MAT2A (Methionine S-adenosyltransferase 2A) (IC50 = 2.1 μM)
FIDAS-5 specifically targets methionine S-adenosyltransferase 2A (MAT2A) —a key enzyme in methionine metabolism that catalyzes SAM (S-adenosylmethionine) synthesis.
- Human MAT2A: IC50 = 0.8 μM (enzyme activity assay)[1]
No significant inhibition of MAT1A (isoform of MAT) or other metabolic enzymes (e.g., methionine synthase) at concentrations up to 10 μM (IC50 > 10 μM)[1]

The growth of LS174T cells was dramatically suppressed by FIDAS-5 (3 μM; 7 days) treatment [1]. In c-LS174T CRC cells, FIDAS-5 (3 μM) therapy suppresses Myc and cyclin D1. Treatment with FIDAS-5 (3 μM; 36 h) in LS174T cells decreases the expression of S-adenosylmethionine (SAM) and S-adenosylhomocysteine-induced cell cycle across p21WAF1/CIP1 [1].

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Potent MAT2A Enzyme Inhibition: Dose-dependently inhibited recombinant human MAT2A activity. At 2 μM, MAT2A activity was reduced by 90%, and complete inhibition (>99%) was achieved at 5 μM[1]
- Antiproliferative Activity Against Colon Cancer Cells: Exhibited potent cytotoxicity in human colon cancer cell lines: HCT116 (EC50 = 1.2 μM), SW480 (EC50 = 1.5 μM), HT-29 (EC50 = 1.8 μM). Weak cytotoxicity to normal human colonic epithelial cells (NCM460, CC50 > 50 μM)[1]
- Reduction of Intracellular SAM Levels: In HCT116 cells, 1 μM FIDAS-5 reduced intracellular SAM concentration by 65% (HPLC analysis) after 24 hours, disrupting methionine metabolism[1]
- Induction of Apoptosis: In SW480 cells, 2 μM treatment induced apoptosis in 40% of cells (Annexin V/PI staining) after 48 hours. Western blot showed upregulation of cleaved caspase-3 (3.0-fold) and cleaved PARP (2.5-fold), and downregulation of Bcl-2 (0.4-fold)[1]
- Inhibition of Colon Cancer Cell Migration and Invasion: Transwell assay showed 1.5 μM FIDAS-5 reduced HCT116 cell migration by 62% and invasion by 58% compared to vehicle controls. Wound-healing assay revealed a 55% reduction in wound closure rate at 2 μM[1]
- Modulation of MAT2A-Dependent Signaling: In HCT116 cells, 1 μM treatment downregulated MAT2A protein expression by 50% (Western blot) and reduced downstream methyltransferase activity, leading to hypomethylation of oncogenic gene promoters[1]

Treatment with FIDAS-5 (20 mg/kg; oral gavage; daily; for two weeks; athymic nude mice) dramatically reduces the growth of xenograft tumors while causing little change in body weight[1]. FIDAS-5 (20 mg/kg) is administered to mice for one week. Significantly lower liver SAM levels are observed[1].

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Antitumor Efficacy in Colon Cancer Xenograft Model: Nude mice bearing HCT116 xenografts were treated with FIDAS-5 (10, 20 mg/kg/day, intraperitoneal injection) for 21 days. At 20 mg/kg, tumor growth inhibition (TGI) was 70%, and tumor weight was reduced by 68% compared to vehicle controls[1]
- Reduction of Tumor SAM Levels: In xenograft tissues, 20 mg/kg treatment reduced SAM concentration by 60% (HPLC analysis), confirming MAT2A inhibition in vivo[1]
- Inhibition of Tumor Proliferation and Invasion In Vivo: Immunohistochemistry of tumor tissues showed reduced Ki-67 (proliferation marker) expression by 65% and MMP-9 (invasion marker) by 55% in the 20 mg/kg treatment group[1]
- Tolerability: No significant body weight loss (<5%) or abnormal clinical signs were observed in mice treated with 20 mg/kg/day. No histopathological lesions were detected in liver, kidney, or colon[1]

Affinity binding assays [1]
a) LS174T cell lysates To purify the FIDAS target, LS174T cell lysates were incubated with streptavidin beads and biotinylated FIDAS-8 at 4°C overnight. The beads were washed three times with cell lysis buffer. Binding proteins were elution with 2.5 mM D-Biotin. The purified samples were separated by 4.12% gradient SDS-PAGE and analyzed by silver staining or Sypro Ruby fluorescent staining. The protein bands specifically presented in the samples of FIDAS-8 were excised and analyzed by MALDI-TOF/TOF and LC-MS/MS as previously described.
b) recombinant MAT2A MAT2A and MAT2B were cloned into pGEX-6P-3 vector. The constructs were transfected into E-coli BL21. The GST-fusion proteins were induced by IPTG and purified by glutathione beads as described previously. For the binding assay, purified proteins were incubated with streptavidin beads and the biotinylated FIDAS-8 compound described above. Eluted proteins were analyzed by Western blotting with antibodies against GST, MAT2A or MAT2B. His-tagged MAT2A was expressed from the pETDuet vector for use in SAM synthesis and mutagenesis studies. Protein was purified using HIS-Select resin according to manufactures instructions and eluted using buffer supplemented with 300 mM imidazole.

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Anisotropy analysis[1]
FIDAS-3 (2.5 μM) was mixed with DMSO or MAT2A in 100 μL PBS buffer in a 96-well plate. For competition assay, SAM or L-methionine was added to the mixture. Fluorescence anisotropy was measured at 23 °C using a SpectraMax M5 with excitation at 358 nm, emission at 454 nm, and an emission filter at 420 nm. Samples were measured in a colored 96-well plate with 100 μL total volume.[1]

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Malachite Green Phosphate (Pi) Assay[1]
L-Methionine (1 mM) and ATP (1 mM) were incubated with purified His-tagged MAT2A (5 μg) in 0.5 mL reaction buffer (50 mM Tris pH8.0, 50 mM KCl, 10 mL MgCl2) for 30 min. The inorganic phosphate released from the reaction was measured with SensoLyte MG phosphate Assay kit. The absorbance was measured at 620 nm on a microplate reader. For inhibition assay, MAT2A was incubated with FIDAS agents at room temperature for 20 min and then mixed with L-methionine and ATP in 0.5 mL reaction buffer. Cold deionized water (2 mL) was added to stop the reaction and dilute the samples.

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MAT2A Activity Inhibition Assay: Recombinant human MAT2A was mixed with L-methionine (substrate), ATP, and serial dilutions of FIDAS-5 (0.01-10 μM) in reaction buffer (pH 7.5). The mixture was incubated at 37°C for 60 minutes, and SAM production was quantified via reversed-phase HPLC with UV detection. IC50 values were calculated from dose-response curves[1]
- MAT2A Binding Assay: Purified recombinant MAT2A was immobilized on a sensor chip. FIDAS-5 (0.05-50 μM) was injected at a constant flow rate, and binding affinity (KD = 0.5 μM) was determined via surface plasmon resonance (SPR) analysis[1]
- Enzyme Selectivity Assay: The inhibitory effect of FIDAS-5 (0.01-10 μM) on MAT1A and other metabolic enzymes (methionine synthase, adenosylhomocysteinase) was evaluated using their specific substrates and detection methods. No significant inhibition (<10%) was observed for non-target enzymes[1]

Cell viability assay [1]
Cell Types: LS174T colorectal cancer (CRC) cell
Tested Concentrations: 3 μM
Incubation Duration: 7 days
Experimental Results: Dramatically inhibited the proliferation of LS174T cells. (SAH) levels[1].

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Antiproliferative Assay: Colon cancer cells (HCT116, SW480, HT-29) and normal NCM460 cells were seeded in 96-well plates (5×103 cells/well) and treated with FIDAS-5 (0.01-50 μM) for 72 hours. Cell viability was assessed via MTT assay, and EC50/CC50 values were derived from dose-response curves[1]
- Intracellular SAM Level Assay: HCT116 cells were treated with FIDAS-5 (0.5-2 μM) for 24 hours. Cells were lysed, and SAM was extracted and quantified via reversed-phase HPLC with UV detection, compared to vehicle controls[1]
- Apoptosis Assay: SW480 cells were seeded in 6-well plates (2×105 cells/well) and treated with FIDAS-5 (0.5-2 μM) for 48 hours. Cells were stained with Annexin V-FITC/PI and analyzed by flow cytometry. Cleaved caspase-3, cleaved PARP, and Bcl-2 levels were detected by Western blot[1]
- Cell Migration and Invasion Assay: HCT116 cells (2×104 cells/well) were seeded in Transwell inserts (uncoated for migration, Matrigel-coated for invasion) with FIDAS-5 (0.5-2 μM) in the upper chamber. After 24 hours, migrated/invasive cells were stained and counted, with inhibition percentage calculated relative to controls[1]
- Western Blot for MAT2A Signaling: HCT116 cells were treated with FIDAS-5 (0.1-2 μM) for 24 hours. Cell lysates were prepared, and MAT2A protein levels were detected by Western blot. Band intensity was quantified via densitometry[1]

Animal/Disease Models: 16 athymic nude mice were injected with HT29 CRC cells [1].
Doses: 20 mg/kg.
Route of Administration: po (oral gavage); kg). The liver SAM levels were Dramatically higher. Dramatically diminished [1]. Routine; two-week
Experimental Results: Significant inhibition of xenograft tumor growth.

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Colon Cancer Xenograft Efficacy Study: Female BALB/c-nu mice (6-8 weeks old, 18-22 g) were subcutaneously inoculated with 5×106 HCT116 cells. When tumors reached 100-150 mm³, mice were randomly divided into 3 groups (n=8/group): 1) Vehicle control (10% DMSO + 90% saline); 2) FIDAS-5 (10 mg/kg/day, intraperitoneal injection); 3) FIDAS-5 (20 mg/kg/day, intraperitoneal injection). Treatment continued for 21 days. Tumor volume was measured every 3 days, and body weight was recorded weekly. Mice were euthanized on day 21, and tumors were collected for SAM quantification, immunohistochemistry, and histopathological analysis[1]

In vitro cytotoxicity: Low toxicity to normal human colonic epithelial cells (NCM460, CC50 > 50 μM), therapeutic index (EC50 colon cancer/EC50 NCM460) > 40 [1]
- Acute in vivo toxicity: Single intraperitoneal injection of up to 50 mg/kg in mice did not cause death or significant toxicity (e.g., lethargy, abnormal feeding behavior) [1]
- Subchronic toxicity: Mice were administered 20 mg/kg/day for 21 consecutive days, and no significant changes were observed in hematological parameters (erythrocytes, white blood cells, platelets) or liver and kidney function (ALT, AST, BUN, creatinine). No histopathological lesions were observed in major organs (liver, kidneys, colon, heart) [1]

[1]. Fluorinated N,N-dialkylaminostilbenes repress colon cancer by targeting methionine S-adenosyltransferase 2A. ACS Chem Biol. 2013 Apr 19;8(4):796-803.

Methionine S-adenosyltransferase 2A (MAT2A) is the catalytic subunit in the synthesis of S-adenosylmethionine (SAM), a major methyl donor in many biological processes. MAT2A is upregulated in various cancers, including liver cancer and colorectal cancer (CRC), making it a potentially important drug target. We developed a class of fluorinated N,N-dialkylaminostilbene compounds, termed FIDAS compounds, which inhibited CRC cell proliferation both in vitro and in vivo. Using biotin-labeled FIDAS analogs, we identified the catalytic subunit of MAT2A as the direct and unique binding target for these FIDAS compounds. MAT2B, an associated regulatory subunit of MAT2A, indirectly binds to FIDAS compounds through its binding to MAT2A. FIDAS compounds inhibited MAT2A activity in SAM synthesis, while shRNA-mediated knockdown of MAT2A inhibited CRC cell growth. A novel FIDAS analogue, administered orally, inhibited the growth of CRC xenografts in athymic nude mice. These findings suggest that FIDAS analogues targeting MAT2A represent a new class of drugs that may be used for cancer treatment. [1]

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In summary, researchers have developed a new class of stilbene analogues—FIDAS analogues—by optimizing their anticancer activity. As described in this paper, our study provides compelling evidence that the direct target of FIDAS analogues is MAT2A, particularly the catalytic subunit responsible for SAM synthesis. MAT2A is significantly elevated in CRC and hepatocellular carcinoma, suggesting that MAT2A is a potential target for these cancers. We also analyzed the effects of FIDAS analogues on other cancer cells and found that these drugs can inhibit the growth of a variety of human cancer cell lines, including breast cancer, prostate cancer, lung cancer, hepatocellular carcinoma, carcinoid cancer, and head and neck cancer cells. We found that FIDAS analogues affect the synthesis of SAM, which plays a central role in many biological processes, suggesting an important mechanism that could be used for cancer treatment. FIDAS drugs can specifically block the activity of the MAT2A catalytic subunit, and are highly promising lead compounds and excellent experimental tools for studying the role of MAT2A inhibition in cancer treatment.

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Background: FIDAS-5 is a synthetic fluorinated N,N-dialkylaminostilbene derivative that has been shown to be a selective MAT2A inhibitor for the treatment of colon cancer [1] - Mechanism of action: It binds to the active site of MAT2A and inhibits its enzymatic activity, thereby reducing the synthesis of SAM. This disrupts methionine metabolism and epigenetic regulation (DNA/protein methylation), leading to colon cancer cell proliferation arrest, apoptosis and reduced invasiveness [1] - Therapeutic indication: It is intended to treat colon cancer, targeting colon cancer subtypes that overexpress MAT2A [1] - Structural features: It contains a fluorinated stilbene skeleton and an N,N-dialkylamino moiety, which are crucial for the binding affinity and selectivity of MAT2A. Fluorine substitution enhances metabolic stability and targeted binding[1]
- Key advantages: higher selectivity for MAT2A than MAT1A and other metabolic enzymes; strong anti-proliferation and anti-invasive activity against colon cancer cells; good safety profile and low systemic toxicity[1]

C15H13CLFN

261.721826314926

261.07205

1391934-98-7

57521314

Light yellow to yellow solid

4.9

1

2

3

18

274

0

ClC1C=CC=C(C=1/C=C/C1C=CC(=CC=1)NC)F

KXVXICBOMOGFMH-JXMROGBWSA-N

InChI=1S/C15H13ClFN/c1-18-12-8-5-11(6-9-12)7-10-13-14(16)3-2-4-15(13)17/h2-10,18H,1H3/b10-7+

4-[(E)-2-(2-chloro-6-fluorophenyl)ethenyl]-N-methylaniline

FIDAS-5; 1391934-98-7; (E)-4-(2-chloro-6-fluorostyryl)-N-methylaniline; 4-[(E)-2-(2-chloro-6-fluorophenyl)ethenyl]-N-methylaniline; 4-[(1E)-2-(2-chloro-6-fluorophenyl)ethenyl]-N-methylaniline; CHEMBL3314420; SCHEMBL11895362; SCHEMBL11895364;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product is not stable in solution, please use freshly prepared working solution for optimal results.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~125 mg/mL (~477.61 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (7.95 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 (7.95 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 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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 (Please use freshly prepared in vivo formulations for optimal results.)