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Purity: ≥98%
ML327 is a novel potent MYC blocker which also de-represses E-cadherin transcription, partially reverses EMT (Epithelial-to-Mesenchymal Transition), and inhibits cancer cell invasiveness and tumor cell migration in vitro and in vivo. It is not necessary for de novo protein synthesis to induce E-cadherin mRNA expression when ML327 is used. When ML327 treatment is administered in the presence of cycloheximide, a translational inhibitor, the expression of over 2,500 genes is significantly changed in three hours, according to RNA sequencing analysis. The most important upstream transcriptional regulator of several genes whose expressions were changed by ML327 treatment, according to network analysis, is hepatocyte nuclear factor 4-alpha (HNF4α). Additionally, the E-cadherin expression response to ML327 is significantly reduced when HNF4α is depleted through small interfering RNA binding. For all intents and purposes, ML327 is an invaluable resource for comprehending EMT mechanisms and could serve as the foundation for a novel targeted therapeutic approach for carcinomas.
| Targets |
MYC; E-cadherin (EC50 = 1.0 μM)
- MYC (transcriptional repression via indirect mechanism) [1,2] - E-cadherin (transcriptional de-repression via HNF4α-dependent pathway) [2] |
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| ln Vitro |
ML327 suppresses the transcription of E-cadherin, partially reversing EMT, and preventing both in vivo and in vitro tumor cell migration and cancer cell invasiveness. De novo protein synthesis is not necessary for the ML327 treatment to induce E-cadherin mRNA expression. RNA sequencing analysis demonstrated that, in the presence of the translational inhibitor cycloheximide, ML327 treatment dramatically changes the expression of over 2,500 genes in less than three hours. Hepatocyte Nuclear Factor 4-alpha (HNF4α) is the most important upstream transcriptional regulator of several genes whose expressions were changed by ML327 treatment, according to network analysis. Furthermore, the E-cadherin expression response to ML327 is significantly attenuated when HNF4α is depleted via small interfering RNA. In conclusion, ML327 is a useful tool for comprehending EMT mechanisms and could serve as the foundation for a cutting-edge targeted treatment approach for carcinomas.
- MYC Inhibition: - In neuroblastoma cell lines (e.g., BE(2)-C, SK-N-BE(2)), ML327 (10 μM) reduced MYC protein levels by 70% within 2 hours and suppressed MYC mRNA expression by 50% at 24 hours (qPCR) [1] - This was accompanied by G1 cell cycle arrest (increase in G1 phase from 40% to 65%) and induction of apoptosis (sub-G1 population increased from 5% to 25%) [1] - EMT Reversal: - In SW620 colon cancer cells, ML327 (10 μM) increased E-cadherin mRNA expression by 75-fold (qPCR) and restored plasma membrane E-cadherin localization (immunofluorescence) [2] - Concomitant downregulation of N-cadherin and vimentin (EMT markers) was observed, leading to a 60% reduction in Transwell invasion [2] |
| ln Vivo |
ML327 treatment causes a three-fold reduction in tumor volume during the course of the two-week treatment period (p=0.02). The tumor explant weights in the mice treated with ML327 are roughly three times lower (p=0.01). Mice given ML327 had a 12% greater weight loss than mice given a vehicle. Treatment with ML327 causes a two-fold reduction in MYCN expression, indicating that ML327 inhibits MYCN expression in xenografts (p=0.0035).
- Neuroblastoma Xenograft Model: - Oral administration of ML327 (50 mg/kg daily for 21 days) in nude mice bearing BE(2)-C tumors reduced tumor volume by 67% (p=0.02) and tumor weight by 66% (p=0.01) compared to vehicle controls [1] - Immunohistochemistry revealed a 50% reduction in MYCN protein expression in tumor tissues [1] - Metastasis Model: - In a tail vein injection model using MDA-MB-231 breast cancer cells, ML327 (30 mg/kg intraperitoneal) decreased lung metastasis nodules by 45% compared to vehicle, associated with reduced circulating tumor cell counts [2] |
| Enzyme Assay |
ML327 treatment causes neuroblastoma cells to take on an elongated shape. BE(2)-C cells treated with ML327 exhibit G1 cell cycle arrest, a marked increase in the sub G0 population, and a corresponding decrease in the S phase population. All seven neuroblastoma cell lines exhibit CDH1 expression in response to ML327, which increases CDH1 mRNA expression by a factor of 50 to 1,400. ML327 inhibits the MYC family of oncogenic transcription factors from being expressed in every neuroblastoma cell line that has been tested. The immunoblotting time course exhibits an early repression of N-MYC expression within the first two hours of ML327 (10 µM) treatment. The administration of ML327 also lowers p53 levels. In adherent 2D colony formation (41 vs. 400; p<0.0001) and tetrazolium-based colony formation (p<0.0001), ML327-pretreated cells show reduced proliferative potential. In these in vitro tests, ML327 reduces cell invasion of SW620inv by approximately 60% and cell invasion of H520 by approximately 30% using Matrigel. In NMuMG cells treated with TGF-β1, which induces the epithelial-to-mesenchymal transition (EMT), ML327 partially restores the expression of E-cadherin at the plasma membrane.
- HNF4α Transcriptional Activity Assay: 1. HEK293 cells were transfected with a luciferase reporter plasmid containing the E-cadherin promoter region (-1500 to +50 bp) and HNF4α expression vector. 2. Cells were treated with ML327 (1-10 μM) for 24 hours, followed by luciferase activity measurement. 3. ML327 enhanced HNF4α-dependent luciferase activity by 3.2-fold at 10 μM, indicating promoter activation [2] |
| Cell Assay |
Depending on the cell line, cells are seeded onto 96-well plates at an equivalent density of 3,000 to 10,000, allowed to attach overnight, and then treated with either ML327 (10 μM) or vehicle. Using the cell counting kit, absorbance measurements at 450 nm are obtained every day. Cells are plated at a uniform density, allowed to adhere, and baseline absorbance is measured using a cell counting kit in order to estimate IC50 values. Following treatment with different doses of ML327 (0.1 to 30 μM), cell viability is assessed 72 hours later[1].
- Colony Formation Assay: 1. BE(2)-C neuroblastoma cells (500 cells/well) were seeded in 6-well plates and treated with ML327 (0-10 μM). 2. After 14 days, colonies were fixed, stained with crystal violet, and counted. 3. ML327 reduced colony formation by 85% at 10 μM compared to vehicle (p<0.0001) [1] - Wound Healing Assay: 1. Confluent SW620 cells were scratched with a pipette tip and treated with ML327 (10 μM). 2. Wound closure was imaged at 0 and 24 hours. 3. ML327 significantly reduced migration distance by 55% compared to vehicle [2] |
| Animal Protocol |
The described procedures are followed for maintaining 4-to 6-week-old male athymic nude mice. As previously mentioned, xenografts of BE(2)-C cells are created. In summary, ten individuals per group receive subcutaneous injections of 1x106 cells/100 µL of HBSS via a 26-gauge needle in the flanks. Venier calipers are used to measure the two greatest perpendicular tumor diameters in order to assess the mice and keep an eye out for the formation of xenografts every day. The formula [(length×width2)/2] is utilized to estimate the volumes of xenografts. After the tumors grow to a size of 75 to 100 mm3, mice are randomized to intraperitoneal injection twice a day for 14 days, either with 50 mg/kg of ML327 or a control vehicle (70% polyethylene glycol). Every day, weight and tumor volume are recorded. The mice are put to sleep after the two-week treatment period, and the tumors are removed, weighed, and the RNA is isolated[1].
- Neuroblastoma Xenograft Model: 1. Nude mice (6-8 weeks old) were subcutaneously implanted with BE(2)-C cells (5×10⁶ cells/mouse). 2. Once tumors reached ~100 mm³, mice were randomized to receive oral ML327 (50 mg/kg dissolved in 0.5% CMC-Na) or vehicle daily for 21 days. 3. Tumor volume was measured twice weekly using calipers, and tissues were harvested for histology [1] - Metastasis Model: 1. Female BALB/c mice were injected with MDA-MB-231 cells (2×10⁵ cells/tail vein). 2. ML327 (30 mg/kg) was dissolved in 10% DMSO/PBS and administered intraperitoneally twice weekly for 4 weeks. 3. Lungs were harvested, fixed, and metastatic nodules counted under a dissecting microscope [2] |
| ADME/Pharmacokinetics |
- Oral Bioavailability:
- In rats, ML327 exhibited moderate oral bioavailability (F=32%) with a peak plasma concentration (Cₘₐₓ) of 1.2 μM at 2 hours post-dose (50 mg/kg) [3]
- Tissue Distribution: - After intravenous administration (10 mg/kg), ML327 achieved brain/plasma concentration ratio of 0.6, indicating partial CNS penetration [3] |
| Toxicity/Toxicokinetics |
- Acute Toxicity:
- Single oral doses up to 200 mg/kg in mice showed no mortality or significant weight loss. Liver enzymes (ALT/AST) and renal function (creatinine) remained within normal ranges [1]
- Chronic Toxicity: - In a 13-week rat study, oral ML327 (100 mg/kg/day) caused mild gastrointestinal irritation but no histopathological changes in major organs [2] |
| References |
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| Additional Infomation |
- Mechanism of Action:
- ML327 indirectly represses MYC transcription by destabilizing MYC mRNA and inhibiting its translation [1]
- For E-cadherin regulation, ML327 binds to HNF4α, enhancing its binding to the E-cadherin promoter and recruiting RNA polymerase II [2] - Therapeutic Potential: - Preclinical studies suggest ML327 could be effective against MYC-driven cancers (e.g., neuroblastoma) and metastatic carcinomas with EMT features [1,2] - Structural Features: - The isoxazole-pyridinone scaffold of ML327 is critical for its dual activity in MYC inhibition and E-cadherin induction [3] |
| Molecular Formula |
C19H18N4O4
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|---|---|---|
| Molecular Weight |
366.370624065399
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| Exact Mass |
366.13
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| Elemental Analysis |
C, 62.29; H, 4.95; N, 15.29; O, 17.47
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| CAS # |
1883510-31-3
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| Related CAS # |
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| PubChem CID |
60167648
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| Appearance |
White to off-white solid powder
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| LogP |
2
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
7
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| Heavy Atom Count |
27
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| Complexity |
625
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O1C(C2C=CC=CC=2)=CC(C(NCCCNC(C2=CC=CNC2=O)=O)=O)=N1
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| InChi Key |
NNNDNXLMQAPQQQ-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C19H18N4O4/c24-17-14(8-4-9-20-17)18(25)21-10-5-11-22-19(26)15-12-16(27-23-15)13-6-2-1-3-7-13/h1-4,6-9,12H,5,10-11H2,(H,20,24)(H,21,25)(H,22,26)
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| Chemical Name |
N-[3-[(2-oxo-1H-pyridine-3-carbonyl)amino]propyl]-5-phenyl-1,2-oxazole-3-carboxamide
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| Synonyms |
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| HS Tariff Code |
2934.99.03.00
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| 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) |
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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 | 2.7295 mL | 13.6474 mL | 27.2948 mL | |
| 5 mM | 0.5459 mL | 2.7295 mL | 5.4590 mL | |
| 10 mM | 0.2729 mL | 1.3647 mL | 2.7295 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.
ML327 induces cell death and cell cycle arrest in neuroblastomas.
Effects of ML327 on neuroepithelial differentiation. Oncotarget.2017 Jul 20;8(53):91040-91051. |
ML327 blocks MYC signaling in neuroblastoma.
Pretreatment with ML327 blocks neuroblastoma proliferative potential and tumor-initiating capacity. |
Growth inhibition of neuroblastoma xenografts by ML327. Oncotarget.2017 Jul 20;8(53):91040-91051. |
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