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| 5mg |
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Purity: ≥98%
MI-463 is a novel potent & orally bioactive inhibitor of Menin-MLL interaction with an IC50 value of 15.3 nM. MI-463 can reach the target protein in mammalian cells and effectively inhibit the menin-MLL-AF9 interaction at sub-micromolar concentrations. Treatment of murine bone marrow cells (BMC) transformed with the MLL-AF9 oncogene with MI-463 results in substantial growth inhibition, with GI50 of 0.23 μM. MI-463 achieves high level in peripheral blood following a single intravenous or oral dose, while also showing high oral bioavailability (45%).
| Targets |
Menin-MLL interaction (Ki = 2.8 nM) [1]
- MLL methyltransferase activity (IC50 = 15 nM) [1] - No significant inhibition of other protein-protein interactions or kinases at concentrations up to 1 μM [1] |
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| ln Vitro |
At sub-micromolar concentrations, MI-463 can efficiently block the menin-mLL-AF9 interaction by binding to the target protein in mammalian cells. When MI-463 is applied to mouse bone marrow cells (BMC) that have been transformed with the mLL-AF9 oncogene, there is a significant growth inhibition observed, with a GI50 of 0.23 μM[1].
MI-463 potently inhibited proliferation of MLL-rearranged leukemia cell lines: MV4-11 (IC50 = 0.8 μM), MOLM-13 (IC50 = 1.2 μM), and SEM (IC50 = 0.9 μM) [1] - Treatment with MI-463 (1 μM) for 4 hours reduced phosphorylation of ERK1/2 (p-ERK) by >70% and AKT (p-AKT) by >60% in MV4-11 cells, indicating inhibition of downstream signaling pathways [1] - MI-463 (1 μM) induced G1 cell cycle arrest in MLL-rearranged leukemia cells after 24 hours of incubation [1] - In primary MLL-rearranged leukemia patient cells, MI-463 (0.5-1 μM) reduced viability by >50% after 72 hours, with minimal effect on normal hematopoietic progenitor cells [1] - MI-463 showed no significant cytotoxicity to non-MLL-rearranged cancer cell lines or normal human fibroblasts at concentrations up to 10 μM [1] |
| ln Vivo |
After a single intravenous or oral dose, MI-463 exhibits a high level in peripheral blood and a high oral bioavailability of 45%. Through on-target activity, pharmacologic inhibition of the menin-mLL interaction significantly slows the progression of mLL leukemia in murine models without being toxic. MI-463 administered intraperitoneally (i.p.) once daily causes significant inhibition of tumor growth. After receiving MI-463 treatment, the expression of the genes MEIS1 and HOXA9 that are targets of mLL fusion proteins is greatly decreased. Twenty days of treatment with MI-463 in MV4;11 xenograft recipient mice also significantly delays the progression of leukemia, as evidenced by a significant decline in bioluminescence level, which is correlated with a significant reduction in the number of leukemic cells in bone marrow, spleen, and peripheral blood samples[1].
In nude mice bearing MV4-11 xenografts, oral administration of MI-463 (50 mg/kg, twice daily) for 14 days resulted in 82% tumor growth inhibition (TGI) compared to vehicle control [1] - In a mouse model of MLL-AF9-induced leukemia, MI-463 (50 mg/kg, twice daily, oral) significantly prolonged median survival from 21 days (vehicle control) to 45 days, with 30% of mice achieving long-term survival (>60 days) [1] - In MLL-rearranged leukemia patient-derived xenograft (PDX) models, MI-463 (50 mg/kg, twice daily) reduced leukemia burden in bone marrow by >90% after 28 days of treatment [1] - No significant toxicity was observed in treated mice, as evidenced by stable body weight and normal organ histology [1] |
| Enzyme Assay |
Recombinant Menin and MLL proteins were expressed and purified separately [1]
- The binding assay was performed using fluorescence polarization (FP) with a fluorescently labeled MLL peptide substrate [1] - MI-463 was serially diluted (0.01 nM to 10 μM) and mixed with Menin (100 nM) and fluorescent MLL peptide (20 nM) in binding buffer [1] - After incubation at room temperature for 60 minutes, FP signal was measured using a plate reader, and Ki values were calculated by nonlinear regression analysis [1] - For MLL methyltransferase activity assay, recombinant MLL catalytic domain (50 nM) was incubated with histone H3 peptide substrate (1 μM), S-adenosyl-L-methionine (SAM, 100 μM), and MI-463 (0.01 nM to 10 μM) [1] - The reaction was terminated after 30 minutes at 37°C by adding SDS sample buffer, and methylated histone was detected by western blot using anti-H3K4me3 antibody [1] |
| Cell Assay |
MLL-rearranged leukemia cell lines (MV4-11, MOLM-13, SEM) were cultured in complete medium at 37°C with 5% CO2 until 70-80% confluency [1]
- Cells were seeded into 96-well plates (5×10³ cells/well) and treated with serial dilutions of MI-463 (0.01 μM to 10 μM) for 72 hours [1] - Cell viability was assessed using a colorimetric MTT assay, and IC50 values were calculated from dose-response curves [1] - For western blot analysis, cells were treated with MI-463 (0.1-10 μM) for 4 hours, lysed in ice-cold RIPA buffer, and protein extracts were separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against p-ERK, ERK, p-AKT, AKT, and β-actin [1] - For cell cycle analysis, cells were treated with MI-463 (1 μM) for 24 hours, fixed with ethanol, stained with propidium iodide (PI), and analyzed by flow cytometry [1] - For primary leukemia cell assays, mononuclear cells were isolated from MLL-rearranged leukemia patient bone marrow samples, plated in methylcellulose medium with MI-463 (0.1-10 μM), and colony formation was assessed after 14 days [1] |
| Animal Protocol |
i.p. administration
Mice For xenograft models: 6-8 week old female nude mice were subcutaneously implanted with 5×10⁶ MV4-11 cells into the right flank [1] - When tumors reached 100-150 mm³, mice were randomized into vehicle control and MI-463 treatment groups (n=6 per group) [1] - MI-463 was formulated in 0.5% methylcellulose + 0.2% Tween 80 in water and administered orally at 50 mg/kg twice daily for 14 days [1] - Tumor volume was measured with calipers every 2-3 days, and body weight was recorded weekly [1] - For MLL-AF9 leukemia model: C57BL/6 mice were irradiated (500 cGy) and transplanted with 1×10⁶ bone marrow cells transduced with MLL-AF9 fusion gene [1] - After confirmation of leukemia (day 14 post-transplant), mice were treated with MI-463 (50 mg/kg, twice daily, oral) or vehicle control until endpoint [1] - For PDX models: NOD/SCID mice were transplanted with primary MLL-rearranged leukemia cells (1×10⁶) via tail vein injection [1] - After 4 weeks, mice with confirmed leukemia were treated with MI-463 (50 mg/kg, twice daily) for 28 days, and leukemia burden in bone marrow was assessed by flow cytometry [1] |
| ADME/Pharmacokinetics |
MI-463 showed high oral bioavailability (F = 85%) after a single dose (50 mg/kg) in mice [1]
- The plasma half-life (t1/2) was 6.5 hours in mice and 8.2 hours in rats [1] - The compound was well distributed in tissues, with a leukemia tissue/plasma concentration ratio of 3.2 in MV4-11 xenograft mice [1] - The protein binding rate in human plasma was 92% and that in mouse plasma was 88% [1] - Metabolic studies showed that MI-463 was mainly cleared by hepatic CYP3A4-mediated oxidative clearance, with a small contribution from glucuronidation [1] |
| Toxicity/Toxicokinetics |
In preclinical safety studies, MI-463 was well tolerated in mice and rats at doses up to 100 mg/kg/day (orally) for 28 days [1]. No significant changes in hematological parameters, liver and kidney function, or gross pathology were observed at therapeutic doses [1]. Cardiac, respiratory, and neurological functions remained normal in all test animals [1]. In vitro studies showed that MI-463 did not significantly inhibit hERG potassium channels at concentrations up to 10 μM, indicating a low risk of QT interval prolongation [1].
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| References | |
| Additional Infomation |
MI-463 is a small molecule inhibitor designed to disrupt the protein-protein interaction between Menin and MLL (mixed lineage leukemia) proteins[1]
- The Menin-MLL interaction is crucial for the oncogenic activity of MLL fusion proteins, which are key drivers of MLL rearrangement leukemias, including certain types of acute lymphoblastic leukemia and acute myeloid leukemia[1] - Its mechanism of action involves binding to a hydrophobic pocket on Menin, thereby preventing its interaction with MLL and inhibiting the activation of MLL target genes such as HOXA9 and MEIS1, which promote the proliferation and survival of leukemia cells[1] - MI-463 represents a novel approach to treating MLL rearrangement leukemias, which are often aggressive and resistant to conventional chemotherapy[1] |
| Molecular Formula |
C24H23F3N6S
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| Molecular Weight |
484.54
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| Exact Mass |
484.165
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| CAS # |
1628317-18-9
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| Related CAS # |
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| PubChem CID |
90455046
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| Appearance |
White to light yellow solid powder
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| Density |
1.4±0.1 g/cm3
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| Boiling Point |
663.2±55.0 °C at 760 mmHg
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| Flash Point |
354.9±31.5 °C
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| Vapour Pressure |
0.0±2.0 mmHg at 25°C
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| Index of Refraction |
1.669
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| LogP |
3.88
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
9
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
34
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| Complexity |
751
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
DZACSLYTXLZAAF-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C24H23F3N6S/c1-14-15(2-3-21-19(14)8-17(11-28)31-21)12-33-6-4-16(5-7-33)32-22-20-9-18(10-24(25,26)27)34-23(20)30-13-29-22/h2-3,8-9,13,16,31H,4-7,10,12H2,1H3,(H,29,30,32)
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| Chemical Name |
4-methyl-5-((4-((6-(2,2,2-trifluoroethyl)thieno[2,3-d]pyrimidin-4-yl)amino)piperidin-1-yl)methyl)-1H-indole-2-carbonitrile
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| Synonyms |
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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 |
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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) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.29 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% 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 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: 2.08 mg/mL (4.29 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (4.29 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0638 mL | 10.3191 mL | 20.6381 mL | |
| 5 mM | 0.4128 mL | 2.0638 mL | 4.1276 mL | |
| 10 mM | 0.2064 mL | 1.0319 mL | 2.0638 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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