WDR5 (Ki < 1 nM); WDR5-MLL1 interaction (IC50 = 0.9 nM); MLL1 (IC50 = 0.32 µM)
The primary target of MM-401 is WDR5 (WD repeat domain 5), where it binds with high affinity to disrupt the MLL1-WDR5 interaction. MM-401 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 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 complex assembly and MLL1-WDR5 interaction, MM-401 can specifically reduce MLL1 activity (IC50 value: 0.32μM) [1]. Specifically, MLL1-dependent H3K4 methylation in cells is inhibited by MM-401 (20 μM; 48 hours) [1]. Similar alterations to the MLL-AF9 transcriptome are caused by MM-401 and MLL1 deletion [1]. By causing cell cycle arrest and death, MM-401 (10, 20, 40 μM; 48 h) specifically suppresses the development of MLL leukemia cells [1].

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In vitro studies demonstrate that MM-401 exerts anti-leukemic effects by specifically inhibiting MLL1 methyltransferase activity. In MLL-rearranged leukemia cell lines, MM-401 (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, with GI50 not determined. In MLL-AF9 cells, MM-401 (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 treatment are highly similar to those observed upon MLL1 knockout. MLL leukemia blasts isolated from patients are also sensitive to MM-401, while non-MLL leukemia cells are not.

Available studies demonstrate that MM-401 exhibits in vivo anti-leukemic activity by targeting the MLL1-WDR5 interaction in animal models. Derivatives of MM-401 with improved pharmacokinetic properties and in vivo bioavailability have been shown to suppress tumor growth and extend survival in mouse models of MLL leukemia. Although the pharmacokinetic properties of MM-401 itself limit its direct in vivo application as a tool compound, its derivatives effectively suppress the expansion of MLL leukemia cells in mice upon intravenous administration, without observed toxicity to normal hematopoietic function.

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 to determine binding affinity (KD < 1 nM). Fluorescence Polarization Competition Assay: Incubate WDR5 protein with fluorescently labeled MLL1 peptide (FAM-WIN peptide) and varying concentrations of MM-401, measure changes in fluorescence polarization to calculate an IC50 of 0.9 nM for disrupting the WDR5-MLL1 interaction. In Vitro Histone Methyltransferase Assay: Pre-incubate the MLL1 core complex (MLL1-WDR5-ASH2L-RbBP5) with varying concentrations of MM-401, 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 at a 1:2 molar ratio, crystallize by the hanging-drop vapor diffusion method, and determine the co-crystal structure at 2.1 Å resolution to confirm the binding mode.

Apoptosis analysis [1]
Cell Types: mouse MLL-AF9 and Hoxa9/Meis1 cells
Tested Concentrations: 0, 20, 40 μM
Incubation Duration: 48 h
Experimental Results: Specific induction of MLL-AF9 cell apoptosis.

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Cell cycle analysis [1]
Cell Types: mouse MLL-AF9 and Hoxa9/Meis1 Cell
Tested Concentrations: 10, 20, 40 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: Significant G1/ was induced in MLL-AF9 cells in a concentration-dependent manner S Stasis.

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RT-PCR[1]
Cell Types: MLL-AF9 Cell
Tested Concentrations: 20 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: H3K4me, the expression of 5 Hox A genes was Dramatically diminished, 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 (0-40 μM) or control MM-NC-401 for 3 days, measure cell viability using the CellTiter-Glo luminescent assay, and calculate GI50 values. Cell Cycle Analysis: After treatment with MM-401 (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 (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 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 (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 (e.g., MM-589) by intravenous injection on a daily schedule for several consecutive weeks. 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 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 a cyclic peptide tool compound, the pharmacokinetic properties of MM-401 have been improved through structural optimization. Compared to the parent compound MM-101, MM-401 exhibits enhanced metabolic stability through the cyclization strategy. Available MM-401 derivatives (e.g., MM-589) have demonstrated improved pharmacokinetic properties and in vivo bioavailability. MM-401 is soluble in DMSO and is commonly used as a stock solution for in vitro experiments. The compound should be stored at -20°C, dry and protected from light. For in vivo applications, it is recommended to use salt forms with higher solubility (e.g., MM-401 TFA salt) with appropriate formulation vehicles.

Based on available research data, MM-401 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 specifically inhibits the proliferation of MLL-rearranged leukemia cells without significant cytotoxicity toward non-MLL leukemia cells or normal bone marrow cells. Specifically, no growth inhibition was observed in K562, HL60, and U937 non-MLL leukemia cells upon MM-401 treatment, with GI50 not determined. Furthermore, MM-401 treatment does not induce significant apoptosis or differentiation abnormalities in normal bone marrow cells. According to supplier information, MM-401 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 comprehensively characterizes our recently developed inhibitor MM-401, which targets the activity of the 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) in other mixed lineage leukemia (MLL) families, revealing the unique regulatory properties of the MLL1 complex. Using MM-401 and its enantiomer control MM-NC-401, we demonstrated that inhibiting MLL1 methyltransferase activity specifically suppresses MLL cell proliferation by inducing cell cycle arrest, apoptosis, and myeloid differentiation, without toxicity to normal bone marrow cells or non-MLL cells. More importantly, transcriptomic 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 in MLL1-dependent leukemia. We envision broad applications for MM-401 in both basic and translational research. [1]

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In summary, the highly selective MLL1 inhibitors we report here have broad application prospects in basic research and lay the foundation for the development of effective treatments in the clinical field in the future. We envision the following research directions that may emerge in the future: First, with the development of the chemical probe MM-401, the essential functions of H3K4 methylation in various biological contexts can be studied. Compared with gene knockout models, the targeted inhibition of pharmacological compounds such as MM-401 produces small but specific perturbations to the MLL1 complex, thereby determining the function of its methyltransferase activity. Second, given that MLL1 and H3K4me regulate several key targets in MLL leukemia (e.g., Hoxa9, Myc, and Bcl2) (Figure 7C), it is crucial to compare the gene pathways dependent on MLL1 and MLL fusion proteins to determine whether they are highly overlapping or belong to different pathways that are essential for the progression of MLL leukemia. Third, it is crucial to test whether MLL1 inhibitors have the potential to treat a variety of diseases other than MLL leukemia. Specifically, inhibiting MLL1 activity may help treat acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) with wild-type MLL1 alleles and Hoxa9 overexpression (Ayton and Cleary, 2001; Dou and Hess, 2008). It may also be applicable to AML with MLL1 amplification and tandem duplication. In the future, testing MM-401 or its derivatives in a variety of human diseases will provide more in-depth insights. [1]

C29H46N8O5

586.726146221161

586.359

C, 59.37; H, 7.90; N, 19.10; O, 13.63

1442106-10-6

MM-401 TFA;1442106-11-7

71586081

Typically exists as solid at room temperature

0.9

7

6

8

42

977

4

CC[C@H]1C(=O)N[C@@H](C(=O)NCCCC[C@@](C(=O)N[C@H](C(=O)N1)CCCN=C(N)N)(C)NC(=O)C(C)C)C2=CC=CC=C2

SILRGLDFBXVGOQ-ZMROOPMESA-N

InChI=1S/C29H46N8O5/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/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)/t20-,21-,22+,29+/m0/s1

N-[6(S)-Ethyl-9(S)-(3-guanidino-propyl)-12(R)-methyl-2,5,8,11-tetraoxo-3(R)-phenyl-1,4,7,10tetraaza-cyclohexadec-12-yl]-isobutyramide

MM 401; MM-401; 1442106-10-6; MM-401; CHEMBL3798088; isobutyryl-D-aMeLys(1)-Arg-Abu-D-Phg-(1); 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; CID 71586081; SCHEMBL15004652; BDBM50164787; MM401

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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