MLL1-WDR5 PPI (IC50 = 2.4 nM)
MM-102 TFA: MLL1/WDR5 protein-protein interaction (Ki < 1 nM) [1]

In leukemia cells carrying the MLL1-AF9 fusion gene, MM-102 (HMTase Inhibitor IX) reduces MLL1 methyltransferase activity as well as MLL-1-induced HoxA9 and Meis-1 gene expression. Leukemia cells containing MLL1 fusion proteins also experienced reduced cell proliferation and death. With an IC50 of 0.4-0.9 μM in the HMT experiment, MM-102 (TFA) exhibits the most inhibitory action and the highest binding affinity for WDR5[1]. Leukemia cell lines carrying the MLL1-AF4 and MLL1-ENL fusion proteins, MV4;11 and KOPN8, respectively, are inhibited in their ability to proliferate by MM-102 (HMTase inhibitor IX) in a dose-dependent manner [1]. MM-102, also known as HMTase Inhibitor IX, totally suppresses cell growth in these cell lines at 75 μM, with an IC50 of 25 μM[1]. Leukemia cells are effectively and selectively inhibited in their proliferation and induced to undergo apoptosis by MM-102 (HMTase inhibitor IX). Leukemia cells are not significantly affected by MLL1 fusion protein or wild-type MLL1 protein [1].

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1. In a fully reconstituted in vitro H3K4 methyltransferase assay, MM-102 TFA functions as a potent antagonist of MLL1 enzymatic activity [1]
2. In cultured mouse renal proximal tubular cells (RPTCs) exposed to cisplatin (20 μM), treatment with MM-102 TFA (50 μM) for 1 h followed by 24 h of cisplatin exposure inhibits apoptosis, reduces phosphorylation of p53, preserves E-cadherin expression, represses levels of MLL1, WDR5 and H3K4me3, blocks cisplatin-triggered DNA damage response (DDR) as shown by dephosphorylation of ATM, ATR, Chk1 and Chk2 proteins, depression of γ-H2AX, and restrains cell cycle arrest (decreased expression of p21 and phospho-histone H3 at serine 10) [2]
3. In leukemia cells harboring MLL1 fusion proteins, MM-102 TFA specifically inhibits cell growth and induces apoptosis [1]

MM-102 attenuates AKI after cisplatin administration in mice[2]
To investigate the role of MLL1/WDR5 in cisplatin-induced AKI, mice were treated with MM102, an inhibitor of the MLL1/WDR5 complex, or vehicle 2 h before cisplatin administration (20 mg/kg, intraperitoneally injection). MM102 was then given daily for three consecutive days. Blood samples and kidney tissue were collected 72 h after cisplatin injection. Blood urea nitrogen (BUN) and serum creatinine (SCr) were used as measures of renal function. As shown in Fig. 1A, BUN levels in cisplatin group were much higher than that in control group (6.217 ± 0.374 vs. 2.420 ± 0.470 mmol/L) (***P < 0.001); MM102 treatment reduced the cisplatin-boosted BUN to 3.172 ± 0.114 mmol/L (**P < 0.01). Similarly, SCr was 68.126 ± 10.217 μmol/L in cisplatin-alone group (Fig. 1B), higher than that in the control group (10.322 ± 2.135 μmol/L) (**P < 0.01); MM102 treatment significantly reduced SCr to 20.922 ± 4.016 μmol/L (**P < 0.01); MM102 alone had little effect on either BUN or SCr.

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MM-102 reduces apoptosis, along with reduced p53 phosphorylation and retained E-cadherin expression in vivo[2]
IF staining indicated that neutrophil gelatinase-associated lipocalin (NGAL, an early biomarker of AKI) was increased in kidneys exposed to cisplatin relative to sham-operated kidneys. Administration of MM102 dramatically reduced NGAL expression in cisplatin-injured kidneys (Fig. 2A, B). Consistently, TdT-mediated dUTP-X nick-end labeling (TUNEL) staining displayed increased number of apoptotic cells in injured kidney and MM102 largely inhibited this response (Fig. 2A, C). Moreover, increased expression of NGAL and cleavage of caspase-3 (C-cas3, a recognized marker of apoptosis) in the kidney after cisplatin administration were detected by immunoblot analysis; treatment with MM102 returned these changes to base levels.

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1. In bone marrow cells transduced with MLL1-AF9 fusion construct, treatment with MM-102 TFA for 96 h effectively decreases the expression of HoxA9 and Meis-1 (two critical MLL1 target genes in MLL1 fusion protein mediated leukemogenesis) [1]
2. In cisplatin-induced acute kidney injury (AKI) mice model (cisplatin dose of 20 mg/kg), administration of MM-102 TFA (15 mg/kg) intraperitoneally 2 h before cisplatin injection and then daily for three consecutive days improves renal function (reduced blood urea nitrogen (BUN) and serum creatinine (Scr)), attenuates tubular injury and apoptosis, represses MLL1, WDR5, and H3K4me3 levels, dephosphorylates p53, preserves E-cadherin, inhibits cisplatin-triggered DDR (dephosphorylation of ATM, ATR, Chk1, Chk2; depression of γ-H2AX) and restrains cell cycle arrest (decreased p21 and phospho-histone H3 at serine 10) [2]

Competitive Binding Assay[1]
Binding affinities of all the synthesized compounds were determined using a fluorescence-polarization (FP)-based competitive binding assay; the details of this assay have been described earlier.

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In Vitro Histone Methyltransferase (HMT) Assay[1]
The HMT assay was performed in 50 mM HEPES pH 7.8, 100 mM NaCl, 1.0 mM EDTA, and 5% glycerol at 22 °C. Each reaction contained 1.5 μCi of the co-factor,3H-S-adenosylmethionine. H3 10-residue peptide was used as the substrate at 50 μM. Compounds were added at concentrations ranging from 0.125 to 128 μM and incubated with the pre-assembled WDR5/RbBP5/ASH2L complex at a final concentration of 0.5 μM for each protein for 2–5 min. Reactions were initiated by addition of the MLL1 protein at a final concentration of 0.5 μM and allowed to proceed for 30 min before preparing scintillation counting. To count samples, reactions were spotted on separate squares of P81 filter paper (Whatman) and precipitated by submerging in freshly prepared 50 mM sodium bicarbonate buffer with pH 9.0. After washing and drying, samples were vortexed in Ultima Gold scintillation fluid and counted. As a negative control, assays were performed using 0.5 μM MLL1/WDR5/RbBP5/ASH2L complex assembled with the non-interacting mutant, WDR5D107A.

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1. For the H3K4 methyltransferase activity assay of the reconstituted MLL1 core complex, a scintillation counter assay was employed to measure the inhibitory effect of MM-102 TFA on MLL1 enzymatic activity; the compound was tested in the assay system to evaluate its capacity to antagonize MLL1-mediated histone methylation [1]
2. A fluorescence polarization (FP)-based binding assay was used to determine the competitive binding curves of MM-102 TFA for WDR5, so as to assess the binding affinity of the compound to the target protein [1]

qRT-PCR Analysis of HOXA9 and MEIS-1 Genes[1]
Murine MLL1-AF9 transformed bone marrow cells were obtained by transducing normal murine bone marrow cells with MLL1-AF9 oncogene according to the procedures described by Tan et al.22 MM-102 and C-MM-102 were dissolved in DMSO. The transformed cells were treated with MM-102 (25 μM, 50 μM), C-MM-102 (50 μM), and Mock (0.2% DMSO), giving a final concentration of 0.2% DMSO in all the samples. Total RNA was isolated from MLL1-AF9 transduced mouse bone marrow cells after 96 h treatment using Trizol and the RNEASY kit according to the protocol described earlier.23 The cDNA was generated using random priming with the SuperScript III kit. Real-time PCR amplifications of HoxA9, Meis1, and GAPDH genes were carried out with primers specific for each gene in the presence of SYBR dye. Relative quantification of each gene transcript was carried out as described in our previous work.10 The results were presented as relative expression to Mock treatment after normalizing to an internal loading control (e.g., GAPDH or total input RNA).

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Cell Growth and Apoptosis Studies of Leukemia Cell Lines[1]
MV4;11, KOPN8, and K562 cells were a generous gift from Dr. Jolanta Grembecka (University of Michigan). MV4;11, KOPN8, and K562 cells were cultured in RPMI 1640 medium (ATCC) supplemented with 10% fetal bovine serum and 100 U/L penicillin-streptomycin and incubated at 37 °C under 5% CO2. Cells were seeded into 12-well plates for suspension at a density of 5 × 105 per well (1 mL) and treated with either vehicle control (DMSO, 0.2%) or MM-102 for 7 days. The medium was changed every 2 days, and compounds were resupplied.

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1. For leukemia cell-related assays, leukemia cells harboring MLL1 fusion proteins were treated with MM-102 TFA, and then cell growth status was monitored for 7 days to evaluate the anti-proliferative effect; apoptosis was detected after 96 h of treatment to assess the pro-apoptotic activity of the compound [1]
2. For bone marrow cell assays, bone marrow cells transduced with MLL1-AF9 fusion construct were treated with MM-102 TFA for 96 h, and then the expression levels of HoxA9 and Meis-1 were detected by PCR and normalized to GAPDH expression to evaluate the regulatory effect of the compound on MLL1 target genes [1]
3. For RPTCs assays, RPTCs were pre-treated with MM-102 TFA (50 μM) for 1 h and then exposed to cisplatin (20 μM) for an additional 24 h; cell viability was detected by CCK8 assay, apoptosis was assessed by TUNEL staining, and the expression levels of related proteins (cleaved caspase3, phospho-p53, p53, E-cadherin, MLL1, WDR5, H3K4me3, ATM, ATR, Chk1, Chk2, p21, phospho-histone H3 at serine 10) were detected by western blot; in some experiments, cells were also transfected with siRNAs targeting MLL1, WDR5, E-cadherin or p53 before drug treatment and cisplatin exposure to further explore the mechanism [2]

Animals models of AKI and treatment[2]
Male C57BL/6J mice aged 6–8 weeks and weighing 20–25 g were purchased from the Jackson Laboratory. The mice were randomly divided into four groups: (1) control, (2) MM-102, (3) cisplatin, and (4) MM-102 plus cisplatin. Cisplatin was intraperitoneally injected at the dose of 20 mg/kg. MM-102 (15 mg/kg) dissolved in solvent containing 10% DMSO and 90% corn oil was administered intraperitoneally 2 h before the cisplatin injection and then given daily for three consecutive days. The dose of MM-102 was selected according to a previous report. For the control and cisplatin-alone groups, mice were injected with an equivalent amount of solvent. Mice in the control and MM-102 groups were injected with an equal volume of a normal saline solution. All the mice were euthanized 72 h after cisplatin injection. Blood samples and kidney tissues were collected for further analysis. All experimental protocols were performed according to the National Institutes of Health Guidelines on the Care and Use of Laboratory Animals and approved by the Lifespan Animal Welfare Committee. The authorization number for the use of laboratory animals is 5074-19.

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1. For the acute leukemia-related in vivo experiment, bone marrow cells transduced with MLL1-AF9 fusion construct were used, and the treatment duration of MM-102 TFA for this cell model was 96 h to detect the expression of target genes [1]
2. For the cisplatin-induced AKI mice model, MM-102 TFA was administered via intraperitoneal injection at a dose of 15 mg/kg; the administration was carried out 2 h before cisplatin (20 mg/kg, intraperitoneal injection) injection, and then the same dose was given daily for three consecutive days; all mice were euthanized 72 h after cisplatin injection, and blood samples and kidney tissues were collected for subsequent detection of renal function indexes (BUN, Scr), pathological section staining (PAS staining), immunoblot analysis of related proteins and immunofluorescent staining of target proteins [2]

[1]. High-affinity, small-molecule peptidomimetic inhibitors of MLL1/WDR5 protein-protein interaction. J Am Chem Soc. 2013, 135(2), 669-682.

[2]. Histone methyltransferase MLL1 drives renal tubular cell apoptosis by p53-dependent repression of E-cadherin during cisplatin-induced acute kidney injury. Cell Death Dis . 2022 Sep 6;13(9):770.

Mixed-lineage leukemia 1 (MLL1) is a histone H3 lysine 4 (H3K4) methyltransferase, and targeting MLL1 enzyme activity has been proposed as a novel therapeutic strategy for acute leukemia carrying MLL1 fusion proteins. The MLL1/WDR5 protein-protein interaction is crucial for MLL1 enzyme activity. In this study, we designed a large number of peptide mimics targeting the MLL1/WDR5 interaction based on the minimum binding motif of MLL1, -CO-ARA-NH-. Our study designed high-affinity peptide mimics that exhibited potent antagonism of MLL1 activity in a fully recombinant in vitro H3K4 methyltransferase activity assay with a binding affinity (K_i < 1 nM) to WDR5. Cocrystal structure determination of two potent peptide mimics with WDR5 revealed the structural basis for their high-affinity binding to WDR5. Evaluation of one of the peptide mimics, MM-102, in bone marrow cells transfected with the MLL1-AF9 fusion construct showed that the compound effectively reduced the expression of HoxA9 and Meis-1, two key target genes in the MLL1 fusion protein-mediated leukemia process. MM-102 also specifically inhibited the growth of leukemia cells carrying the MLL1 fusion protein and induced their apoptosis. Our study is the first to validate the concept of designing small molecule inhibitors of WDR5/MLL1 protein-protein interaction as a novel treatment for acute leukemia carrying the MLL1 fusion protein. [1] Mixed lineage leukemia 1 (MLL1) is a histone H3 lysine 4 (H3K4) methyltransferase that interacts with WD repeat domain 5 (WDR5) to regulate cell survival, proliferation and senescence. The role of MLL1 in the pathogenesis of acute kidney injury (AKI) is unclear. In this study, we found that cisplatin-induced upregulation of MLL1, WDR5, and trimethylated H3K4 (H3K4me3) expression in mouse AKI renal tubular cells, along with increased p53 phosphorylation and decreased E-cadherin expression. Administration of the selective MLL1/WDR5 complex inhibitor MM102 improved renal function, reduced renal tubular damage and apoptosis, while inhibiting the expression of MLL1, WDR5, and H3K4me3, dephosphorylating p53, and maintaining E-cadherin expression. In cultured mouse proximal renal tubular cells (RPTCs), cisplatin treatment, MM102 treatment, or transfection with MLL1 or WDR5 siRNA all inhibited apoptosis and p53 phosphorylation while maintaining E-cadherin expression. Inhibition of p53 with Pifithrin-α reduced cisplatin-induced apoptosis but did not affect the expression of MLL1, WDR5, and H3K4me3. Interestingly, silencing E-cadherin counteracts the cytoprotective effects of MM102 but has no effect on p53 phosphorylation. These results suggest that MLL1/WDR5 activates p53, thereby inhibiting E-cadherin and ultimately leading to apoptosis during cisplatin-induced acute kidney injury (AKI). Further studies showed that MM102 effectively inhibits cisplatin-induced DNA damage response (DDR), manifested as dephosphorylation of ataxia-telangiectasia mutant protein (ATM) and ATM and Rad-3-related protein (ATR), dephosphorylation of checkpoint kinases 1 and 2 (Chk1 and Chk2), inhibition of γ-H2AX, and inhibition of cell cycle arrest (manifested as decreased p21 expression in vitro and in vivo and reduced phosphorylation of histone H3 at serine 10). Overall, we found that MLL1 is a novel DDR regulator that drives cisplatin-induced RPTC apoptosis and AKI by modulating the MLL1/WDR5/ATR/ATM-Chk-p53-E-cadherin axis. Targeting the MLL1/WDR5 complex may have the potential to treat AKI. [2]

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1. MM-102 TFA is a high-affinity small peptide mimic inhibitor designed based on the minimal binding motif derived from MLL1 -CO-ARA-NH- to target the MLL1/WDR5 protein-protein interaction; the co-crystal structure of the MM-102 TFA complex with WDR5 elucidates the structural basis for its high-affinity binding to WDR5. [1]
2. In cisplatin-induced acute kidney injury (AKI), MM-102 TFA exerts its protective effect by modulating the MLL1/WDR5-/ATR/ATM-Chk-p53-E-cadherin axis. Among them, MLL1/WDR5 activates p53 to inhibit E-cadherin, thereby leading to apoptosis. MM-102 TFA can block this pathway, thereby reducing renal tubular cell apoptosis[2]. 3. The relevant technology of MM-102 TFA has been licensed to Ascentage Group; one of the researchers, Wang Shaomeng, is the co-founder of Ascentage Group, holds shares in the company and serves as an advisor[1].

C37H50F5N7O6

783.828226566315

783.374

C, 56.70; H, 6.43; F, 12.12; N, 12.51; O, 12.25

1883545-52-5

MM-102;1417329-24-8

71520620

White to off-white solid powder

7

12

16

55

1170

1

CCC(CC)(C(=O)N[C@@H](CCCN=C(N)N)C(=O)NC1(CCCC1)C(=O)NC(C2=CC=C(C=C2)F)C3=CC=C(C=C3)F)NC(=O)C(C)C.C(=O)(C(F)(F)F)O

ZRKTWBXVGMHWHM-YCBFMBTMSA-N

InChI=1S/C35H49F2N7O4.C2HF3O2/c1-5-34(6-2,43-29(45)22(3)4)31(47)41-27(10-9-21-40-33(38)39)30(46)44-35(19-7-8-20-35)32(48)42-28(23-11-15-25(36)16-12-23)24-13-17-26(37)18-14-24;3-2(4,5)1(6)7/h11-18,22,27-28H,5-10,19-21H2,1-4H3,(H,41,47)(H,42,48)(H,43,45)(H,44,46)(H4,38,39,40);(H,6,7)/t27-;/m0./s1

N-[bis(4-fluorophenyl)methyl]-1-[[(2S)-5-(diaminomethylideneamino)-2-[[2-ethyl-2-(2-methylpropanoylamino)butanoyl]amino]pentanoyl]amino]cyclopentane-1-carboxamide;2,2,2-trifluoroacetic acid

MM-102 TFA; MM-102 trifluoroacetic acid; MM-102; MM 102; MM102;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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 (~127.58 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.19 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 (3.19 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 (3.19 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.)