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| Targets |
MDM2-MDMX RING domain heterocomplex E3 ligase activity. MEL24 is structurally similar to MEL23 and was identified as a validated Mdm2 inhibitor. The compound has an EC50 of 3.0 μg/mL (9.2 μM) in the cell-based Mdm2(wt)-luciferase auto-ubiquitination assay [1].
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| ln Vitro |
In Vitro: In cell-based assays using Mdm2(wt)-luciferase and Mdm2(C464A)-luciferase stable cell lines, MEL24 (5.33 μg/mL, 2 h) increased luminescence of the wild-type but not the mutant cell line, indicating specific inhibition of Mdm2 E3 ligase activity. EC50 was 3.0 μg/mL (9.2 μM). In RKO and HCT116 cells, MEL24 (5 μg/mL, 6 h) increased Mdm2 and p53 protein levels to similar levels as MG132 treatment. MEL24 also prevented etoposide-induced MdmX degradation in MCF7 cells [1].
In p53-null H1299 cells, MEL24 (5 μg/mL, 6 h) stabilized Flag-Mdm2 protein, while doxorubicin had no effect, indicating p53-independent Mdm2 stabilization. MEL24 did not induce p53 Ser15 phosphorylation or histone H2A.X Ser139 phosphorylation, indicating no genotoxic stress response. MEL24 increased p53 levels in RKO cells (wild-type p53) but not in RKO-E6 cells (where p53 is degraded by HPV-E6), confirming specificity for Mdm2-mediated p53 regulation [1]. In vitro ubiquitination assays showed that MEL24 inhibited ubiquitination of Mdm2 and p53 in cell-based systems. In Mdm2(wt)-luciferase cells transfected with HA-ubiquitin, MEL24 (10 μg/mL) significantly reduced Mdm2-ubiquitin conjugates. In H1299 cells co-transfected with Flag-Mdm2, p53, and HA-ubiquitin, MEL24 (10 μg/mL) inhibited p53 ubiquitination [1]. MEL24 has an ED50 value of 9.2 μM and acts on 293T cells [1]. In HCT116 cells (p53 wild type), MEL24 (15 μM, 6 hours) activates p53, raises Mdm2, p53, and MdmX levels, and prevents the degradation of endogenous MdmX [1]. |
| Enzyme Assay |
Enzyme Assay: In vitro ubiquitination assays were performed using purified full-length Flag-Mdm2 and HA-MdmX proteins. Reactions contained E1, UbcH5C, ³²P-labeled ubiquitin, and ATP. MEL24 was tested at increasing concentrations. The products were resolved by SDS-PAGE and analyzed by autoradiography. MEL24 inhibited the E3 ligase activity of the Mdm2-MdmX hetero-complex [1].
For the cell-based high-throughput screen, Mdm2(wt)-luciferase stable 293T cells were seeded at 7,500 cells/well in 384-well plates. After 24 h, compounds (final concentration 5.33 μg/mL) were added for 2 h, followed by lysis with luminescence buffer and reading on a Victor3 Plate Reader. Counter-screen used Mdm2(C464A)-luciferase cells [1]. |
| Cell Assay |
Cell Assay: For the Mdm2-luciferase assay, 293T cells stably expressing Mdm2(wt)-luciferase or Mdm2(C464A)-luciferase were seeded in 384-well plates (7,500 cells/well). After 24 h, cells were treated with MEL24 for 2 h, lysed with luminescence buffer, and luminescence was measured. EC50 was calculated using GraphPad Prism [1].
For Western blotting, cells (RKO, HCT116, H1299, MCF7) were lysed in buffer containing 50 mM Tris-HCl pH 7.5, 200 mM NaCl, 0.5% NP-40, and protease inhibitors. Proteins were separated by SDS-PAGE, transferred to nitrocellulose, and probed with antibodies against Mdm2, p53, MdmX, and actin [1]. For cell viability assays comparing MEL23 and MEL24, NALM6 cells were treated with a dilution series of each compound for 72 h, and resazurin was added for 2 h before OD600 measurement. The IC50 of MEL24 in NALM6 cells was 4.58 μM (from the Frontiers in Oncology paper, not from the primary MEL24 papers) [2]. For ubiquitination assays, Mdm2(wt)-luciferase cells were transfected with HA-ubiquitin (5 μg). After 8 h, cells were treated with 10 μg/mL MEL24 or DMSO for 16 h, then additionally with 10 μM MG132 for 3 h. Cells were lysed and immunoprecipitated with anti-HA affinity matrix, followed by Western blotting with anti-Mdm2 antibody. For p53 ubiquitination, H1299 cells were transfected with Flag-Mdm2 (5 μg), p53 (0.5 μg), and HA-ubiquitin (2 μg), then treated similarly [1]. |
| Toxicity/Toxicokinetics |
MEL24 did not induce p53 Ser15 phosphorylation or histone H2A.X Ser139 phosphorylation in RKO cells at 5 μg/mL for 6 h, indicating that it does not cause genotoxic stress. In p53-null H1299 cells, MEL24 (5 μg/mL, 6 h) stabilized Flag-Mdm2 without affecting cell viability under normal culture conditions [1].
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| References | |
| Additional Infomation |
MEL24 is a small molecule containing tetrahydro-beta-carboline and barbituric acid moieties, structurally similar to MEL23 with slightly different substituents. It was identified as a validated Mdm2 E3 ligase inhibitor from a screen of 270,080 compounds using a novel cell-based Mdm2 auto-ubiquitination assay. Both the tetrahydro-beta-carboline and barbituric acid moieties are necessary for activity. MEL24, like MEL23, preferentially inhibits the E3 ligase activity of the MDM2-MDMX heterocomplex. The compound increases Mdm2 and p53 protein levels in wild-type p53 cells, and increases Mdm2 levels in p53-null cells, indicating p53-independent activity. MEL24 does not induce DNA damage response markers, distinguishing it from genotoxic agents like doxorubicin [1][2].
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| Molecular Formula |
C17H18N4O3
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| Molecular Weight |
326.349823474884
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| Exact Mass |
326.137
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| CAS # |
642072-48-8
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| PubChem CID |
71451689
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| Appearance |
White to off-white solid powder
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| LogP |
0.5
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
1
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| Heavy Atom Count |
24
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| Complexity |
556
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1(=O)N(C)C(=O)C(C2C3=C(CCN2)C2=C(N3)C=CC=C2)C(=O)N1C
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| InChi Key |
FOUOSAVQDWJOGI-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C17H18N4O3/c1-20-15(22)12(16(23)21(2)17(20)24)14-13-10(7-8-18-14)9-5-3-4-6-11(9)19-13/h3-6,12,14,18-19H,7-8H2,1-2H3
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| Chemical Name |
1,3-dimethyl-5-(2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-1,3-diazinane-2,4,6-trione
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| Synonyms |
MEL24; MEL-24
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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 |
| 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 | 3.0642 mL | 15.3210 mL | 30.6419 mL | |
| 5 mM | 0.6128 mL | 3.0642 mL | 6.1284 mL | |
| 10 mM | 0.3064 mL | 1.5321 mL | 3.0642 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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