IC50: 64.5 μM (METTL3-METTL14)[1]
METTL3-METTL14 (methyltransferase-like 3 and 14 complex). STM2120 targets the METTL3-METTL14 heterodimer complex, which is the primary enzyme responsible for catalyzing N⁶-methyladenosine (m⁶A) modification of RNA. By inhibiting this complex, STM2120 disrupts the methylation of adenosine residues in RNA, affecting RNA stability, splicing, and translation. The compound has an IC₅₀ of 64.5 μM against METTL3-METTL14. However, it is significantly less potent than STM2457 and is primarily used as a negative control in studies investigating METTL3 function.

STM2120 (0.04-50 μM; 72 h) does not affect the proliferation of MOLM-13 cells. To investigate the therapeutic potential of targeting the enzymatic activity of METTL3 as an anti-leukaemic strategy, we developed the small molecule STM2457. A high throughput screen (HTS) of 250,000 diverse drug-like compounds was carried out. STM1760 [half maximum inhibitory concentration (IC50) = 51.7 μM] was one of only two non-S-adenosyl methionine (SAM) related hits arising from the HTS (Extended Data Fig. 1a, b). After optimisation of potency, in vitro ADME and in vivo pharmacokinetic properties, we identified STM2457 (Fig. 1a). STM2120 (IC50 = 64.5 μM) was identified as structurally related, but 1,000 fold less active compared to STM2457 (Fig. 1b and Extended Data Fig. 1a)[1].

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STM2120 shows weak inhibitory activity against METTL3-METTL14 in biochemical enzyme assays, with an IC₅₀ of 64.5 μM. In cell-based assays, STM2120 (0.04-50 μM; 72 h) does not affect the proliferation of MOLM-13 cells. This lack of cellular activity is consistent with its role as a negative control, as it is approximately 1,000-fold less active than the potent METTL3 inhibitor STM2457. The compound serves as a valuable tool to distinguish specific METTL3 inhibition effects from non-specific effects in cellular studies.

STM2120 is not intended for in vivo therapeutic use, serving primarily as a negative control tool compound. Its weak activity (IC₅₀ = 64.5 μM) and 1,000-fold lower potency compared to STM2457 make it unsuitable for achieving meaningful in vivo METTL3 inhibition. It is used in research settings to validate that observed biological effects are specifically due to METTL3 inhibition rather than off-target effects of more potent compounds.

METTL3/14 RF/MS methyltransferase assay[1]
The enzymatic assay was established to determine IC50 values for the inhibition of RNA methyltransferase activity. The enzyme used was full-length his-tagged METTL3 co-expressed with full length FLAG-tagged METTL14 in a baculovirus expression system. The enzyme complex was purified using standard affinity chromatography. Enzymatic reactions were performed at room temperature in 384-well plates using a final reaction volume of 20 μL containing 20 mM TrisCl pH 7.6, 1 mM DTT, 0.01% Tween-20. 5 nM final concentration of METTL3/14 was pre-incubated with various compound concentrations for 10 minutes, followed by addition of 0.2 μM final concentration synthetic RNA substrate (5’P-uacacucgaucuggacuaaagcugcuc-3’) and 0.5 μM final concentration S-adenosyl methionine (SAM). The reaction was incubated for further 60 minutes at room temperature, and then quenched by the addition of 40 μL 7.5% TCA with internal standard. After termination, plates were sealed, centrifuged and stored at 4°C until analysis. METTL3 activity was assessed using the RapidFire™ mass spectrometry (RF/MS) platform to measure the S-adenosyl homocysteine (SAH) product. Stopped and stable assay plates were analyzed on the Agilent RF300 integrated autosampler/solid-phase extraction (SPE) system coupled to an ABSciex 4000 mass spectrometer for the quantification of the SAH and normalized to the ratio of signal of two internal standards. The mass transition for the product (SAH) was 384.9/135.9 Da. Transitions of the internal standard were used for normalization of matrix effects.[2]

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Non-cell-based assays for STM2120 involve METTL3/14 methyltransferase activity measurements using the RapidFire™ mass spectrometry (RF/MS) platform. The enzymatic assay uses full-length his-tagged METTL3 co-expressed with FLAG-tagged METTL14 in a baculovirus system. Reactions are performed in 384-well plates containing 20 mM TrisCl pH 7.6, 1 mM DTT, 0.01% Tween-20, with 5 nM METTL3/14 pre-incubated with compound for 10 minutes, followed by addition of 0.2 μM RNA substrate and 0.5 μM SAM. After 60 minutes, reactions are quenched with 7.5% TCA and analyzed for SAH product by RF/MS.

Drug and Proliferation Assays[1]
All suspension cells were plated in 96-well plates in triplicate at 5,000–10,000 cells per well and treated for 72 hours with vehicle or the indicated concentrations of STM2457 and STM2120 (0.04-50 μM). On day 4, an equal volume for all wells was split using fresh media and compound, such that the resulting cell density in each well matched the initial seeding density. Plates were measured on day 6 using CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay in order to calculate the relative cell proliferation. All the compounds were dissolved in DMSO.

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Cell-based assays for STM2120 typically use MOLM-13 leukemia cells to assess its effects on cell proliferation. Cells are treated with STM2120 at concentrations ranging from 0.04 to 50 μM for 72 hours, and proliferation is measured using standard cell viability assays. The compound shows no effect on MOLM-13 cell proliferation, confirming its inactivity as a negative control. Additional cell-based studies may involve assessing m⁶A RNA methylation levels following compound treatment to confirm lack of METTL3 inhibition in cellular contexts.

In vivo studies of STM2120 are not commonly performed due to its use as a negative control compound with weak activity. The compound is typically not administered to animals for therapeutic or pharmacological studies. Instead, it serves as a reference compound in biochemical and cell-based assays to validate the specificity of METTL3 inhibition by more potent compounds such as STM2457.

STM2120 has a molecular weight of 333.34 g/mol and a molecular formula of C₁₈H₁₅N₅O₂. It has a LogP of 1.4 and a tPSA of 79.1 Ų. The compound is soluble in DMSO (100 mg/mL, ~300 mM). It appears as an off-white to light yellow solid. Storage recommendations: powder at -20°C for 3 years or 4°C for 2 years; in solvent at -80°C for 6 months or -20°C for 1 month. Purity is ≥98%.

Specific toxicity data for STM2120 are limited. As a research compound used primarily as a negative control, comprehensive toxicological studies have not been reported. The compound shows no effect on MOLM-13 cell proliferation at concentrations up to 50 μM, suggesting low cytotoxicity in this cell line. Standard laboratory safety practices should be followed when handling this compound, including the use of personal protective equipment. It is intended for research use only and is not for human consumption.

[1]. Small-molecule inhibition of METTL3 as a strategy against myeloid leukaemia. Nature. 2021 May;593(7860):597-601.

N6-methyladenosine (m6A) is an abundant intraRNA modification^1,2, primarily catalyzed by the METTL3-METTL14 methyltransferase complex^3,4. The m6A methyltransferase METTL3 is closely related to the occurrence and maintenance of acute myeloid leukemia (AML), but the therapeutic potential of targeting this enzyme remains unclear^5-7. This article reports the identification and characterization of STM2457, a highly efficient and selective first-in-class METTL3 catalytic inhibitor, and resolves the crystal structure of the STM2457-METTL3-METTL14 complex. STM2457 treatment of tumors inhibits AML cell growth and promotes cell differentiation and apoptosis. These cellular effects are accompanied by a selective reduction in m6A levels on known leukemic mRNAs and a decrease in the expression of these mRNAs, consistent with translational defects. We demonstrated that pharmacological inhibition of METTL3 in vivo led to transplant failure and prolonged survival in various mouse models of acute myeloid leukemia (AML), particularly targeting key stem cell subpopulations of AML. Overall, these results suggest that inhibiting METTL3 is a potential strategy for treating AML and provide proof of concept for targeting RNA-modifying enzymes as a promising approach to anticancer therapy. [1]

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STM2120 is a METTL3-METTL14 inhibitor developed as a negative control for the potent METTL3 inhibitor STM2457. It was identified from a high-throughput screen of 250,000 drug-like compounds, where STM1760 (IC₅₀ = 51.7 μM) was one of only two non-SAM-related hits. STM2120 (IC₅₀ = 64.5 μM) is structurally related but 1,000-fold less active than STM2457. METTL3 is the catalytic subunit of the m⁶A methyltransferase complex, and its inhibition is being explored as an anti-leukemic strategy. The compound is for research use only.

C18H15N5O2

333.344002962112

333.122574

2762286-04-2

168355665

Typically exists as Off-white to light yellow solids at room temperature

1.4

1

4

3

25

708

0

O=C(C1=CC(N2C=CC=CC2=N1)=O)NCC1=CN2C(C)=CC=CC2=N1

BOTBQIXFHWFWHV-UHFFFAOYSA-N

InChI=1S/C18H15N5O2/c1-12-5-4-7-15-20-13(11-23(12)15)10-19-18(25)14-9-17(24)22-8-3-2-6-16(22)21-14/h2-9,11H,10H2,1H3,(H,19,25)

N-[(5-methylimidazo[1,2-a]pyridin-2-yl)methyl]-4-oxopyrido[1,2-a]pyrimidine-2-carboxamide

STM-2120; EX-A8309; DA-67811; STM2120; EXA8309; DA67811;

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)

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

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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