WDR5 (Ki < 1 nM); WDR5-MLL1 interaction (IC50 = 0.9 nM); MLL1 (IC50 = 0.32 µM);
The primary target of MM-401 TFA is WDR5 (WD repeat domain 5), where it binds with high affinity to disrupt the MLL1-WDR5 interaction. MM-401 TFA exhibits a binding affinity for WDR5 with Ki < 1 nM and an IC50 of 0.9 nM for disrupting the WDR5-MLL1 interaction. By occupying the MLL1-binding interface on WDR5, MM-401 TFA prevents the assembly of the MLL1 core complex, thereby specifically inhibiting MLL1 histone methyltransferase activity (IC50 of 0.32 μM). Importantly, this targeting strategy does not affect the methyltransferase activities of other MLL family members (including MLL2, MLL3, MLL4, SETD1A, and SETD1B), revealing a unique regulatory feature of the MLL1 complex.

MM-401 inhibits the WDR5-MLL1 interaction with an IC50 value of 0.9 nM while maintaining a strong binding affinity to WDR5 with a Ki value of < 1 nM[1]. By preventing the MLL1-WDR5 interaction and consequently the complex assembly, MM-401 can specifically inhibit MLL1 activity (IC50 value of 0.32µM)[1]. MLL1-dependent H3K4 methylation in cells is particularly inhibited by MM-401 (20 μM; 48 h)[1]. Similar alterations to the MLL-AF9 transcriptome are induced by MM-401 as by the MLL1 deletion[1]. MM-401 (10, 20, 40 μM; 48 h) specifically prevents MLL leukemia cells from growing by causing apoptosis and cell cycle arrest[1].

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In vitro studies demonstrate that MM-401 TFA exerts anti-leukemic effects by specifically inhibiting MLL1 methyltransferase activity. In MLL-rearranged leukemia cell lines, MM-401 TFA (10-40 μM, 48 hours) induces G1/S cell cycle arrest and apoptosis in a concentration-dependent manner, without significant inhibitory effects on non-MLL leukemia cells (such as K562, HL60, U937) or normal bone marrow cells. In MLL-AF9 cells, MM-401 TFA (20 μM, 48 hours) significantly reduces H3K4me3 levels and downregulates the expression of MLL1 target genes including Hoxa9 and Hoxa10. RNA sequencing analysis shows that the gene expression changes induced by MM-401 TFA treatment are highly similar to those observed upon MLL1 knockout. Wright-Giemsa staining indicates that MM-401 TFA treatment induces myeloid differentiation in MLL leukemia cells.

As a tool compound, in vivo studies of MM-401 TFA are primarily conducted using its structurally optimized derivatives. Available studies demonstrate that MM-401 derivatives targeting the MLL1-WDR5 interaction exhibit in vivo anti-leukemic activity in animal models. It has been shown that upon intravenous administration, MM-401 derivatives effectively suppress the expansion of MLL leukemia cells in mice and prolong survival, without observed toxicity to normal hematopoietic function. However, the pharmacokinetic properties of MM-401 TFA itself limit its direct in vivo application, and its in vivo administration typically requires appropriate formulation vehicles to improve bioavailability.

Histone Methytransferase Assays[1]
The HMT assay was performed as described previously (Dou et al., 2005). For inhibitor studies, compounds at various concentrations were incubated first with the pre-assembled complex and reactions were initiated by addition of substrates. For kinetic analyses, the reaction progression curve was established to determine the linear range of the reaction at room temperature. For Lineweaver-Burk curve, reactions (0.5µM enzyme complex and 50µM substrates) were initiated and quenched after 4 minutes by addition of β-mercaptoethanol at a final concentration of 178µM.

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Crystal Structures[1]
WDR5//MM-401 or WDR5/MM-NC-401 binary complex was obtained by mixing WDR5 and compounds at molar ratio 1: 2. The complex was crystallized by hanging-drop-vapor-diffusion at 22°C. Details see supplemental information.

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WDR5 Protein Expression and Purification: Express recombinant human WDR5 protein and purify by affinity chromatography and size-exclusion chromatography. Bio-Layer Interferometry: Using the Octet RED system, immobilize biotinylated WDR5 on streptavidin sensors and incubate with varying concentrations of MM-401 TFA to determine binding affinity (Ki < 1 nM). Fluorescence Polarization Competition Assay: Incubate WDR5 protein with fluorescently labeled MLL1 peptide (FAM-WIN peptide) and varying concentrations of MM-401 TFA, measure changes in fluorescence polarization to calculate an IC50 of 0.9 nM. In Vitro Histone Methyltransferase Assay: Pre-incubate the MLL1 core complex (MLL1-WDR5-ASH2L-RbBP5) with varying concentrations of MM-401 TFA, initiate the reaction by adding substrates (histone H3 and [³H]-SAM), measure [³H]-methyl incorporation by scintillation counting, and calculate an IC50 of 0.32 μM. Co-crystallization Structure Determination: Mix WDR5 and MM-401 TFA at a 1:2 molar ratio, crystallize by the hanging-drop vapor diffusion method at 22°C, and determine the co-crystal structure to confirm the binding mode.

Apoptosis Analysis[1]
Cell Types: Murine MLL-AF9 and Hoxa9/Meis1 cells
Tested Concentrations: 10, 20, 40 μM
Incubation Duration: 48 h
Experimental Results: Specifically induced apoptosis of MLL-AF9 cells.

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Cell Cycle Analysis[1]
Cell Types: Murine MLL-AF9 and Hoxa9/Meis1 cells
Tested Concentrations: 10, 20, 40 μM
Incubation Duration: 48 h
Experimental Results: Induced prominent G1/S arrest in MLL-AF9 cells in a concentration dependent manner.

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RT-PCR[1]
Cell Types: MLL-AF9 cells
Tested Concentrations: 20 μM
Incubation Duration: 48 h
Experimental Results: Dramatically diminished H3K4me, expression of 5 Hox A genes, especially Hoxa9 and Hoxa10.

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Assays for Cell Viability, Wright-Giemsa staining, apoptosis, cell cycle and cell differentiation [1]
Inhibitors were diluted from stock to culture media containing 0.1% DMSO final concentration. For viability assays, cells were cultured at 1×105/ml and passaged every 2 days. Viability was determined using the CellTitreGlo® Kit according to the manufacturer’s directions. Luminescence was monitored on a Molecular Dynamics plate reader. For staining, cells treated with 10, 20 and 40µM /MM-401, or 40µM MM-NC-401 or DMSO vehicle for 4 days were diluted to 2.5×105/ml in 1× PBS and fixed to glass slides by cytospin followed by Wright-Giemsa staining. Cell images were taken at 40× magnification by light microscopy. Apoptosis, cell cycle and cell differentiation analyses were performed using standard protocols (see supplemental information).

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Real Time-PCR, RNA-seq and CHIP assays [1]
MLL1-AF9 cells were cultured for 2 days in the presence of /MM-401 or MM-NC-401. At the end of treatment, cells were harvested by centrifugation at 300×g and washed with 1xPBS. RNAs for duplicated biological samples were extracted by a standard protocol. 10ng RNAs were used for Illumina sequencing library. Four RNA seq samples were multiplexed and loaded into one lane in Hi-seq sequencer. RNA-seq analyses were described in supplemental information. CHIP assays were performed as previously described.

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Cell Culture: Culture MLL-rearranged leukemia cells (e.g., MLL-AF9-transduced mouse bone marrow cells, ML-2, KOPN-8, MV4-11) and non-MLL leukemia cells (K562, HL60, U937) in RPMI-1640 medium containing 10-20% fetal bovine serum at 37°C in a 5% CO₂ incubator. Cell Viability Assay: Seed cells in 96-well plates (1×10⁵/mL), treat with varying concentrations of MM-401 TFA (0-40 μM) or control MM-NC-401 for 3 days, and measure cell viability using the CellTiter-Glo luminescent assay. Cell Cycle Analysis: After treatment with MM-401 TFA (10, 20, 40 μM) for 48 hours, fix cells with 70% ethanol, stain with propidium iodide, and analyze cell cycle distribution by flow cytometry. Apoptosis Detection: After treatment with MM-401 TFA (10, 20, 40 μM) for 48 hours, stain cells with Annexin V-FITC/PI and detect apoptosis rate by flow cytometry. Wright-Giemsa Staining: After 4 days of MM-401 TFA treatment, perform cytospin, stain with Wright-Giemsa, and observe morphological changes and myeloid differentiation markers under light microscopy. RT-PCR and Western Blot: After treatment with MM-401 TFA (20 μM) for 48 hours, extract RNA or protein to detect Hoxa9, Hoxa10 gene expression and H3K4me3 levels.

Animal Models: Establish disseminated leukemia models by intravenous injection of MLL-rearranged leukemia cells (e.g., MLL-AF9 cells) into immunodeficient mice (e.g., NSG mice) via the tail vein. Dosing Regimen: Administer MM-401 derivatives by intravenous injection on a daily schedule for several consecutive weeks. Due to the solubility and stability limitations of MM-401 TFA itself, appropriate formulation is typically required for in vivo administration. Efficacy Assessment: Monitor tumor burden by bioluminescent imaging, record survival, and detect the percentage of leukemia cells in peripheral blood and bone marrow by flow cytometry. Toxicity Assessment: Monitor animal body weight changes, behavioral performance, and peripheral blood cell counts to evaluate effects on normal hematopoietic function. Data Analysis: Compare survival differences and tumor burden changes between treatment and placebo groups, and calculate median survival using Kaplan-Meier analysis.

As the trifluoroacetate salt form of a cyclic peptide compound, the pharmacokinetic properties of MM-401 TFA are similar to those of the parent compound MM-401. Compared to the linear parent compound MM-101, MM-401 exhibits enhanced metabolic stability through the cyclization strategy. This compound has good solubility in DMSO (up to 100 mg/mL), but limited solubility in aqueous buffers. The powder is stable for 2-3 years when stored at -20°C or 4°C protected from light; solutions are stable for 6 months at -80°C protected from light. For in vivo applications, appropriate formulation vehicles are recommended to improve bioavailability. This compound is for research use only and is not for human use.

According to the Safety Data Sheet, MM-401 TFA is not classified as a hazardous substance or mixture, and GHS label elements do not apply. Based on available research data, MM-401 TFA exhibits a favorable safety profile toward normal cells. In comparative studies of MLL leukemia cells, non-MLL leukemia cells, and normal bone marrow cells, MM-401 TFA specifically inhibits the proliferation of MLL-rearranged leukemia cells without significant cytotoxicity toward non-MLL leukemia cells (K562, HL60, U937) or normal bone marrow cells. MM-401 TFA treatment does not induce significant apoptosis or differentiation abnormalities in normal bone marrow cells. This compound has a purity of ≥98% and is intended for research use only, not for human or veterinary applications. Standard laboratory safety practices should be followed when handling.

[1]. Targeting MLL1 H3K4 methyltransferase activity in mixed-lineage leukemia. Mol Cell. 2014 Jan 23;53(2):247-61.

This article fully characterizes our recently developed inhibitor MM-401, which targets the activity of MLL1 H3K4 methyltransferase. MM-401 specifically inhibits MLL1 activity by blocking the MLL1-WDR5 interaction, thereby inhibiting the assembly of the complex. This targeting strategy does not affect histone methyltransferases (HMTs) of other mixed lineage leukemia (MLL) families, revealing the unique regulatory properties of the MLL1 complex. We demonstrated using MM-401 and its enantiomer control MM-NC-401 that inhibiting MLL1 methyltransferase activity specifically inhibits the proliferation of MLL cells by inducing cell cycle arrest, apoptosis and myeloid differentiation, without toxicity to normal bone marrow cells or non-MLL cells. More importantly, transcriptome analysis showed that the gene expression changes induced by MM-401 were similar to those induced by MLL1 deficiency, supporting the view that MLL1 activity plays a dominant role in regulating the transcriptional program of MLL1-dependent leukemia. We envision that MM-401 has broad application prospects in basic and translational research. [1]

C31H47F3N8O7

700.749497652054

700.351

1442106-11-7

MM-401;1442106-10-6

137662115

isobutyryl-D-aMeLys(1)-Arg-Abu-D-Phg-(1).TFA

White to light yellow solid powder

8

11

8

49

1060

4

C(F)(F)(F)C(=O)O.O=C1NCCCC[C@@](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC)C(=O)N[C@]1([H])C1C=CC=CC=1)(C)NC(=O)C(C)C

UCPNHHHBFDAYBP-HXXCMCGZSA-N

InChI=1S/C29H46N8O5.C2HF3O2/c1-5-20-24(39)36-22(19-12-7-6-8-13-19)26(41)32-16-10-9-15-29(4,37-23(38)18(2)3)27(42)35-21(25(40)34-20)14-11-17-33-28(30)31;3-2(4,5)1(6)7/h6-8,12-13,18,20-22H,5,9-11,14-17H2,1-4H3,(H,32,41)(H,34,40)(H,35,42)(H,36,39)(H,37,38)(H4,30,31,33);(H,6,7)/t20-,21-,22+,29+;/m0./s1

N-[(3R,6S,9S,12R)-9-[3-(diaminomethylideneamino)propyl]-6-ethyl-12-methyl-2,5,8,11-tetraoxo-3-phenyl-1,4,7,10-tetrazacyclohexadec-12-yl]-2-methylpropanamide;2,2,2-trifluoroacetic acid

MM-401 TFA; 1442106-11-7; MM-401 (TFA); CHEMBL4099772;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO : 100 mg/mL (142.70 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.57 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 25.0 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (3.57 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 25.0 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (3.57 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 25.0 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.)