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| Targets |
The RUNX1/ETO fusion protein (NHR2 domain) is the specific target. NHR2 mediates tetramerization, which is essential for the aberrant transcriptional activity of the fusion protein. This inhibitor directly disrupts this protein-protein interaction (PPI), representing a strategy to target the oncogenic function of a fusion protein that lacks traditional enzymatic activity.
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| ln Vitro |
RUNX1/ETO is made up of four neural homology regions (NHR1-4) that are produced by the ETO gene and DNA-binding Runt domain 5, which is the result of the RUNX1 gene. RUNX1/ETO tetramerization is brought about by the NHR2 domain. The vitality of RUNX1/ETO-dependent human leukemia SKNO-1 cells is preferentially reduced by RUNX1/ETO tetramer-IN-1 (compound 7.44) at 1 μM and 10 μM; 3, 5, 7 d [1]. U937 cells are not affected in the same way. Compound 7.44, which is the RUNX1/ETO tetramer-IN-1, suppresses the proliferation of RUNX1/ETO-expressing cells (SKNO-1, Kasumi-1, and K562) and induces myeloid differentiation at 25 μM and 50 μM, respectively, in five days. 2.]. RUNX1/ETO tetramer-IN-1 (100 μM; 7 d) causes RUNX1/ETOtr-expressing CD34+ progenitor cells to differentiate and enter a growth arrest [2]. Compound 7.44, also known as RUNX1/ETO tetramerization-IN-1, exhibits favorable physicochemical and ADME characteristics, as well as high water solubility, significant stability in buffer and plasma, minimal hepatic clearance in vitro, and water solubility of 60 μg/mL[3]. CYP2B6, 2C9, 2C19, and 3A4 may be inhibited by RUNX1/ETO tetramer-IN-1 (1 μM and 10 μM) [3]. RUNX1/ETO tetramer-IN-1 (compound 8) (50 μM; 16 hours) impacts the JNK pathway in cells and inhibits c-Jun N-terminal kinase (JNK) [4].
At concentrations of 1 and 10 microM, the inhibitor preferentially reduces the viability of RUNX1/ETO-dependent human leukemia SKNO-1 cells. At 50 microM for 5 days, it induces myeloid differentiation in Kasumi-1 and K562 cells, indicating a direct anti-leukemic effect. It effectively restores the expression of genes that are downregulated by the RUNX1/ETO fusion. |
| ln Vivo |
RUNX1/ETO tetramerized-IN-1 (compound 7.44) (200-250 μg/kg; i.p.; 5 times per week; 130 days) delays tumor growth of mouse RUNX1/ETO cells [2].
In a mouse model, this compound delays tumor growth at a dose of 200-250 microg/kg, administered intraperitoneally five times a week for 130 days. The study demonstrated that targeting the RUNX1/ETO tetramerization is a viable therapeutic approach for RUNX1/ETO-dependent leukemia in vivo. |
| Enzyme Assay |
While specific protocols for this compound are not detailed, the binding affinity for NHR2 is typically measured using fluorescence polarization (FP) or AlphaScreen assays. Purified recombinant NHR2 domain protein is incubated with a fluorescently labeled tracer peptide that binds to NHR2. Compound is added in competition, and the decrease in polarization or signal is measured to calculate EC50.
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| Cell Assay |
Cell viability assay [1]
Cell Types: RUNX1/ETO-dependent human leukemia SKNO-1 and U937 cells Tested Concentrations: 1 μM and 10 μM Incubation Duration: 3, 5, 7 days Experimental Results: Specific inhibition of SKNO-1 cell growth. Cell viability assay[3] Cell Types: RUNX1/ETO Pharmacokinetic Properties of Tetramer-IN-1 Tested Concentrations: Incubation Duration: Experimental Results: Kinetic Solubility (99% PBS, 1% DMSO) 177 µM Plasma Protein Binding ( Mouse plasma, 60 minutes) 98.4% Plasma stability (mouse plasma, 0–240 minutes) No degradation Hepatocyte stability (mouse hepatocytes) 2.5 µL/min/million cells Chemical stability in PBS (0 –4 hrs (hours)) No degradation The cellular activity of RUNX1/ETO tetramerization-IN-1 is typically evaluated using SKNO-1 and Kasumi-1 cell lines. Cells are seeded in 96-well plates and treated with the compound at various concentrations for 3-7 days. Cell viability is measured using MTT or CellTiter-Glo assays. Myeloid differentiation is assessed by flow cytometry after 5 days of treatment with 50 microM compound, using antibodies against surface markers CD11b and CD14. |
| Animal Protocol |
Animal/Disease Models: NSG immunodeficient mice (NOD.Cg-Prkdcscid Il2rgtm1WjI/SzJ) injected with Kasumi-1 cells [2]
Doses: 200-250 μg/Kg Route of Administration: intraperitoneal (ip) injection; 5 times a week for 130 days. Experimental Results: Reduces the spread of leukemia cells, with 75% of mice still alive on day 130 after treatment. In a standard protocol, 6-8 week old female immunodeficient (e.g., NSG) mice are injected subcutaneously or via tail vein with SKNO-1 cells to establish a disseminated leukemia model. Once the tumor is established, the compound is administered intraperitoneally at 200-250 microg/kg, five days per week. Tumor burden is monitored via bioluminescence imaging if cells are engineered with luciferase, or via human CD45+ cell counts in peripheral blood by flow cytometry. |
| ADME/Pharmacokinetics |
The compound exhibits high water solubility (60 microg/mL), significant stability in both aqueous buffer and plasma, and low in vitro hepatic clearance. It potentially inhibits cytochrome P450 enzymes CYP2B6, 2C9, 2C19, and 3A4, suggesting a risk for drug-drug interactions.
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| Toxicity/Toxicokinetics |
Specific toxicological data for this compound are not detailed in the available literature. However, its ability to target a fusion protein specific to cancer cells may provide a favorable therapeutic window. Standard toxicological endpoints in animal studies typically include monitoring body weight, overall behavior, and histopathological examination of key organs to assess potential off-target effects.
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| References |
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| Additional Infomation |
The RUNX1/ETO gene fusion is a hallmark of AML with t(8;21)(q22;q22). This inhibitor is significant as it directly targets the tetramerization interface of the fusion protein, a protein-protein interaction previously considered "undruggable." As of the latest updates, it is a valuable experimental research tool for understanding leukemogenesis and has not progressed to clinical trials.
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| Molecular Formula |
C18H14O7
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| Molecular Weight |
342.299565792084
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| Exact Mass |
342.074
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| CAS # |
88755-39-9
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| PubChem CID |
294348
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| Appearance |
White to off-white solid powder
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| Density |
1.472g/cm3
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| Boiling Point |
598.6ºC at 760 mmHg
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| Flash Point |
223.2ºC
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| Index of Refraction |
1.641
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| LogP |
2.585
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
25
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| Complexity |
522
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O=C(C(CC(C1=CC=C2OCOC2=C1)=O)C3=CC=C4OCOC4=C3)O
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| InChi Key |
PPEBBOHQEAQKSW-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C18H14O7/c19-13(11-2-4-15-17(6-11)25-9-23-15)7-12(18(20)21)10-1-3-14-16(5-10)24-8-22-14/h1-6,12H,7-9H2,(H,20,21)
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| Chemical Name |
2,4-bis(1,3-benzodioxol-5-yl)-4-oxobutanoic acid
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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) |
DMSO: ~100 mg/mL (~292.1 mM; with ultrasonication)
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| Solubility (In Vivo) |
Solubility in Formulation 1: 2.5 mg/mL (7.30 mM) in 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), Clear solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of DMSO stock solution (25.0 mg/mL) to 400 μL of PEG300 and mix well; then add 50 μL of Tween-80 and mix well; finally add 450 μL of physiological saline and 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: 2.5 mg/mL (7.30 mM) 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; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of DMSO stock solution (25.0 mg/mL) to 900 μL of 20% SBE-β-CD saline and mix well. 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 3: 2.5 mg/mL (7.30 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), Clear solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of DMSO stock solution (25.0 mg/mL) to 900 μL of corn oil and mix well. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.9214 mL | 14.6071 mL | 29.2141 mL | |
| 5 mM | 0.5843 mL | 2.9214 mL | 5.8428 mL | |
| 10 mM | 0.2921 mL | 1.4607 mL | 2.9214 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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