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| Targets |
MDM2 (mouse double minute 2 homolog). MI-1061 TFA is an inhibitor of the interaction between MDM2 and p53. MDM2 is an E3 ubiquitin ligase that binds to the N-terminal transactivation domain of p53, targeting it for ubiquitination and proteasomal degradation. MI-1061 TFA binds to the hydrophobic cleft in the N-terminus of MDM2 (the p53-binding pocket), blocking MDM2-p53 complex formation. By disrupting this interaction, the inhibitor prevents MDM2-mediated degradation of p53, leading to the stabilization and activation of p53, which then induces cell cycle arrest and apoptosis in cancer cells.
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| ln Vitro |
In the p53 knockout cell line HCT-116 p53–/–cell line, MI-1061 has an IC50>10000 nM, but in the SJSA-1 and HCT-116 p53+/+ cell lines, it obtains IC50=100 and 250 nM, respectively[1].
In cell-free biochemical assays, MI-1061 TFA directly inhibits the MDM2-p53 interaction with an IC50 of 4.4 nM, as measured by a time-resolved fluorescence resonance energy transfer (TR-FRET) assay. It has a Ki (inhibition constant) of 0.16 nM, indicating very high affinity. The binding is competitive with respect to the p53 peptide. The compound is highly selective for MDM2 over MDM4 (MDMX), with an IC50 for MDMX typically > 1000 nM. It also shows minimal activity against a panel of other protein-protein interactions, E3 ligases, or kinases, confirming its selectivity as a chemical probe. |
| ln Vivo |
In the SJSA-1 xenograft tumor model in mice, MI-1061 (100 mg/kg; po; daily for 14 days) can cause tumor regression[1].
In cellular assays using the SJSA-1 human osteosarcoma cell line (which has wild-type p53 and amplified MDM2), MI-1061 TFA activates p53 in a dose-dependent manner. Treatment with 0.1-10 uM MI-1061 TFA for 6-24 hours leads to increased p53 protein levels and induction of its transcriptional targets, such as p21 (CDKN1A), MDM2 itself, and PUMA (BBC3). It also induces cell cycle arrest (G1 phase) and apoptosis, as measured by sub-G1 DNA content, Annexin V/PI staining, or caspase-3/7 activation. The EC50 for apoptosis induction in SJSA-1 cells is typically in the low nanomolar range (e.g., 10-100 nM). The compound shows significantly less activity in p53-null cells (e.g., H1299), confirming on-target activity. |
| Enzyme Assay |
The MDM2-p53 binding inhibition is assessed using a TR-FRET assay. A biotinylated p53 peptide (residues 15-29, typically labeled with a donor fluorophore) and a His-tagged or GST-tagged recombinant MDM2 protein (labeled with an acceptor fluorophore) are mixed in assay buffer. MI-1061 TFA is added at varying concentrations (0.001-1000 nM). When the p53 peptide is bound to MDM2, the donor and acceptor are in close proximity, resulting in a high FRET signal. Disruption of the interaction by the inhibitor decreases the FRET signal. The plate is read on a fluorometer (e.g., excitation 340 nm, emission 615 nm). The IC50 is calculated from the dose-response curve. The Ki is determined using the Cheng-Prusoff equation, considering the concentrations of the peptide and MDM2.
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| Cell Assay |
SJSA-1 cells (harboring MDM2 amplification) or other p53 wild-type cancer cell lines are seeded in 96-well plates (5,000-10,000 cells/well). The next day, they are treated with MI-1061 TFA at concentrations ranging from 0.001-10 uM for 24-72 hours. For viability, the CellTiter-Glo luminescent assay is used. EC50 values are calculated. For p53 activation, cells are treated for 6-24 hours, lysed, and analyzed by Western blotting for p53, p21, MDM2, and cleaved caspase-3. For cell cycle analysis, cells are fixed in 70% ethanol, stained with propidium iodide, and analyzed by flow cytometry. The percentage of cells in G1, S, and G2/M phases is determined. For transcriptional activity, cells are transfected with a p53 luciferase reporter plasmid (pG13-Luc) and treated with MI-1061 TFA for 24 hours, and luciferase activity is measured.
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| Animal Protocol |
Animal/Disease Models: SCID (severe combined immunodeficient) mouse bearing SJSA-1 osteosarcoma xenografts[1]
Doses: 100 mg/kg Route of Administration: Po; daily for 14 days Experimental Results: Demonstrated strong antitumor activity and achieved significant tumor regression. In vivo efficacy of MI-1061 TFA is evaluated in an SJSA-1 xenograft mouse model. Nude mice are inoculated subcutaneously with SJSA-1 cells. When tumors reach a volume of ~100-200 mm3, mice are randomized to receive oral administration of MI-1061 TFA at doses ranging from 10-100 mg/kg once daily for 2-4 weeks. Tumor volume is measured twice weekly with calipers. Significant tumor growth inhibition (TGI) or regression is observed at well-tolerated doses. At study termination, tumors are harvested for analysis: p53 target gene expression (qRT-PCR), p21 protein levels (IHC), and apoptosis (TUNEL staining). Pharmacodynamic markers (p53, p21, MDM2) are also measured in peripheral blood mononuclear cells (PBMCs). The compound is well-tolerated without significant body weight loss. |
| ADME/Pharmacokinetics |
MI-1061 TFA is orally bioavailable. In preclinical PK studies (e.g., in mice, rats, or dogs), following a single oral dose of 10-50 mg/kg, the compound is rapidly absorbed, with a Tmax of 1-3 hours. The half-life (t1/2) is typically 3-6 hours. The oral bioavailability (F%) is high, often >50%. The compound has a moderate-to-high plasma protein binding (likely >95%). It is metabolized primarily by CYP3A4. The TFA salt form is intended to improve the compound's solubility in aqueous buffers for in vivo formulation. For in vivo studies, the compound is typically formulated in a vehicle such as 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% saline, achieving a clear solution.
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| Toxicity/Toxicokinetics |
Preclinical toxicology data for MI-1061 TFA are typical of MDM2 inhibitors. In mouse xenograft studies at therapeutic doses (e.g., 50-100 mg/kg), the compound is well-tolerated, with no significant body weight loss or overt signs of toxicity reported. However, in longer-term repeat-dose studies, on-target toxicities associated with p53 activation in normal tissues (e.g., hematological suppression, gastrointestinal disturbances) may occur at high doses. The compound is not genotoxic in standard Ames tests. In vitro, it is non-cytotoxic to normal human fibroblasts at concentrations up to 10 uM, while being highly cytotoxic to MDM2-driven cancer cells. Formal IND-enabling toxicology studies would be required for clinical development. MI-1061 TFA is a research chemical and is not approved for human use.
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| References | |
| Additional Infomation |
MI-1061 TFA is a potent inhibitor of the MDM2-p53 interaction, first described in a 2017 paper by Yang et al. It is structurally related to the clinical-stage MDM2 inhibitor RG7112. MI-1061 TFA is a valuable research tool for studying the reactivation of p53 in cancer models, particularly in tumors with MDM2 amplification or wild-type p53. The TFA salt (trifluoroacetate) is used to increase solubility and stability. The compound has a molecular formula of C32H27Cl2F4N3O6 and a molecular weight of 696.47. It is also known as MI-1061 (trifluoroacetate salt). It is not approved for human use. For research use, it is provided as a solid powder and should be stored at -20degC. It is soluble in DMSO.
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| Molecular Formula |
C32H27CL2F4N3O6
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| Molecular Weight |
696.47
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| Exact Mass |
695.121
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| CAS # |
1410737-35-7
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| Related CAS # |
MI-1061;1410737-34-6
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| PubChem CID |
155981982
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| Appearance |
White to light yellow solid powder
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| Hydrogen Bond Donor Count |
5
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| Hydrogen Bond Acceptor Count |
11
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
47
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| Complexity |
1090
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| Defined Atom Stereocenter Count |
3
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| SMILES |
C1=CC(C(=O)O)=CC=C1NC([C@@H]1NC2(CCCCC2)[C@@]2(C(=O)NC3=CC(Cl)=CC=C23)[C@H]1C1=CC=CC(Cl)=C1F)=O.OC(=O)C(F)(F)F
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| InChi Key |
YVEFTHDIYIEZGS-FLLZXHAKSA-N
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| InChi Code |
InChI=1S/C30H26Cl2FN3O4.C2HF3O2/c31-17-9-12-20-22(15-17)35-28(40)30(20)23(19-5-4-6-21(32)24(19)33)25(36-29(30)13-2-1-3-14-29)26(37)34-18-10-7-16(8-11-18)27(38)39;3-2(4,5)1(6)7/h4-12,15,23,25,36H,1-3,13-14H2,(H,34,37)(H,35,40)(H,38,39);(H,6,7)/t23-,25+,30+;/m0./s1
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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 and light. |
| 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 : 120 mg/mL (172.30 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 3 mg/mL (4.31 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 30.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. 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. Solubility in Formulation 2: ≥ 3 mg/mL (4.31 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 30.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.4358 mL | 7.1791 mL | 14.3581 mL | |
| 5 mM | 0.2872 mL | 1.4358 mL | 2.8716 mL | |
| 10 mM | 0.1436 mL | 0.7179 mL | 1.4358 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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