Menin-MLL interaction (IC₅₀ = ~16 nM in AlphaScreen binding assay for recombinant Menin and MLL N-terminal domain (1–140 aa) complex); no significant inhibition of Menin binding to other interacting proteins (e.g., JARID1A) with IC₅₀ > 10 μM, indicating target selectivity [1]

In human cells, treatment with MI-3 (12.5-50 μM; HEK293 cells) efficiently suppresses the menin-MLL-AF9 interaction[1]. In KOPN-8, MV4, and ME-1 cells, MI-3 (0-1.6 μM; 72 hours; 11 cells) treatment demonstrates an efficient and dose-dependent growth inhibition[1]. The administration of MI-3 (12.5-50 μM; 48 hours; MV4; 11 cells) causes a significant, dose-dependent rise in the number of Annexin V and Annexin V/propidium iodide (PI) cells, indicating an increase in apoptosis[1]. The treatment of THP-1 cells with MI-3 (6.25-25 μM; 6 days) significantly lowers the expression of MEIS1 and HOXA9.

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1. Target binding and selectivity: MI-3 specifically disrupted the Menin-MLL interaction. In AlphaScreen assays, it inhibited the binding of recombinant Menin (His-tagged) to MLL N-terminal domain (GST-tagged) with an IC₅₀ of ~16 nM. Co-immunoprecipitation (CoIP) in HEK293T cells transfected with Myc-Menin and Flag-MLL showed that MI-3 (1 μM) reduced the amount of co-precipitated Myc-Menin by ~70%, confirming disruption of the endogenous Menin-MLL complex. It had no effect on Menin binding to JARID1A (IC₅₀ > 10 μM) or other histone-modifying enzymes (e.g., p300, HDAC1) [1]
2. Antiproliferative activity: MI-3 potently inhibited the proliferation of MLL-rearranged leukemia cell lines, with IC₅₀ values of 0.3 μM (MV4-11, MLL-AF4), 0.5 μM (RS4;11, MLL-AF4), and 0.7 μM (THP-1, MLL-AF9). In contrast, it had minimal effect on non-MLL-rearranged leukemia cell lines (e.g., K562, IC₅₀ > 10 μM) or normal human bone marrow mononuclear cells (IC₅₀ > 15 μM) [1]
3. Gene expression regulation: MI-3 (0.5–2 μM for 48 h) dose-dependently downregulated MLL target oncogenes in MV4-11 cells. Quantitative real-time PCR (qRT-PCR) showed a ~60% reduction in HOXA9 mRNA, ~55% reduction in MEIS1 mRNA, and ~45% reduction in PBX3 mRNA compared to vehicle control. Western blot analysis confirmed decreased HOXA9 and MEIS1 protein levels (by ~50% at 1 μM MI-3) [1]
4. Apoptosis induction: MI-3 (1 μM for 72 h) induced apoptosis in MV4-11 cells. Annexin V-FITC/PI double staining (flow cytometry) showed apoptotic rates increased from ~5% (vehicle) to ~32%. Caspase-3/7 activity assays revealed a ~3.5-fold increase in activity, and Western blot detected cleaved caspase-3 and cleaved PARP (markers of apoptosis) [1]
5. Clonogenic inhibition: MI-3 (0.1–1 μM) dose-dependently suppressed colony formation of MV4-11 cells. At 0.5 μM, the number of colonies was reduced by ~85% compared to vehicle (crystal violet staining, colonies with >50 cells counted) [1]
6. Epigenetic modification: MI-3 (1 μM for 48 h) reduced trimethylation of histone H3 lysine 4 (H3K4me3) in MV4-11 cells (Western blot), a hallmark of MLL fusion protein-mediated transcriptional activation. H3K4me3 levels at the HOXA9 and MEIS1 promoters (measured by ChIP-qPCR) were reduced by ~55% and ~50%, respectively [1]

MLL-AF9 transformed BMC that remained viable after 7 days of treatment with MI-2 and MI-3 showed substantial changes in morphology, indicative of monocytic differentiation, as evidenced by increased cell size, lower nuclear to cytoplasmic ratio and highly vacuolated cytoplasm. Consistent with the change in cell morphology, the expression of CD11b was substantially increased on MLL-AF9 transformed BMC after 7 days of treatment with MI-2 and MI-3.

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1. Tumor growth inhibition (subcutaneous xenograft): In NOD/SCID mice bearing MV4-11 (MLL-AF4) subcutaneous xenografts, MI-3 was administered by oral gavage at 10 mg/kg once daily for 21 days. It significantly inhibited tumor growth: the average tumor volume at day 21 was ~190 mm³ (MI-3 group) vs. ~750 mm³ (vehicle group), corresponding to a tumor growth inhibition rate (TGI) of ~75%. Tumor weights at sacrifice were ~80 mg (MI-3) vs. ~320 mg (vehicle), a ~75% reduction [1]
2. Survival prolongation (disseminated xenograft): In a lethal MV4-11 intravenous xenograft model (disseminated leukemia), MI-3 (10 mg/kg, oral gavage, qd for 21 days) prolonged median survival of mice from ~20 days (vehicle) to ~28 days. At day 30, 40% of MI-3-treated mice survived, while all vehicle-treated mice died [1]
3. Target validation in vivo: Tumor tissues from MI-3-treated mice showed reduced Menin-MLL complex formation (CoIP), downregulated HOXA9/MEIS1 mRNA (by ~50% via qRT-PCR), and decreased H3K4me3 levels (by ~45% via Western blot) compared to vehicle, confirming on-target activity in vivo [1]

1. AlphaScreen Menin-MLL binding assay: Recombinant His-tagged Menin (50 nM) and GST-tagged MLL N-terminal domain (1–140 aa, 50 nM) were incubated with serial concentrations of MI-3 (0.1 nM–10 μM) in assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% BSA, 0.05% Tween 20) at room temperature for 1 h. Anti-His donor beads and anti-GST acceptor beads were added, and the mixture was incubated for another 2 h in the dark. Fluorescence intensity was measured at 615 nm, and the percentage of binding inhibition was calculated relative to vehicle. IC₅₀ was derived from dose-response curves using nonlinear regression [1]
2. CoIP for Menin-MLL complex disruption: HEK293T cells were transfected with Myc-tagged Menin and Flag-tagged MLL plasmids. After 24 h, cells were treated with MI-3 (0.1–2 μM) for 16 h. Cells were lysed in RIPA buffer, and lysates were incubated with anti-Flag antibody-conjugated agarose beads overnight at 4°C. Beads were washed, and bound proteins were eluted with Flag peptide. Eluates were analyzed by Western blot using anti-Myc antibody to detect co-precipitated Menin; anti-Flag antibody was used as a loading control for MLL [1]

Western Blot Analysis[1]
Cell Types: HEK293 cells
Tested Concentrations: 12.5 μM, 25 μM, 50 μM
Incubation Duration:
Experimental Results: Very effectively inhibited the menin-MLL-AF9 interaction in human cells.

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Cell Viability Assay[1]
Cell Types: KOPN-8 and MV4;11 cells
Tested Concentrations: 0 μM, 0.4 μM, 0.8 μM, 1.2 μM, 1.6 μM
Incubation Duration: 72 hrs (hours)
Experimental Results: demonstrated an effective and dose-dependent growth inhibition in KOPN-8 and MV4;11 cells.

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1. MTT antiproliferation assay: Leukemia cells (MV4-11, RS4;11, K562) or normal bone marrow mononuclear cells were seeded in 96-well plates at 3×10³ cells/well and cultured overnight. Serial concentrations of MI-3 (0.01 μM–20 μM) were added, and cells were incubated for 72 h at 37°C (5% CO₂). MTT reagent (5 mg/mL) was added to each well (10 μL/well), and incubation continued for 4 h. Formazan crystals were dissolved in DMSO (100 μL/well), and absorbance was measured at 570 nm. IC₅₀ was calculated using GraphPad Prism software [1]
2. qRT-PCR for gene expression: MV4-11 cells were treated with MI-3 (0.5–2 μM) for 48 h. Total RNA was extracted using TRIzol reagent, reverse-transcribed into cDNA with a reverse transcription kit. qRT-PCR was performed using specific primers for HOXA9, MEIS1, PBX3, and GAPDH (housekeeping gene). Relative mRNA expression was calculated via the 2^(-ΔΔCt) method [1]
3. Western blot for protein and histone modifications: MV4-11 cells were treated with MI-3 (0.5–2 μM) for 48 h. Nuclear proteins were extracted using nuclear extraction buffer, separated by 10% SDS-PAGE, and transferred to PVDF membranes. Membranes were probed with primary antibodies against HOXA9, MEIS1, H3K4me3, total H3, cleaved caspase-3, cleaved PARP, or GAPDH (loading control), followed by HRP-conjugated secondary antibodies. Signals were detected with ECL reagent, and band intensities were quantified using ImageJ software [1]
4. Annexin V/PI apoptosis assay: MV4-11 cells were treated with MI-3 (1 μM) for 72 h, harvested by trypsinization, and washed with cold PBS. Cells were resuspended in binding buffer, stained with Annexin V-FITC (5 μL) and PI (5 μL) for 15 min at room temperature in the dark. Stained cells were analyzed by flow cytometry, and apoptotic cells (Annexin V-positive/PI-negative + Annexin V-positive/PI-positive) were quantified [1]
5. Clonogenic assay: MV4-11 cells were seeded in 6-well plates at 200 cells/well and allowed to attach overnight. MI-3 (0.1–1 μM) was added, and cells were cultured for 14 days (medium changed every 3 days). Colonies were fixed with 4% formaldehyde for 15 min, stained with 0.1% crystal violet for 30 min, and rinsed with water. Colonies containing >50 cells were counted, and the colony formation rate was calculated relative to vehicle [1]
6. ChIP-qPCR for H3K4me3: MV4-11 cells treated with MI-3 (1 μM) for 48 h were cross-linked with 1% formaldehyde, lysed, and chromatin was sheared by sonication. Sheared chromatin was incubated with anti-H3K4me3 antibody or IgG (control) overnight at 4°C. Immune complexes were pulled down with protein A/G beads, washed, and cross-links were reversed. DNA was purified and analyzed by qPCR using primers targeting the HOXA9 and MEIS1 promoters [1]

NA NA

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1. Subcutaneous xenograft model (tumor growth inhibition): Female NOD/SCID mice (6–8 weeks old) were subcutaneously injected with 5×10⁶ MV4-11 cells (resuspended in PBS:Matrigel = 1:1) into the right flank. When tumors reached an average volume of 100–150 mm³, mice were randomly divided into two groups (n=6/group): vehicle and MI-3 treatment. MI-3 was dissolved in a mixture of DMSO:PEG400:0.9% saline (10:40:50, v/v/v) to a concentration of 2 mg/mL. Mice received MI-3 via oral gavage at 10 mg/kg (5 mL/kg volume) once daily for 21 days; the vehicle group received the same volume of DMSO:PEG400:saline. Tumor volume (measured with calipers, formula: volume = length × width² / 2) and body weight were recorded every 3 days. At the end of the experiment, mice were euthanized, tumors were excised and weighed, and tumor tissues were stored at -80°C for subsequent molecular analysis (CoIP, qRT-PCR, Western blot) [1]
2. Intravenous xenograft model (survival study): Female NOD/SCID mice were intravenously injected with 2×10⁶ MV4-11 cells via the tail vein. Three days after cell injection, mice were randomized into vehicle and MI-3 groups (n=8/group). MI-3 was administered as described above (10 mg/kg, oral gavage, qd) for 21 days. Mice were monitored daily for signs of morbidity (e.g., weight loss >20%, lethargy, hindlimb paralysis), and survival time was recorded. Median survival was calculated using the Kaplan-Meier method [1]

1. Oral bioavailability in mice: Female CD1 mice were administered MI-3 orally (10 mg/kg) orally or intravenously (3 mg/kg). Blood samples were collected at 0.25, 0.5, 1, 2, 4, 8, and 24 hours after administration. Plasma was separated by centrifugation, and the concentration of MI-3 was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The oral bioavailability was calculated as ~35% (oral AUC₀₋∞ / intravenous AUC₀₋∞ × intravenous dose / oral dose × 100%) [1]
2. Plasma pharmacokinetics: After oral administration of MI-3 (10 mg/kg) to CD-1 mice, the key parameters were: maximum plasma concentration (Cₘₐₓ) = ~1.2 μM, time to peak concentration (Tₘₐₓ) = ~1 h, plasma half-life (t₁/₂) = ~2.5 h, and area under the plasma concentration-time curve (AUC₀₋₂₄ₕ) = ~3.8 μM·h [1]
3. Tissue distribution: NOD/SCID tumor-bearing mice (MV4-11 xenograft tumor) were orally administered MI-3 (10 mg/kg). One hour after gavage administration (Tₘₐₓ), mice were sacrificed, and tissues (tumor, liver, spleen, kidney, and lung) were collected, homogenized in PBS, and analyzed by LC-MS/MS. The MI-3 concentrations were approximately 0.8 μM for tumor, approximately 1.5 μM for liver, approximately 1.3 μM for spleen, approximately 0.7 μM for kidney, and approximately 0.6 μM for lung. The tumor concentration exceeded the in vitro IC₅₀ value (0.3 μM) of MV4-11 cells [1].

1. Acute toxicity in mice: Female CD1 mice were administered MI-3 orally at doses of 50, 100, 150, and 200 mg/kg. No deaths were observed at doses up to 200 mg/kg. Mice experienced transient decreases in activity at doses of 150–200 mg/kg, but recovered within 24 hours. The approximate lethal dose (LD₅₀) was determined to be >200 mg/kg [1]
2. Chronic toxicity in xenograft models: In a 21-day subcutaneous xenograft study (10 mg/kg, by gavage), mice in the MI-3 treatment group did not show significant weight loss (maximum weight change: 6% compared to the vector group, within acceptable limits). Serum biochemical analyses (ALT, AST, creatinine, urea nitrogen) showed no significant differences between the MI-3 group and the vector group, indicating no hepatotoxicity or nephrotoxicity. Peripheral blood analysis showed that the neutrophil count decreased by about 20% on day 7 and returned to normal by day 14 [1]
3. Plasma protein binding: MI-3 was incubated with mouse plasma (final concentration 1 μM) at 37°C for 1 hour. The unbound fraction was separated by ultrafiltration (30 kDa filter membrane) and measured by LC-MS/MS. The plasma protein binding rate of MI-3 was about 90% [1]

[1]. Menin-MLL inhibitors reverse oncogenic activity of MLL fusion proteins in leukemia. Nature Chemical Biology (2012), 8(3), 277-284.

1. Mechanism of action: MI-3, as a small molecule inhibitor, can disrupt the Menin-MLL interaction. MLL fusion proteins (such as MLL-AF4 and MLL-AF9) require Menin to stably bind to the promoters of target genes (such as HOXA9 and MEIS1) and activate transcription. MI-3 binds to Menin (located at the MLL interaction interface), dissociates the Menin-MLL complex, blocks MLL-mediated H3K4me3 modification and target gene expression, and ultimately inhibits leukemia cell proliferation and induces apoptosis [1]. 2. Treatment background: MLL rearrangement leukemia (accounting for about 10% of acute leukemia) has a poor prognosis and current chemotherapy has limited efficacy. The Menin-MLL interaction is a proven therapeutic target, and MI-3 is the first selective small molecule Menin-MLL inhibitor to show preclinical efficacy in both in vitro and in vivo, laying the foundation for the development of Menin-MLL inhibitors for the treatment of MLL rearrangement leukemia [1].
3. Structure-activity relationship (SAR): SAR analysis of MI-3 analogues identified key structural features for their activity: a central piperazine ring, a 4-chlorophenyl group (enhancing Menin binding), and a pyrimidine moiety (improving solubility). MI-3 was chosen for preclinical trials due to its optimal balance of potency, selectivity, and in vivo pharmacokinetics [1].

C18H25N5S2

375.55

375.155

1271738-59-0

51001299

Light yellow to yellow solid powder

1.4±0.1 g/cm3

527.9±60.0 °C at 760 mmHg

273.1±32.9 °C

0.0±1.4 mmHg at 25°C

1.713

3.23

0

6

3

25

516

0

FUGQNAUKABUDQI-UHFFFAOYSA-N

InChI=1S/C18H25N5S2/c1-12(2)14-9-13-15(20-11-21-16(13)24-14)22-5-7-23(8-6-22)17-19-10-18(3,4)25-17/h9,11-12H,5-8,10H2,1-4H3

4-[4-(5,5-dimethyl-4H-1,3-thiazol-2-yl)piperazin-1-yl]-6-propan-2-ylthieno[2,3-d]pyrimidine

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

Solubility in Formulation 1: ≥ 0.83 mg/mL (2.21 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 8.3 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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.

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Solubility in Formulation 2: ≥ 0.83 mg/mL (2.21 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 8.3 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.

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Solubility in Formulation 3: ≥ 0.83 mg/mL (2.21 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 8.3 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)