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| Targets |
Mdm2-MdmX RING domain interaction. MMRi64 disrupts the Mdm2-MdmX RING-RING interaction, inhibiting the E3 ligase activity of the Mdm2-MdmX heterodimer complex toward Mdm2 and p53 substrates. It does not inhibit Mdm2 RING domain homodimer E3 activity or NEDD4-1 autoubiquitination [1].
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| ln Vitro |
In Vitro: In biochemical assays, MMRi64 (10 μM) effectively inhibited MdmX-stimulated Mdm2 autoubiquitination and Mdm2-MdmX-mediated p53 polyubiquitination, similar to MMRi6. In pulldown experiments using recombinant FLAG-MdmX and HA-Mdm2 RING proteins, MMRi64 effectively inhibited Mdm2-MdmX interaction in vitro [1].
In HCT-8 colon cancer cells, MMRi64 (0.31-5 μM) induced time- and concentration-dependent p53 accumulation and Mdm2 induction, as well as significant downregulation of MdmX. In pre-B acute lymphoblastic leukemia NALM6 cells, MMRi64 (1-10 μM) activated p53 in time- and concentration-dependent manners and strongly reduced Mdm2 expression (in contrast to HCT-8 cells) and MdmX levels [1]. In NALM6 cells, MMRi64 (1 μM) induced strong PUMA expression but only transient p21 induction that decreased below basal levels by 24 h. PARP cleavage was evident at 8 h and further increased at 24 h, indicating activation of the intrinsic apoptosis pathway. In contrast, Nutlin3a at the same concentration induced stronger p53 accumulation and PUMA induction but stronger p21 expression, and PARP cleavage was barely detectable at 24 h [1]. In wt-p53 Emu-myc mouse lymphoma cells, MMRi64 (0.1-2 μM) induced p53 accumulation at concentrations as low as 0.1 μM and PARP cleavage at ~0.5 μM, while p53-null Emu-myc cells showed no PARP cleavage. Flow cytometry showed that MMRi64 at 0.5 and 1 μM for 48 h induced 7.3% and 20% sub-G1 populations, respectively, while Nutlin3a at 0.5, 1 and 2 μM induced only 0.4%, 0.8% and 3.0% sub-G1 populations, respectively [1]. In growth inhibition assays, p53 contributed to a maximal ~10% more growth inhibition by MMRi64 in HCT116 cells compared to HCT116-p53-/- cells. Combination of MMRi64 (0.2-0.4 μM) with Nutlin3a (2 μM) synergistically induced apoptosis, increasing sub-G1 populations from 2.5% (MMRi64 alone) and 1.3% (Nutlin3a alone) to 8.7% and 16% for the two combinations, respectively [1]. |
| Enzyme Assay |
Enzyme Assay: FRET-based in vitro ubiquitination assay was used to measure MdmX-stimulated Mdm2 autoubiquitination. Pre-reaction mixture contained 40 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 2 mM DTT, 5 mM ATP, 20 nM E1, 350 nM E2 (UbcH5), 25 nM HA-tagged Mdm2, 200 nM MdmX. MMRi64 (10 μM) was added and reaction was started by adding HA-ubiquitin and ubiquitin cryptate. After incubation at 37°C for 1.5 h, FRET signals were measured. For validation, in vitro ubiquitination assays were performed with 100 nM HA-Mdm2, 200 nM MdmX, and 100 nM p53 (for p53 assay) in the presence of 10 μM compound at 30°C for 1 h, followed by SDS-PAGE and WB. NEDD4-1 autoubiquitination (200 nM) served as specificity control [1].
For pulldown experiments, HA-Mdm2 RING domain (500 nM) and Flag-MdmX (250 nM) with 10 μM MMRi64 were incubated in NP40 buffer for 30 min, then diluted and pulled down with anti-FLAG M2 beads. Bound proteins were eluted with 3×Flag peptides and detected by WB with anti-HA antibody [1]. Docking analysis using DOCK6 program with the 3-D structure of Mdm2-MdmX RING domains indicated that MMRi64 binds to the MdmX RING domain, interfering with its interaction with the Mdm2 RING domain [1]. |
| Cell Assay |
Cell Assay: For growth inhibition assays, HCT116 and HCT116-p53-/- cells were treated with indicated concentrations of MMRi64 for 72 h and growth inhibition was measured by MTT method [1].
For Western blot analysis, HCT-8, NALM6, and Emu-myc cells were treated with MMRi64 at indicated concentrations and times. Whole-cell lysates were analyzed for p53, Mdm2, MdmX, PUMA, p21, PARP, cleaved caspase 3, and tubulin (loading control) using specific antibodies [1]. For flow cytometry apoptosis analysis, NALM6 cells were treated with MMRi64 or Nutlin3a alone or in combination for 48 h, fixed, stained with propidium iodide, and subjected to flow cytometric analysis to quantify sub-G1 populations [1]. |
| References | |
| Additional Infomation |
MMRi64 is an analog of MMRi6, a small molecule inhibitor identified through high-throughput screening of a 55,230-compound diversity library using a FRET-based E3 ligase activity assay. Both compounds specifically target the Mdm2-MdmX RING-RING interaction, a previously unexplored interface for drug development. Unlike Nutlin3a (an Mdm2-p53 binding inhibitor), MMRi64 induces selective activation of the apoptotic arm of the p53 pathway (PUMA induction) with minimal induction of the growth-arrest effector p21. MMRi64 also downregulates Mdm2 and MdmX in leukemia cells, contributing to its pro-apoptotic effects. The compound synergizes with Nutlin3a to induce apoptosis in lymphoma cells. The chemical structure of MMRi64 is shown in Figure 3d of the original paper [1].
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| Molecular Formula |
C22H17CL2N3O
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| Molecular Weight |
410.295882940292
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| Exact Mass |
409.07
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| Elemental Analysis |
C, 64.40; H, 4.18; Cl, 17.28; N, 10.24; O, 3.90
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| CAS # |
430458-66-5
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| PubChem CID |
2907163
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| Appearance |
White to off-white solid powder
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| LogP |
5.9
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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 |
4
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| Heavy Atom Count |
28
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| Complexity |
510
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| Defined Atom Stereocenter Count |
0
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| SMILES |
ClC1C(=CC=CC=1C(C1C=CC2=CC=CN=C2C=1O)NC1C=C(C)C=CN=1)Cl
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| InChi Key |
HQICAVDTVBACIN-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C22H17Cl2N3O/c1-13-9-11-25-18(12-13)27-21(15-5-2-6-17(23)19(15)24)16-8-7-14-4-3-10-26-20(14)22(16)28/h2-12,21,28H,1H3,(H,25,27)
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| Chemical Name |
7-[(2,3-dichlorophenyl)-[(4-methylpyridin-2-yl)amino]methyl]quinolin-8-ol
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| Synonyms |
MMRi64; MMRi-64; MMRi 64;
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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 | 2.4372 mL | 12.1862 mL | 24.3724 mL | |
| 5 mM | 0.4874 mL | 2.4372 mL | 4.8745 mL | |
| 10 mM | 0.2437 mL | 1.2186 mL | 2.4372 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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