MAT2A (Methionine S-adenosyltransferase 2A) (IC50 = 2.1 μM)
FIDAS-5 specifically targets methionine adenosyltransferase 2A (MAT2A), a key enzyme in the methionine metabolism pathway. It functions as a competitive inhibitor of MAT2A, effectively competing with the natural substrate S-adenosylmethionine (SAM) for binding to the enzyme's active site. FIDAS-5 demonstrates potent inhibition of MAT2A with an IC50 of 2.1 microM. MAT2A is frequently upregulated in various cancers, including liver, colon, and lung cancers, to support elevated SAM demand for transmethylation reactions needed for rapid cell proliferation. By inhibiting MAT2A, FIDAS-5 disrupts SAM-dependent methylation of DNA, RNA, proteins, and lipids, thereby interfering with cancer cell growth and survival.

Treatment of LS174T cells with FIDAS-5 (3 μM; 7 days) dramatically reduced their ability to proliferate [1]. Myc and cyclin D1 are inhibited in c-LS174T CRC cells by FIDAS-5 (3 μM). The FIDAS-5 FIDAS-5 (3 μM; 36 h) treatment decreases cell cycle expression across p21WAF1/CIP1 in LS174T cells stimulated by S-adenosylmethionine (SAM) and S-adenosylhomocysteine [1].

---

In vitro studies have shown that FIDAS-5 effectively competes with SAM for MAT2A binding, leading to reduced enzyme activity. The compound demonstrates anticancer effects in a variety of cancer cell lines through mechanisms that likely involve altered methylation status of key regulatory molecules. By decreasing SAM availability, FIDAS-5 disrupts normal methylation patterns, which can lead to growth arrest and cell death in MAT2A-overexpressing cancer cells. The compound shows selectivity for cancer cells over normal cells where MAT2A expression is lower, although comparative IC50 values across different cell lines are not provided in the available datasheets.

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].

---

FIDAS-5 HCl is characterized as an orally bioactive compound with demonstrated anticancer effects in preclinical models. Following oral administration, the compound can achieve sufficient systemic exposure to exert its MAT2A inhibitory activity. The in vivo efficacy of FIDAS-5 is attributed to the reduction of SAM pools within tumors, leading to disruption of methylation reactions critical for cancer cell proliferation and survival. Detailed in vivo efficacy data, including tumor growth inhibition rates in specific xenograft models, are not provided in the available datasheets.

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.

---

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]

---

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.

---

For non-cellular MAT2A enzyme inhibition assays, recombinant human MAT2A enzyme is expressed and purified. The enzyme activity is measured by incubating MAT2A (0.1-0.5 microg/reaction) with varying concentrations of FIDAS-5 or FIDAS-5 HCl in assay buffer (50 mM Tris-HCl, pH 7.5, 100 mM KCl, 10 mM MgCl2, and 1 mM DTT). The reaction is initiated by the addition of substrate L-methionine and ATP (both at saturating concentrations, e.g., 100 microM and 200 microM, respectively). After 30-60 minutes at 37degC, the reaction is terminated by heating or by adding a stop solution. SAM production is quantified using HPLC with UV detection, LC-MS/MS, or a coupled enzyme assay (e.g., using SAM-dependent methyltransferases and fluorometric detection). IC50 values are calculated from dose-response curves. A known MAT2A inhibitor (e.g., SAM itself at high concentrations) or vehicle control is included as a reference. The competitive nature of inhibition can be assessed by varying SAM concentrations while measuring enzyme activity in the presence of FIDAS-5 to generate Lineweaver-Burk plots.

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].

---

For in vitro cellular assays, human cancer cell lines with high MAT2A expression, such as HCT-116 (colon cancer), HepG2 (hepatocellular carcinoma), or A549 (lung cancer), are cultured in appropriate media (e.g., DMEM or RPMI-1640) supplemented with 10% FBS and antibiotics. Cells are seeded in 96-well plates and allowed to attach overnight. Serial dilutions of FIDAS-5 HCl (typically 0.1-100 microM) are added to the cells, and incubation is continued for 48-72 hours. Cell viability is assessed using a colorimetric MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) or a luminescent CellTiter-Glo assay. The half-maximal inhibitory concentration (IC50) is calculated from dose-response curves. To assess effects on cellular SAM levels, cells treated with FIDAS-5 for 24-48 hours are harvested, lysed, and SAM concentration is measured using LC-MS/MS or an enzyme-linked immunosorbent assay (ELISA). To evaluate global DNA methylation status, genomic DNA is extracted, hydrolyzed, and analyzed by LC-MS/MS to quantify the ratio of 5-methylcytosine (5-mC) to cytosine. Protein lysates can also be analyzed by Western blotting using antibodies specific for symmetrically or asymmetrically methylated histone residues (e.g., H3K4me3, H3K9me3, H4R3me2, H3R8me2).

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.

---

For in vivo pharmacology studies, BALB/c nude mice (6-8 weeks old, female) are subcutaneously implanted with human cancer cells such as HCT-116 (3-5 × 10⁶ cells in 100 microL PBS) into the right flank region. Once tumors reach an average volume of approximately 100-150 mm3 (typically 7-14 days post-implantation), mice are randomly assigned to treatment and control groups (n = 6-8 per group). FIDAS-5 HCl is formulated in a suitable vehicle, such as 0.5% carboxymethyl cellulose (CMC) or 10% Cremophor EL in saline, and administered orally by gavage at doses of 25-100 mg/kg once daily. A control group receives vehicle alone. Tumor dimensions are measured every 2-3 days using digital calipers, and tumor volume is calculated using the formula: length × width2 × 0.5. Body weight is monitored as a general indicator of toxicity. After 3-4 weeks of treatment or when tumors in the control group reach ethical endpoint size (typically 1500-2000 mm3), mice are euthanized. Tumors are excised, weighed, and processed for downstream analyses: (1) SAM levels are measured by LC-MS/MS; (2) Histone methylation patterns are assessed by Western blotting; (3) Global DNA methylation is analyzed; (4) Tumor sections are stained with H&E for histopathology and with Ki67 antibody for proliferation index, and TUNEL assay for apoptosis.

FIDAS-5 HCl is characterized as an orally bioavailable MAT2A inhibitor with drug-like physicochemical properties. The compound has a molecular weight of approximately 261.7 as the HCl salt. It is soluble in DMSO and can be formulated in vehicles suitable for oral administration. Detailed pharmacokinetic parameters such as half-life (t½), maximum plasma concentration (Cmax), time to peak concentration (Tmax), area under the concentration-time curve (AUC), volume of distribution (Vd), and oral bioavailability (%F) are not provided in the available datasheets. As a methionine metabolism inhibitor, its PK profile likely includes good absorption followed by distribution to tissues with high MAT2A expression, including the liver and tumors. The compound may undergo hepatic metabolism and biliary excretion.

Specialized toxicology studies for FIDAS-5 HCl have not been published in standard research compound datasheets. However, as a MAT2A inhibitor that reduces cellular SAM pools, potential toxicity may arise from disruption of normal methylation reactions essential for cell function. Rapidly dividing cells (e.g., gastrointestinal epithelium, bone marrow, hair follicles) may be particularly sensitive, similar to other antimetabolite anticancer agents. In preclinical animal studies reported to date, FIDAS-5 HCl appears to be well-tolerated at efficacious doses (e.g., 25-50 mg/kg oral daily) with no overt signs of acute toxicity or significant body weight loss. Formal Good Laboratory Practice (GLP) toxicology studies, including single-dose and repeat-dose toxicity in two species, genotoxicity, and safety pharmacology evaluations, have not been conducted as the compound remains in early-stage discovery research.

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

FIDAS-5 HCl (CAS# 1391934-98-7) is a research-grade MAT2A inhibitor intended for laboratory use only and is not an approved therapeutic drug. The compound has a molecular weight of approximately 261.7 (free base) and a molecular formula of C1₅H13FN2 (free base). It functions as a competitive inhibitor of MAT2A by binding to the SAM binding site. By targeting the methionine metabolism pathway, FIDAS-5 represents a strategy to disrupt SAM-dependent methylation reactions, which are essential for cancer cell proliferation. This compound has not yet advanced into clinical trials and remains at the preclinical discovery stage. Its primary research applications include studying the role of MAT2A and SAM metabolism in cancer biology, investigating the therapeutic potential of targeting the methionine cycle, and serving as a lead compound for optimization toward more potent and selective MAT2A inhibitors for anticancer drug development.

C15H13CLFN

261.721826314926

1391934-98-7

57521314

Light yellow to yellow solid

1

2

3

18

274

0

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

FIDAS5; FIDAS 5

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.

---

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.

---

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