Mdm2-MdmX RING domain interaction. MMRi6 specifically inhibits the E3 ligase activity of the Mdm2-MdmX heterodimer complex without affecting Mdm2 RING domain homodimer E3 activity or NEDD4-1 autoubiquitination. The compound disrupts the protein-protein interaction between Mdm2 and MdmX RING domains [1].

In Vitro: In biochemical assays, MMRi6 (10 μM) effectively inhibited MdmX-stimulated Mdm2 autoubiquitination and Mdm2-MdmX-mediated p53 polyubiquitination, but did not inhibit NEDD4-1 autoubiquitination, indicating specificity for the Mdm2-MdmX E3 complex. MMRi6 and its analog MMRi64 effectively inhibited Mdm2-MdmX interaction in vitro as demonstrated by pulldown experiments using recombinant FLAG-MdmX and HA-Mdm2 RING proteins. In contrast to MMRi6, the Mdm2-p53 binding inhibitor Nutlin3a had no effect on Mdm2-MdmX-mediated p53 polyubiquitination at the same concentration [1].
In HCT-8 colon cancer cells, MMRi6 (5 μM for 8 h) induced p53 protein accumulation. Its analog 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. In contrast to Nutlin3a, MMRi64 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 increased at 24 h, indicating activation of the intrinsic apoptosis pathway. 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, MMRi6 showed IC50 values of ~0.5 μM in wt-p53 Emu-myc lymphoma cells and ~3 μM in p53-null Emu-myc cells (6-fold difference). In HCT116 colon cancer cells, p53 contributed to a maximal ~10% more growth inhibition by MMRi64 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].

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MMRi6 (10 μM) inhibited Mdm2 autoubiquitination and Mdm2-MdmX-mediated p53 ubiquitination in vitro [1]. MMRi6 (5 μM, 8 h) induced p53 protein accumulation in HCT-8 cells and wt-p53 Emu-myc lymphoma cells [1]. MMRi6 (0.5 μM, 24 h) induced PARP lysis in wt-p53 Emu-myc lymphoma cells [1]. MMRi6 (0.5-1 μM, 72 h) inhibited the growth of wt-p53 and p53-null Emu-myc lymphoma cells, with IC50 values of approximately 0.5 μM and 3 μM, respectively [1].

Enzyme Assay: FRET-based in vitro ubiquitination assay was adapted for HTS. Pre-reaction mixture one 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. Compounds (8 nL each) were added by robot pin tool. Reaction was started by adding pre-mixture two containing 250 nM HA-ubiquitin and 50 nM ubiquitin cryptate (2 μL/well). After incubation at 37°C for 1.5 h, reaction was terminated with detection buffer (50 mM phosphate buffer pH 7.0, 0.1% BSA, 0.1 M EDTA, 0.8 M KF, 20 nM XL665-conjugated anti-HA antibody). FRET signals were measured at 615 nm (donor) and 665 nm (acceptor) after 1 h at room temperature. Z'-factor was determined to be 0.52, indicating a suitable HTS assay [1].
For in vitro validation, Mdm2 autoubiquitination and p53 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. For pulldown experiments, HA-Mdm2 RING domain (500 nM) and Flag-MdmX (250 nM) with 10 μM compound 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 MMRi62 and MMRi64 bind to the MdmX RING domain (gold color), interfering with its interaction with the Mdm2 RING domain (light green) [1].

Cell Assay: For growth inhibition assays, Emu-myc lymphoma cells (wt-p53 and p53-null) were cultured with 0.5 and 1 μM MMRi6 for 72 h, and viable cells were counted by trypan blue exclusion. For HCT116 and HCT116-p53-/- cells, cells were treated with indicated drug concentrations 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 MMRi6 or 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].

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Western Blot Analysis[1]
Cell Types: HCT-8 cells, wt-p53 Emu-myc lymphoma cells and p53-null Emu-myc lymphoma cells
Tested Concentrations: 0.5, 5, 10 μM
Incubation Duration: 8, 24 h
Experimental Results: Induced p53 protein accumulation in HCT-8 cells and in wt-p53 Emu-myc lymphoma cells. Induced PARP cleavage in wt-p53 Emu-myc lymphoma cells but not in p53-null Emu-myc lymphoma cells.

[1]. https://pubmed.ncbi.nlm.nih.gov/26720344/

MMRi6 is a small molecule inhibitor identified through high-throughput screening of a 55,230-compound diversity library (ChemBridge DIVERSet) using a FRET-based E3 ligase activity assay. The compound specifically targets the Mdm2-MdmX RING-RING interaction, a previously unexplored interface for drug development. Among seven specific MMRis identified, MMRi6 and its analog MMRi64 were characterized as disruptors of Mdm2-MdmX interaction. 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. This study reveals that targeting the Mdm2-MdmX RING-RING interaction represents a novel strategy for p53-based cancer therapy [1].

C21H15CL2N3O

396.269302606583

709009-15-4

White to light yellow solid powder

ClC1C2=CC=CN=C2C(=C(C=1)C(C1C=CC=C(C=1)Cl)NC1C=CC=CN=1)O

MMRi6; MMRi-6

2934.99.9001

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 and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~126.18 mM; with sonication)

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.)