tumor tissues into the flank. To preserve histopathological and genomic characteristics of clinical CRC tumors and recapitulate the differential responses of CRC tumors to FASN inhibitors, all established PDX models were treated at G1 with exception of the Pt 2387 case, which was treated at G2. When tumors reached 100 mm3, the mice were randomized according to animal weight and tumor size. For evaluation as monotherapy, treatment (n = 5) and control (n = 5) groups were given TVB-3664 (3–6 µg/kg) vs vehicle (30% PEG400) by oral gavage daily for 4–6 weeks. Tumors were measured once per week by a digital caliper and tumor volume was calculated using the formula: TV = width2 × length × 0.52 as previously described. Tumor weight was measured at the end of the experiment. Blood samples were collected through cardiac puncture at the time of sacrifice. Mice were fasted and then injected i.p. with 2 g kg–1 body mass of 13C6-glucose 16 h prior to sacrifice as previously described, to allow SIRM tracing of cellular metabolites of tumors. 1 h after injection, blood samples were collected and separated into plasma and blood cells by centrifugation (4° C, 3,500 × g, 15 min). Mice were euthanized and tumor tissue was collected for IHC and flash frozen immediately in liquid N2 for protein and metabolic analysis.[1]
In vivo animal experiments for TVB-3664 are conducted using mouse xenograft models of cancer. Mice bearing subcutaneous tumors are treated orally with the compound, and tumor volume is measured over time. The compound's effects on tumor metabolism and signaling pathways are assessed by analyzing tumor tissue.

Pharmacokinetic (PK) properties of TVB-3664 indicate it is orally available and has high bioavailability. The compound is designed to achieve sufficient concentrations in the target tissues to inhibit FASN activity. Specific PK parameters, such as half-life and clearance, are determined in preclinical studies via LC-MS/MS analysis of plasma samples.

Toxicology (toxicology) data for TVB-3664 are being evaluated in preclinical studies. As a FASN inhibitor, its safety profile is important for its development as a therapeutic agent. The compound is generally well-tolerated at therapeutic doses. Its effects on normal lipid metabolism are being carefully evaluated.

[1]. Preclinical evaluation of novel fatty acid synthase inhibitors in primary colorectal cancer cells and a patient-derived xenograft model of colorectal cancer. Oncotarget. 2018 May 15;9(37):24787-24800.

[2]. FASN Inhibition and Taxane Treatment Combine to Enhance Anti-tumor Efficacy in Diverse Xenograft Tumor Models through Disruption of Tubulin Palmitoylation and Microtubule Organization and FASN Inhibition-Mediated Effects on Oncogenic Signaling and Gene Expression. EBioMedicine. 2017 Feb;16:51-62.

Fatty acid synthase (FASN) is a key enzyme in de novo liposynthesis and is highly expressed in various cancers, including colorectal cancer (CRC); elevated FASN expression is associated with poor prognosis. The potent FASN inhibitor (TVB) developed by 3-V Biosciences has shown antitumor activity both in vitro and in vivo and demonstrated good tolerability in a phase I clinical trial. However, the CRC characterization associated with FASN inhibition response is not fully elucidated. We evaluated the effect of TVB-3664 on tumor growth in 9 CRC patient-derived xenografts (PDXs) and investigated the molecular and metabolic changes associated with the CRC response to FASN inhibition. The sensitivity of CRC cells and PDXs to FASN inhibition differed significantly. TVB-3664 treatment showed significant efficacy (tumor volume reduction) in 30% of cases. The antitumor activity of TVB-3664 was associated with a significant reduction in the adenine nucleotide pool and alterations in lipid composition, including a significant decrease in fatty acids and phospholipids, and an increase in lactosylceramide and sphingomyelin in a PDX model sensitive to FASN inhibition. Furthermore, Akt, Erk1/2, and AMPK are major oncogenic pathways associated with TVB alterations. In summary, we demonstrate that a novel TVB inhibitor exhibits antitumor activity in CRC, and this activity is associated with reduced activation of the Akt and Erk1/2 oncogenic pathways and significant alterations in tumor lipid composition. Further understanding of the genetic and metabolic characteristics of FASN-sensitive tumors may contribute to the selection and individualized treatment of CRC patients.
Reference: https://pubmed.ncbi.nlm.nih.gov/29872506/
Other information: TVB-3664 is a research compound being developed as a potential anti-cancer agent. It is available from chemical suppliers for preclinical research purposes.

C25H23F3N4O2

468.48

468.177

C, 64.10; H, 4.95; F, 12.17; N, 11.96; O, 6.83

2097262-58-1

129101638

White to off-white solid powder

1.36±0.1 g/cm3(Predicted)

665.7±55.0 °C(Predicted)

3.8

1

7

5

34

770

0

CC1=CC(=C(C=C1C2=C(NC(=N2)C(F)(F)F)COC)C(=O)N3CC(C3)C4=CC=C(C=C4)C#N)C

YFEOVRCUSPPGFZ-UHFFFAOYSA-N

InChI=1S/C25H23F3N4O2/c1-14-8-15(2)20(9-19(14)22-21(13-34-3)30-24(31-22)25(26,27)28)23(33)32-11-18(12-32)17-6-4-16(10-29)5-7-17/h4-9,18H,11-13H2,1-3H3,(H,30,31)

4-(1-(5-(4-(methoxymethyl)-2-(trifluoromethyl)-1H-imidazol-5-yl)-2,4-dimethylbenzoyl)azetidin-3-yl)benzonitrile

TVB3664; TVB 3664; TVB-3664

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 : ~10 mg/mL (~21.35 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.34 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.44 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 3: ≥ 2.08 mg/mL (4.44 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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 (Please use freshly prepared in vivo formulations for optimal results.)