MLL1-WDR5 PPI (IC50 = 2.4 nM)

在携带 MLL1-AF9 融合基因的白血病细胞中，MM-102（HMTase 抑制剂 IX）可降低 MLL1 甲基转移酶活性以及 MLL-1 诱导的 HoxA9 和 Meis-1 基因表达。含有 MLL1 融合蛋白的白血病细胞也经历了细胞增殖减少和死亡。在 HMT 实验中，MM-102 (TFA) 的 IC50 为 0.4-0.9 μM，对 WDR5 表现出最强的抑制作用和最高的结合亲和力[1]。分别携带 MLL1-AF4 和 MLL1-ENL 融合蛋白 MV4;11 和 KOPN8 的白血病细胞系的增殖能力被 MM-102（HMTase 抑制剂 IX）以剂量依赖性方式抑制 [1]。 MM-102，也称为 HMTase Inhibitor IX，在 75 μM 浓度下完全抑制这些细胞系中的细胞生长，IC50 为 25 μM[1]。 MM-102（HMTase 抑制剂 IX）可有效、选择性地抑制白血病细胞的增殖并诱导其凋亡。 MLL1融合蛋白或野生型MLL1蛋白对白血病细胞没有显着影响[1]。

MM-102减轻小鼠顺铂给药后AKI [2]
为了研究MLL1/WDR5在顺铂诱导AKI中的作用，小鼠在顺铂给药前2小时用MLL1/WDR5复合物抑制剂MM102或载药（20 mg/kg，腹腔注射）治疗。然后每天给予MM102，连续三天。注射顺铂后72h采集血液和肾脏组织。血尿素氮（BUN）和血清肌酐（SCr）作为肾功能指标。如图1A所示，顺铂组BUN水平明显高于对照组（6.217±0.374 vs. 2.420±0.470 mmol/L） （***P < 0.001）；MM102治疗使顺铂组BUN降低至3.172±0.114 mmol/L （**P < 0.01）。单用顺铂组SCr为68.126±10.217 μmol/L（图1B），高于对照组（10.322±2.135 μmol/L） （**P < 0.01）；MM102处理显著降低SCr为20.922±4.016 μmol/L (**P < 0.01)；单独使用MM102对BUN或SCr的影响很小。

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MM-102可减少细胞凋亡，同时降低p53磷酸化水平，保留E-cadherin在体内的表达[2]
IF染色显示，相对于假手术肾脏，暴露于顺铂的肾脏中中性粒细胞明胶酶相关脂钙蛋白（中性粒细胞明胶酶相关脂钙蛋白，AKI的早期生物标志物）增加。给药MM102显著降低了顺铂损伤肾脏中NGAL的表达（图2A， B）。与此一致，tdt介导的dUTP-X镍端标记（TUNEL）染色显示损伤肾脏中凋亡细胞数量增加，MM102在很大程度上抑制了这种反应（图2A， C）。此外，通过免疫印迹分析检测到顺铂给药后肾脏中NGAL表达增加，caspase-3 （C-cas3，一种公认的细胞凋亡标志物）的切割；MM102治疗使这些变化恢复到基础水平。

竞争结合试验[1]
采用基于荧光偏振（FP）的竞争结合法测定所有合成化合物的结合亲和力；这个实验的细节已经在前面描述过了。

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体外组蛋白甲基转移酶（HMT）测定[1]
HMT检测在50 mM HEPES pH 7.8、100 mM NaCl、1.0 mM EDTA和5%甘油中进行，温度为22°C。每个反应含有1.5 μCi的辅助因子3h - s -腺苷蛋氨酸。以h310 -残基肽为底物，深度为50 μM。加入浓度为0.125 ~ 128 μM的化合物，并与预组装好的WDR5/RbBP5/ASH2L复合物一起孵育，每个蛋白的终浓度为0.5 μM，孵育2-5分钟。加入终浓度为0.5 μM的MLL1蛋白开始反应，并允许进行30分钟，然后准备闪烁计数。为了对样品进行计数，将反应在P81滤纸 的单独正方形上进行标记，并将其浸入新鲜配制的pH为9.0的50 mM碳酸氢钠缓冲液中沉淀。洗涤和干燥后，样品在Ultima Gold闪烁液中旋转并计数。作为阴性对照，用0.5 μM MLL1/WDR5/RbBP5/ASH2L复合物与非相互作用突变体WDR5D107A组装进行检测。

HOXA9和MEIS-1基因的qRT-PCR分析[1]
用MLL1-AF9致癌基因转染正常小鼠骨髓细胞，获得MLL1-AF9转化的小鼠骨髓细胞，方法由Tan等描述。 MM-102和C-MM-102溶解于DMSO中。转化后的细胞分别用MM-102 (25 μM, 50 μM)、C-MM-102 （50 μM）和Mock （0.2% DMSO）处理，所有样品的终浓度均为0.2% DMSO。用Trizol和RNEASY试剂盒按照前面描述的方法处理96小时后，从MLL1-AF9转导的小鼠骨髓细胞中分离总RNA利用SuperScript III试剂盒随机引物生成cDNA。在SYBR染料存在的情况下，用每种基因特异性引物对HoxA9、Meis1和GAPDH基因进行实时PCR扩增。每个基因转录物的相对定量按照我们之前的工作进行在归一化到内部负载控制（例如，GAPDH或总输入RNA）后，将结果呈现为模拟处理的相对表达。

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白血病细胞系细胞生长和凋亡的研究[1]
MV4;11， KOPN8和K562细胞是来自密歇根大学Jolanta Grembecka博士的慷慨馈赠。MV4；11、KOPN8和K562细胞在添加10%胎牛血清和100 U/L青霉素-链霉素的RPMI 1640培养基（ATCC）中培养，在37℃、5% CO2下培养。将细胞以5 × 105 /孔（1ml）的密度接种于12孔板中悬浮，用载体对照（DMSO, 0.2%）或MM-102处理7天。每2天更换一次培养基，并补充化合物。

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.

[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 the MLL1 enzymatic activity has been proposed as a novel therapeutic strategy for the treatment of acute leukemia harboring MLL1 fusion proteins. The MLL1/WDR5 protein-protein interaction is essential for MLL1 enzymatic activity. In the present study, we designed a large number of peptidomimetics to target the MLL1/WDR5 interaction based upon -CO-ARA-NH-, the minimum binding motif derived from MLL1. Our study led to the design of high-affinity peptidomimetics, which bind to WDR5 with K(i) < 1 nM and function as potent antagonists of MLL1 activity in a fully reconstituted in vitro H3K4 methyltransferase assay. Determination of co-crystal structures of two potent peptidomimetics in complex with WDR5 establishes their structural basis for high-affinity binding to WDR5. Evaluation of one such peptidomimetic, MM-102, in bone marrow cells transduced with MLL1-AF9 fusion construct shows that the compound effectively decreases the expression of HoxA9 and Meis-1, two critical MLL1 target genes in MLL1 fusion protein mediated leukemogenesis. MM-102 also specifically inhibits cell growth and induces apoptosis in leukemia cells harboring MLL1 fusion proteins. Our study provides the first proof-of-concept for the design of small-molecule inhibitors of the WDR5/MLL1 protein-protein interaction as a novel therapeutic approach for acute leukemia harboring MLL1 fusion proteins.[1]

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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 unknown. In this study, we demonstrate that MLL1, WDR5, and trimethylated H3K4 (H3K4me3) were upregulated in renal tubular cells of cisplatin-induced AKI in mice, along with increased phosphorylation of p53 and decreased expression of E-cadherin. Administration of MM102, a selective MLL1/WDR5 complex inhibitor, improved renal function and attenuated tubular injury and apoptosis, while repressing MLL1, WDR5, and H3K4me3, dephosphorylating p53 and preserving E-cadherin. In cultured mouse renal proximal tubular cells (RPTCs) exposed to cisplatin, treatment with MM102 or transfection with siRNAs for either MLL1 or WDR5 also inhibited apoptosis and p53 phosphorylation while preserving E-cadherin expression; p53 inhibition with Pifithrin-α lowered cisplatin-induced apoptosis without affecting expression of MLL1, WDR5, and H3K4me3. Interestingly, silencing of E-cadherin offset MM102's cytoprotective effects, but had no effect on p53 phosphorylation. These findings suggest that MLL1/WDR5 activates p53, which, in turn, represses E-cadherin, leading to apoptosis during cisplatin-induced AKI. Further studies showed that MM102 effectively inhibited cisplatin-triggered DNA damage response (DDR), as indicated by dephosphorylation of ataxia telangiectasia mutated (ATM) and ATM and Rad-3 related (ATR) proteins, dephosphorylation of checkpoint kinase 1 and 2 (Chk1 and Chk2); depression of γ-H2AX; and restrained cell cycle arrest, as evidenced by decreased expression of p21 and phospho-histone H3 at serine 10 in vitro and in vivo. Overall, we identify MLL1 as 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 a therapeutic potential for the treatment of AKI.[2]

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

218

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

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (3.19 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (3.19 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (3.19 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
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