WDR5 (Ki < 1 nM); WDR5-MLL1 interaction (IC50 = 0.9 nM); MLL1 (IC50 = 0.32 µM)

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].

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.

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.

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

Here we report a comprehensive characterization of our recently developed inhibitor MM-401 that targets the MLL1 H3K4 methyltransferase activity. MM-401 is able to specifically inhibit MLL1 activity by blocking MLL1-WDR5 interaction and thus the complex assembly. This targeting strategy does not affect other mixed-lineage leukemia (MLL) family histone methyltransferases (HMTs), revealing a unique regulatory feature for the MLL1 complex. Using MM-401 and its enantiomer control MM-NC-401, we show that inhibiting MLL1 methyltransferase activity specifically blocks proliferation of MLL cells by inducing cell-cycle arrest, apoptosis, and myeloid differentiation without general toxicity to normal bone marrow cells or non-MLL cells. More importantly, transcriptome analyses show that MM-401 induces changes in gene expression similar to those of MLL1 deletion, supporting a predominant role of MLL1 activity in regulating MLL1-dependent leukemia transcription program. We envision broad applications for MM-401 in basic and translational research. [1]

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In summary, the highly selective MLL1 inhibitor we reported here will have broad applications in basic research and lay the foundation for future development of effective therapeutics in clinical arenas. We envision several lines of research may arise in future: First, with development of chemical probe MM-401, the requisite function of H3K4 methylation in various biological contexts can be examined. Compare to genetic knockout models, on-target inhibition by pharmacological compounds such as MM-401 introduces small but specific perturbation to the MLL1 complex, allowing functional attribution to its methyltransferase activity. Second, given that MLL1 and H3K4me regulate several key targets (e.g. Hoxa9, Myc and Bcl2) in MLL leukemia (Figure 7C), it will be important to compare MLL1 and MLL fusion protein dependent gene pathways to see if they largely overlap with each other or they belong to distinct pathways that are important for progression of MLL leukemia. Third, it will be important to test whether MLL1 inhibitor has therapeutic potential for a broad spectrum of diseases beyond MLL leukemia. Specifically, inhibiting MLL1 activity may be useful for treatment of AMLs and myelodysplastic syndrome (MDS) that have wild type MLL1 alleles and Hoxa9 overexpression (Ayton and Cleary, 2001; Dou and Hess, 2008). It may also be applicable for AMLs with MLL1 amplification and tandem duplication. Testing MM-401 or its derivatives on broad spectrum of human diseases will provide greater insights in future. [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.)