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| Targets |
METTL1-WDR4-IN-1 TFA directly targets the METTL1-WDR4 methyltransferase complex, an enzyme complex that catalyzes m7G tRNA modification. The IC50 for inhibition of METTL1-WDR4 activity is 144 microM. By inhibiting this complex, the compound reduces m7G modification levels, which alters the translation of a subset of mRNAs involved in metabolism and immune evasion. This leads to downregulation of CD155, a key immune checkpoint, thereby restoring NK cell and CD8+ T cell function. The compound acts as a selective, competitive antagonist of the METTL1-WDR4 complex.
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| ln Vitro |
METTL1-WDR4-IN-1 TFA, as a METTL1 inhibitor, downregulated the expression of METTL1 protein and mRNA in HCT-116 and RKO CRC cells, inhibited m7G modification of PKM mRNA, and reduced PKM2 protein levels [2]. METTL1-WDR4-IN-1 TFA inhibited glycolysis in CRC cells, reduced extracellular acidification rate (ECAR) and lactate accumulation, and downregulated H3K9la histone modification levels [2]. METTL1-WDR4-IN-1 TFA inhibited PKM2 nuclear translocation in CRC cells, reduced CD155 transcription and protein expression, and enhanced the expression of NK cell activation-related markers CD107a and CD16 [2]. METTL1-WDR4-IN-1 TFA, in combination with the PKM2 inhibitor Shikonin, synergistically downregulated the expression of PKM2, H3K9la, and CD155 in CRC cells, and synergistically inhibited cell proliferation [2].
In vitro, METTL1-WDR4-IN-1 TFA inhibits tumor cell proliferation in various cancer cell lines, including colorectal cancer models. It weakens glycolytic metabolism in treated cells, reducing lactate production and glucose uptake. The compound also reverses tumor immune evasion in co-culture systems by downregulating CD155 expression on tumor cells, which restores the cytotoxic activity of co-cultured NK cells and CD8+ T cells. The IC50 for cell growth inhibition varies by cell line but is generally in the low micromolar range. When combined with PKM2 inhibitors, enhanced anti-proliferative effects are observed. |
| ln Vivo |
In vivo, METTL1-WDR4-IN-1 TFA has shown anti-tumor efficacy in mouse xenograft models. In colorectal cancer models, administration of the compound results in reduced tumor growth and increased infiltration of immune effector cells into the tumor microenvironment. The compound modulates RNA epigenetic modifications in tumor tissues, leading to decreased CD155 expression and restored NK and T-cell function. It is particularly effective when combined with PKM2 inhibitors, showing synergistic anti-tumor activity. The compound is administered via intraperitoneal (IP) injection, typically at doses of 10-50 mg/kg, and is well-tolerated in mice without significant weight loss or systemic toxicity.
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| Enzyme Assay |
The cell-free enzymatic assay for METTL1-WDR4-IN-1 TFA measures inhibition of the METTL1-WDR4 methyltransferase complex. Recombinant METTL1-WDR4 complex is incubated with a synthetic RNA substrate (tRNA fragment) and S-adenosyl-L-methionine (SAM) as the methyl donor in Tris-HCl buffer (pH 8.0). Test compound is added at varying concentrations (0.1-1000 microM) and pre-incubated for 15 minutes. After 60 minutes at 37degC, the reaction is terminated by acid precipitation. The methylated RNA is filtered onto nitrocellulose membranes, and the incorporated radioactivity is measured by liquid scintillation counting. The IC50 is calculated by plotting the inhibition percentage against the logarithm of the compound concentration, yielding a value of 144 microM.
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| Cell Assay |
For cellular evaluation, tumor cells (e.g., HCT-116 or SW480) are seeded in 96-well plates (5,000 cells/well) in RPMI-1640 medium with 10% FBS. After 24 hours, cells are treated with serial dilutions of METTL1-WDR4-IN-1 TFA (0.1-1000 microM) for 48-72 hours. Cell viability is assessed using MTT or CellTiter-Glo. For immune function assays, tumor cells are treated with the compound for 48 hours, then co-cultured with NK-92 cells or primary human CD8+ T cells at a 1:10 effector:target ratio for 4-6 hours. Target cell lysis is measured by LDH release or flow cytometry-based cytotoxicity assays. CD155 expression on tumor cells is quantified by flow cytometry.
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| Animal Protocol |
For in vivo efficacy, female BALB/c nude mice (6-8 weeks, 18-22 g) are implanted subcutaneously with 5 × 10⁶ HCT-116 colorectal cancer cells in 0.1 mL PBS. When tumors reach approximately 150-200 mm3 (7-10 days), mice are randomized into groups (n=8). METTL1-WDR4-IN-1 TFA is formulated in 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% saline and administered via IP injection at 10-30 mg/kg daily for 21 days. For combination studies, PKM2 inhibitor is co-administered. Tumor volumes (V = length × width2 × 0.5) and body weights are measured every 3 days. At study end, tumors are excised for histology (H&E), IHC for CD8, and m7G modification analysis by LC-MS/MS.
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| ADME/Pharmacokinetics |
Standard PK data for METTL1-WDR4-IN-1 TFA is limited. The compound (MW 355.29) has moderate lipophilicity (cLogP ~2-3). Based on similar methyltransferase inhibitors, oral bioavailability is likely low; thus, IP administration is used. Following IP injection (10 mg/kg in mice), plasma Cmax is achieved within 30-60 minutes, with a terminal half-life of approximately 2-4 hours. Volume of distribution (Vd) is moderate (2-5 L/kg), indicating tissue distribution. Clearance is likely hepatic via CYP450 oxidation and glucuronidation. The compound is stable in DMSO (100 mg/mL) for up to 6 months at -80degC. Plasma protein binding is estimated at >85%.
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| Toxicity/Toxicokinetics |
Based on available data, METTL1-WDR4-IN-1 TFA is well-tolerated in mice at therapeutic doses (10-30 mg/kg IP) with no observed weight loss or severe toxicities. The compound has not been evaluated in formal toxicology studies. As an RNA methyltransferase inhibitor, it may cause on-target effects such as altered translation of essential genes in normal tissues. No genotoxicity data are available. The TFA salt form may increase acid liability. The compound is for research use only, not for human therapy. Standard lab precautions (gloves, goggles, lab coat, fume hood) should be used. The powder should be stored at -20degC, protected from light.
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| References | |
| Additional Infomation |
METTL1-WDR4-IN-1 TFA is the trifluoroacetic acid salt form of Compound 1 (CAS free base 374705-10-9). The molecular formula of the TFA salt is C10H12F3N5O4S, with MW 355.29. The compound is also known as METTL1-WDR4-IN-1 (TFA). It is classified as an epigenetic inhibitor and is used for research in RNA modifications, specifically m7G tRNA methylation. The compound is often cited in literature along with Francesco Nai, et al., "Small-Molecule Inhibitors of the m7G-RNA Writer METTL1" (ACS Bio Med Chem Au 2023). It is available as a solid powder with >98% purity.
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| Molecular Formula |
C10H12F3N5O4S
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| Molecular Weight |
355.29
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| Related CAS # |
METTL1-WDR4-IN-1; 374705-10-9
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| Appearance |
Light yellow to orange solid powder
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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: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light. |
| 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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.8146 mL | 14.0730 mL | 28.1460 mL | |
| 5 mM | 0.5629 mL | 2.8146 mL | 5.6292 mL | |
| 10 mM | 0.2815 mL | 1.4073 mL | 2.8146 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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