WDR5 (WD repeat domain 5 protein) – MLL (Mixed Lineage Leukemia) protein-protein interaction. [1]
Binding affinity to WDR5: IC50 = 0.90 ± 0.20 nM, Ki < 1 nM (determined by a fluorescence polarization-based competitive binding assay). [1]
The primary target of MM-589 is WDR5, functioning by blocking the interaction between WDR5 and MLL proteins. WDR5 is a core component of the H3K4 histone methyltransferase complex, interacting with MLL1-MLL4 family members and playing key roles in histone H3K4 trimethylation, chromatin remodeling, and transcriptional activation of target genes. MM-589 binds to WDR5 with extremely high affinity, with an IC50 of 0.90 nM (Ki < 1 nM), competitively occupying the MLL binding interface on WDR5 to specifically inhibit MLL1 methyltransferase activity.

- MLL HMT Activity Inhibition: MM-589 potently inhibits the histone H3 lysine 4 (H3K4) methyltransferase (HMT) activity of the MLL core complex in a cell-free AlphaLISA functional assay, with an IC50 value of 12.7 ± 1.5 nM. [1]
- Enzymatic Selectivity: In a previously published HMT assay, compound MM-589 effectively inhibits MLL HMT activity (IC50 = 12.7 nM) but shows no or minimal effect (up to 100 μM) on the HMT activity of other SET1 family members, including MLL2, MLL3, MLL4, SET1a, and SET1b. [1]
- Cell Growth Inhibition in MLL-translocated Leukemia Cells: MM-589 potently and selectively inhibits the cell growth of human acute leukemia cell lines harboring MLL translocations. [1]
- In MOLM-13 cells (harboring MLL-AF9 fusion), the IC50 for cell growth inhibition is 0.21 ± 0.02 μM after a 4-day treatment (with 10,000 cells/well in 96-well plates). [1]
- In MV4-11 cells (harboring MLL-AF4 fusion), the IC50 for cell growth inhibition is 0.25 ± 0.01 μM after a 4-day treatment (with 10,000 cells/well in 96-well plates). [1]
- The compound displays >30-fold selectivity for these MLL-translocated cell lines over the HL-60 leukemia cell line (lacking MLL translocation), which has an IC50 of 8.56 ± 1.14 μM. [1]
- Long-term Treatment Effect: With a 7-day treatment (starting with 50,000 cells/mL in 24-well plates, with re-dosing on day 4), MM-589 achieves >95% maximum cell growth inhibition in both MOLM-13 and MV4-11 cell lines, compared to approximately 75% maximum inhibition with a 4-day treatment. The IC50 values improve by 2-3 times with the longer treatment. [1]
- Stereospecificity: The enantiomer of MM-589 (Compound 19, where the chiral center in the phenylglycine residue is changed from D- to L-configuration) has an IC50 of 14.3 μM and 35.0 μM in MOLM-13 and MV4-11 cells, respectively, making it >50-100 times less potent than MM-589. [1]

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MM-589 exhibits excellent inhibitory activity in vitro. By fluorescence polarization competition assay, MM-589 binds to WDR5 with an IC50 of 0.90 nM (Ki < 1 nM); in a fully reconstituted MLL core complex histone methyltransferase (HMT) assay, the IC50 for inhibiting HMT activity is 12.7 nM. In human leukemia cell lines harboring MLL translocations, MM-589 (0.01-10 μM) treatment for 4-7 days potently and selectively inhibits cell proliferation. The IC50 values against MV4-11 and MOLM-13 cells are 0.25 μM and 0.21 μM, respectively, with much weaker inhibitory activity against non-MLL leukemia cells (e.g., HL-60, IC50 of 8.6 μM). MM-589 exhibits >40-fold improved growth inhibition potency compared to the previously reported compound MM-401.

MM-589 TFA salt demonstrates in vivo anti-leukemic activity in animal models. As a potent inhibitor of the WDR5-MLL interaction, MM-589 effectively suppresses the expansion of MLL-rearranged leukemia cells in mice. Studies indicate that optimized derivatives of MM-589 with improved pharmacokinetic properties have the potential to be trialed in clinical settings. Compared to MM-401, MM-589 exhibits superior pharmacodynamic profiles in in vivo studies due to its improved cellular permeability and metabolic stability. MM-589 represents the most potent inhibitor of the WDR5-MLL interaction reported to date, and further optimization may yield a new therapy for acute leukemia.

- Fluorescence Polarization (FP)-based Competitive Binding Assay for WDR5: To determine binding affinities of compounds to WDR5, an FP-based competitive binding assay was used. 5 μL of test compound solution in DMSO was added to 120 μL of a preincubated complex solution containing N-terminal His-tagged WDR5 protein (residues 24-334) and a S-FAM labeled tracer in assay buffer (0.1 M phosphate, 25 mM KCl, 0.01% Triton, pH 6.5). The final concentrations of WDR5 and tracer were 4 nM and 0.6 nM, respectively. The plate was incubated at room temperature on a shaker for 3 hours, after which the mP values were measured using a plate reader. Ki values were calculated using a previously described equation. [1]
- Cell-free AlphaLISA MLL HMT Functional Assay: The inhibition of MLL HMT activity was evaluated using a homogeneous AlphaLISA assay. The recombinant MLL core complex (containing MLL, WDR5, ASH2L, RbBP5, DPY30) and recombinant nucleosomes were used. Serial dilutions of the compound (2.5 μL) in assay buffer with 4% DMSO and 5 μL of MLL complex solution were added to a 384-well plate and incubated for 30 minutes with gentle shaking. Then, 2.5 μL of a SAM/Nucleosome mixture was added, with final concentrations of MLL complex, SAM, and nucleosomes being 5 nM, 200 nM, and 3 nM, respectively, in 1% DMSO. The methylation reaction proceeded for 120 minutes in the dark. The reaction was stopped by adding 5 μL of a high-salt stopping solution for 15 minutes. Subsequently, 5 μL of a mixture containing anti-H3K4me1/2 AlphaLISA acceptor beads and biotinylated anti-H3 (C-terminus) antibody in detection buffer was added and incubated for 1 hour. Finally, 5 μL of streptavidin-labeled donor beads were added and incubated for 30 minutes. The plate was read using a microplate reader with excitation at 680 nm and emission at 615 nm. IC50 values were determined by fitting fluorescence intensities versus compound concentrations using a sigmoidal dose-response curve in Graphpad Prism software. [1]

- Cell Viability Assay (4-day treatment): To assess cell growth inhibition, MOLM-13, MV4-11, or HL-60 leukemia cells were seeded at 1 x 10⁴ cells per well in 96-well plates. Cells were treated with various concentrations of the test compound (MM-589 or others) for 4 days. The final concentration of DMSO in the culture media was 0.2%. Cell viability was determined using the WST-8 cell proliferation assay kit, following the manufacturer's instructions. The IC50 values (50% cell growth inhibition) were calculated relative to the DMSO control using Prism software. Experiments were performed three times in triplicate. [1]
- Long-term Cell Viability Assay (7-day treatment): MOLM-13 and MV4-11 cell lines were plated at a density of 5 x 10⁴ cells/mL in 24-well plates (2 mL/well) and treated with relevant concentrations of MM-589. On day 4, cell viability was measured using the WST-8 assay. Then, 10% of viable cells from each well were transferred to freshly prepared medium containing corresponding concentrations of the compound and cultured for an additional 3 days. On day 7, cell viability for each treatment was determined. [1]

Animal Models: Establish disseminated leukemia models by intravenous injection of MLL-rearranged leukemia cells (e.g., MLL-AF9 cells) into immunodeficient mice (NSG or NOD/SCID mice) via the tail vein. Dosing Regimen: Administer MM-589 TFA by intravenous injection. Formulation example: 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% saline. Efficacy Assessment: Monitor tumor burden by bioluminescent imaging, record survival (Kaplan-Meier analysis), and detect the percentage of leukemia cells (hCD45⁺) 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 control groups.

As the trifluoroacetate salt form of a macrocyclic peptidomimetic, MM-589 TFA salt exhibits favorable physicochemical properties. Its molecular formula is C₃₀H₄₅F₃N₈O₇ with a molecular weight of 686.72. Regarding solubility, this compound is soluble in DMSO (25 mg/mL, ultrasonic and warming to 60°C required), with limited solubility in aqueous buffers. Storage conditions: powder is stable for 3 years at -20°C protected from light; solutions are stable for 6 months at -80°C and 1 month at -20°C protected from light. Compared to the linear peptide MM-101, MM-589 exhibits significantly improved metabolic stability and cellular permeability through the cyclization strategy.

According to the Material Safety Data Sheet (MSDS), MM-589 TFA salt is not classified as a hazardous substance or mixture, and GHS label elements do not apply. Regarding carcinogenicity: Not listed as a carcinogen by NTP; not listed by IARC; not regulated by OSHA; not listed by ACGIH. The toxicological effects of this product have not been thoroughly studied. At the cellular level, MM-589 shows no significant toxicity to normal bone marrow cells, selectively inhibiting the proliferation of MLL-rearranged leukemia cells. Standard laboratory safety practices should be followed when handling, and release to the environment should be avoided. MM-589 TFA salt has a purity of ≥98% and is for research use only, not for human or veterinary applications.

[1]. Discovery of a Highly Potent, Cell-Permeable Macrocyclic Peptidomimetic (MM-589) Targeting the WD Repeat Domain 5 Protein (WDR5)-Mixed Lineage Leukemia (MLL) Protein-Protein Interaction. J Med Chem. 2017 Jun 22;60(12):4818-4839.

- MM-589 corresponds to Compound 18 in the study. It was synthesized via a method involving solid-phase peptide synthesis, ring-closing metathesis (RCM), catalytic hydrogenation, and removal of protecting groups. The overall synthetic route is detailed in Schemes 4 and 5 of the paper. [1]
- A cocrystal structure of MM-589 (18) in complex with WDR5 was determined at a resolution of 1.64 Å (PDB code 5VFC). The structure shows that MM-589 binds to the central channel of the WD40 propeller in WDR5 through a conserved network of hydrogen bonds and hydrophobic interactions. The smaller ring size of MM-589 (n=2) compared to earlier analogs (n=4 for compound 2, n=6 for compound 16) forms a more compact structure, facilitating optimal intramolecular hydrogen bonds and reducing conformational flexibility, which contributes to its high binding affinity. [1]
- The study found a good correlation between the potencies of compounds in inhibiting MLL HMT activity (IC50) and inhibiting cell growth (IC50) in MV4-11 and MOLM-13 cell lines. [1]
- The compound is stable in cell culture media for up to 7 days. [1]

C30H45F3N8O7

686.722917318344

686.336

C, 52.47; H, 6.61; F, 8.30; N, 16.32; O, 16.31

2097887-21-1

2097887-20-0;2097887-21-1 (TFA salt);

132585206

White to off-white solid at room temperature

8

11

9

48

1050

4

CC[C@H]1C(=O)N[C@@H](C(=O)NCC[C@@](C(=O)N[C@H](C(=O)N1)CCCNC(=NC)N)(C)NC(=O)C(C)C)C2=CC=CC=C2.C(=O)(C(F)(F)F)O

NARXNZHWAWPJIY-HTCBAMNXSA-N

InChI=1S/C28H44N8O5.C2HF3O2/c1-6-19-23(38)35-21(18-11-8-7-9-12-18)25(40)31-16-14-28(4,36-22(37)17(2)3)26(41)34-20(24(39)33-19)13-10-15-32-27(29)30-5;3-2(4,5)1(6)7/h7-9,11-12,17,19-21H,6,10,13-16H2,1-5H3,(H,31,40)(H,33,39)(H,34,41)(H,35,38)(H,36,37)(H3,29,30,32);(H,6,7)/t19-,20-,21+,28+;/m0./s1

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

MM589 (racemic mixture ) (TFA); MM-589 (racemic mixture ) (TFA)

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)

H2O: 40 mg/mL (58.3 mM)

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.)