MAT2A (Methionine S-adenosyltransferase 2A) (IC50 = 2.1 μM)

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].

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].

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.

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].

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.

[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 for synthesis of S-adenosylmethionine (SAM), the principal methyl donor in many biological processes. MAT2A is up-regulated in many cancers, including liver cancer and colorectal cancer (CRC) and is a potentially important drug target. We developed a family of fluorinated N,N-dialkylaminostilbene agents, called FIDAS agents, that inhibit the proliferation of CRC cells in vitro and in vivo. Using a biotinylated FIDAS analogue, we identified the catalytic subunit of MAT2A as the direct and exclusive binding target of these FIDAS agents. MAT2B, an associated regulatory subunit of MAT2A, binds indirectly to FIDAS agents through its association with MAT2A. FIDAS agents inhibited MAT2A activity in SAM synthesis, and depletion of MAT2A by shRNAs inhibited CRC cell growth. A novel FIDAS agent delivered orally repressed CRC xenografts in athymic nude mice. These findings suggest that FIDAS analogues targeting MAT2A represent a family of novel and potentially useful agents for cancer treatment.[1]

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In summary, researchers developed a novel family of stilbenes analogs, the FIDAS agents, by optimizing their anti-cancer activity. Our studies, as reported here, provide convincing evidence that the direct target of FIDAS analogs is MAT2A, specifically the catalytic subunit responsible for the synthesis of SAM. MAT2A levels are significantly increased in CRC and liver cancers, suggesting that MAT2A is a potentially interesting target for these cancers. We also analyzed the effects of FIDAS agents on other cancer cells, and found that these agents can inhibit the growth of multiple human cancer cell lines, including breast, prostate, lung, liver, carcinoid and head and neck cancer cells. Our finding that FIDAS agents affect SAM synthesis that in turn plays such a central role in numerous biological processes suggests an important mechanism that can be exploited for cancer treatment. The FIDAS agents, which uniquely block the activity of the catalytic subunit of MAT2A, are promising lead candidates and great experimental tools to study the role of MAT2A inhibition in cancer therapy.

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.)