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| Targets |
The target is the SET and MYND domain-containing protein 3 (SMYD3), a histone methyltransferase that methylates histone H3 at lysine 4 (H3K4) and also non-histone proteins. EM-127 is a covalent inhibitor, meaning it forms a permanent chemical bond with its target, leading to sustained inhibition. This is indicated by its KD value of 13 microM.
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| ln Vitro |
In MDA-MB-231 and HCT116 cells, EM127 (5 μM; 24, 48, and 72 hours) exhibits strong anti-proliferative action [1]. When SMYD3 is expressed at low levels or knocked out in MDA-MB-231 cells, EM127 (5 μM; 24, 48, and 72 hours) does not influence expression. However, it can reduce the expression of SMYD3 target genes [1]. In HCT116 and MDA-MB-231 cells, EM127 (1, 3.5, 5 μM; 48, 72 hours) decreases ERK1/2 phosphorylation in a dose- and time-dependent manner [1].
In cellular assays, EM-127 at 5 microM exhibits potent anti-proliferative effects in MDA-MB-231 (breast cancer) and HCT116 (colon cancer) cell lines. It effectively inhibits ERK1/2 phosphorylation in a dose- and time-dependent manner and reduces the transcriptional regulation of SMYD3 target genes. This effect is not observed when SMYD3 is knocked down, confirming target specificity. |
| ln Vivo |
While specific in vivo data for EM-127 are not widely published, its potent in vitro anti-proliferative activity and mechanism of action suggest a potential for in vivo efficacy. As a covalent inhibitor, it would provide sustained target engagement in vivo. Studies would typically involve mouse xenograft models of SMYD3-positive breast or colon cancer to evaluate tumor growth inhibition.
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| Enzyme Assay |
Although specific protocols for EM-127 are not detailed, a typical biochemical assay for a covalent SMYD3 inhibitor involves a time-dependent assay. Purified recombinant SMYD3 enzyme is pre-incubated with the compound for various periods (e.g., 0-60 min), then diluted to a concentration where the compound is below its KD. The remaining methyltransferase activity is measured using a peptide substrate and 3H-SAM, followed by detection of 3H-labeled histone peptide on a filter plate.
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| Cell Assay |
Cell proliferation assay [1]
Cell Types: MDA-MB-231, HCT116 Cell Tested Concentrations: 5 μM Incubation Duration: 24, 48, 72 hrs (hours) Experimental Results: Cell proliferation was Dramatically delayed by 48 hrs (hours). RT-PCR[1] Cell Types: MDA-MB-231 Cell Tested Concentrations: 0.5, 3.5, 5 μM Incubation Duration: 48 hrs (hours) Experimental Results: Dramatically diminished expression of known SMYD3 regulatory genes CDK2 and C-MET. diminished mRNA abundance of extracellular matrix components fibronectin 1 (FN1) and N-cadherin (N-CAD). Western Blot Analysis [1] Cell Types: HCT116, MDA-MB-231 Cell Tested Concentrations: 1, 3.5, 5 μM Incubation Duration: 48, 72 hrs (hours) Experimental Results: At the same concentration, ERK1/2 phosphorylation attenuates and induces PARP processing, Thereby delaying cell proliferation. To evaluate EM-127's cellular activity, MDA-MB-231 or HCT116 cells are seeded in 96-well plates and treated with varying concentrations of EM-127 (e.g., 0.1-10 microM) for 48-72 hours. Cell viability is assessed using a CellTiter-Glo luminescent assay. For mechanistic studies, cells are treated with 5 microM EM-127 for 24-72 hours, lysed, and analyzed by Western blot using antibodies against total-ERK1/2, phospho-ERK1/2, cleaved PARP, and SMYD3 target gene products. |
| Animal Protocol |
No detailed in vivo animal protocols are available. A proposed study would involve establishing MDA-MB-231 xenografts in female nude mice. Once tumors reach a certain size (e.g., 100-150 mm3), mice would be randomized into treatment and control groups. EM-127 would be administered via a suitable route (e.g., intraperitoneal or intravenous) daily or every other day. Tumor volume and body weight would be measured twice weekly. At study termination, tumors would be excised for analysis of SMYD3 target gene expression and ERK1/2 phosphorylation.
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| ADME/Pharmacokinetics |
Specific pharmacokinetic properties of EM-127 are not detailed in the available literature. However, as a covalent inhibitor, its pharmacodynamic effect (duration of target inhibition) is expected to outlast its pharmacokinetic presence. This "hit-and-run" mechanism is a feature of irreversible inhibitors, potentially allowing for durable efficacy with intermittent dosing.
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| Toxicity/Toxicokinetics |
Detailed toxicological data for EM-127 are not specified in the publicly available literature. Initial safety assessments would typically be conducted during in vivo efficacy studies by monitoring body weight, overall animal health, and performing histopathological analysis of major organs. As a covalent modifier, off-target selectivity is a critical factor in its safety profile.
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| References | |
| Additional Infomation |
SMYD3 is a histone methyltransferase overexpressed in various cancers, making it a promising anticancer drug target. As a covalent inhibitor, EM-127 offers prolonged target engagement and the potential for sustained efficacy. As of the latest updates, EM-127 is a research-grade chemical and has not yet been evaluated in clinical trials or approved for sale.
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| Molecular Formula |
C14H18CLN3O3
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| Molecular Weight |
311.76
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| Exact Mass |
311.103
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| CAS # |
1886879-71-5
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| PubChem CID |
118949515
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| Appearance |
White to off-white solid powder
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| LogP |
1.2
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
21
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| Complexity |
408
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1C(C(NC2CCN(C(CCl)=O)CC2)=O)=NOC=1C1CC1
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| InChi Key |
OENTXAYVUCLRJQ-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C14H18ClN3O3/c15-8-13(19)18-5-3-10(4-6-18)16-14(20)11-7-12(21-17-11)9-1-2-9/h7,9-10H,1-6,8H2,(H,16,20)
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| Chemical Name |
N-[1-(2-chloroacetyl)piperidin-4-yl]-5-cyclopropyl-1,2-oxazole-3-carboxamide
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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. |
| 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) |
DMSO : ~25 mg/mL (~80.19 mM)
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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 | 3.2076 mL | 16.0380 mL | 32.0760 mL | |
| 5 mM | 0.6415 mL | 3.2076 mL | 6.4152 mL | |
| 10 mM | 0.3208 mL | 1.6038 mL | 3.2076 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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