PRMT1 (IC50 = 30 nM); PRMT3 (IC50 = 119 nM); PRMT4 (IC50 = 83 nM); PRMT6 (IC50 = 4 nM); PRMT8 (IC50 = 5 nM)

在 MCF7 细胞中，MS023 二盐酸盐（1-1000 nM；48 小时）可抑制 PRMT1 甲基转移酶活性 [1]。在 HEK293 细胞中，MS023 二盐酸盐（1-1000 nM；20 小时）可抑制 PRMT6 甲基转移酶活性 [1]。

当与 PKC412（100 mg/kg，ig）联合给药时，MS023 二盐酸盐（160 mg/kg，ip）可抑制功能性 MLL-r ALL 起始细胞的维持，从而阻断 MLL-r 急性淋巴细胞白血病 (ALL)。分散[2]。

PRMT生化测定[1]
闪烁邻近度测定（SPA）用于评估试验化合物对抑制PRMT催化的甲基转移反应的影响，如前所述。27简言之，氚化的S-腺苷-L-甲硫氨酸被用作甲基的供体。将（3H）甲基化生物素标记肽捕获在链亲和素/闪烁体包被的微孔板中，使掺入的3H甲基和闪烁体接近，从而产生发光，通过追踪TopCount NXT™微孔板闪烁和发光计数器测量的放射性信号（每分钟计数）来量化发光。必要时，使用非氚化SAM来补充反应。在平衡条件下，通过滴定反应混合物中的测试化合物，在底物和辅因子的Km浓度下测定IC50值。

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细胞PRMT1测定[1]
MCF7细胞在补充有10%FBS、青霉素（100单位mL−1）和链霉素（100μg mL−1。将40%汇合的细胞用不同浓度的MS023和化合物4-6以指定浓度或DMSO对照处理48小时。将细胞在100μL总裂解缓冲液（20 mM Tris-HCl pH 8、150 mM NaCl、1 mM EDTA、10 mM MgCl2、0.5%TritonX-100、12.5 U mL−1苯并酶、完全不含EDTA的蛋白酶抑制剂混合物）中裂解。在RT下孵育3分钟后，将SDS添加到最终1%的浓度。在SDS-PAGE上运行裂解物，并如下所述进行免疫印迹以在蛋白质印迹中测定H4R3me2a、精氨酸不对称二甲基化、精氨酰对称二甲基化和精氨酸单甲基化。

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细胞PRMT6测定[1]
HEK293细胞在补充有10%FBS、青霉素（100 U mL−1）和链霉素（100μg mL−1）的DMEM中的12孔板中生长。按照制造商的说明，使用jetPRIME®转染试剂，用FLAG标记的PRMT6或突变体V86K/D88A PRMT6（每孔1μg DNA）转染50%的融合细胞。4小时后移除培养基，并用指示浓度的MS023或DMSO对照处理细胞。20小时后，取出培养基，并在100μL的总裂解缓冲液中裂解细胞。

蛋白质印迹分析[1]
细胞类型： MCF7 和 HEK293 细胞
测试浓度： 1.4、4、12、37、111、333 和 1000 nM
孵育时间：MCF7 细胞为 48 小时； HEK293 细胞 20 小时
实验结果： 有效治疗且浓度依赖性降低 H4R3me2a 的细胞水平 (IC50=9±0.2 nM)。治疗浓度依赖性地降低 H3R2me2a 标记 (IC50=56±7 nM)。

Animal/Disease Models: NOD-scid IL2Rgnull (NSG) mice bearing primary MLL-r ALL cells[2]
Doses: 160 mg/kg
Route of Administration: intraperitoneal (ip)injection; PKC412 (100 mg/kg, ig), MS023 (160 mg/kg, ip ), or a combination for 4 weeks
Experimental Results: Combinatorial treatment extended survival of leukemic mice relative to single treatments.

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In vivo treatment of MLL-r ALL-engrafted mouse model[1]
For studying inducible short hairpin PRMT1 (shPRMT1), SEM cells stably expressing either doxycycline (DOX)-inducible short hairpin control (shCtrl) or PRMT1 short hairpin RNA (shRNA) (shPRMT1) were transplanted into irradiated immunodeficient NOD-scid IL2Rgnull (NSG) mice (1 × 106 cells per mouse). Each group was administered DOX treatment (10 mg/kg) orally for 3 weeks after engraftment. To assess R972/973 function in leukemogeneis, KOCL45 cells transduced with constructs expressing an FLT3 variant were sorted by using red fluorescent protein and injected into mice (1 × 105 cells per mouse), and mouse survival was monitored daily. To assess MS023 effects in vivo, we transplanted primary MLL-r ALL cells into NSG mice (0.5 × 106 cells per mouse). After engraftment, grouped mice were treated with vehicle, PKC412 (100 mg/kg, intragastrically), MS023 (160 mg/kg, intraperitoneally), or a combination for 4 weeks. After treatment, engrafted human cells were identified. Secondary transplantations of whole bone marrow (BM) cells from treated or control mice were then performed. Animal procedures were performed in accordance with federal and state government guidelines and established institutional guidelines and protocols approved by the Institutional Animal Care and Use Committee at City of Hope.

[1]. Eram MS, et al. A Potent, Selective, and Cell-Active Inhibitor of Human Type I Protein Arginine Methyltransferases. ACS Chem Biol. 2016 Mar 18;11(3):772-81.
[2]. Yinghui Zhu, et al. Targeting PRMT1-mediated FLT3 methylation disrupts maintenance of MLL-rearranged acute lymphoblastic leukemia. Blood. 2019 Oct 10;134(15):1257-1268.

Protein arginine methyltransferases (PRMTs) play a crucial role in a variety of biological processes. Overexpression of PRMTs has been implicated in various human diseases including cancer. Consequently, selective small-molecule inhibitors of PRMTs have been pursued by both academia and the pharmaceutical industry as chemical tools for testing biological and therapeutic hypotheses. PRMTs are divided into three categories: type I PRMTs which catalyze mono- and asymmetric dimethylation of arginine residues, type II PRMTs which catalyze mono- and symmetric dimethylation of arginine residues, and type III PRMT which catalyzes only monomethylation of arginine residues. Here, we report the discovery of a potent, selective, and cell-active inhibitor of human type I PRMTs, MS023, and characterization of this inhibitor in a battery of biochemical, biophysical, and cellular assays. MS023 displayed high potency for type I PRMTs including PRMT1, -3, -4, -6, and -8 but was completely inactive against type II and type III PRMTs, protein lysine methyltransferases and DNA methyltransferases. A crystal structure of PRMT6 in complex with MS023 revealed that MS023 binds the substrate binding site. MS023 potently decreased cellular levels of histone arginine asymmetric dimethylation. It also reduced global levels of arginine asymmetric dimethylation and concurrently increased levels of arginine monomethylation and symmetric dimethylation in cells. We also developed MS094, a close analog of MS023, which was inactive in biochemical and cellular assays, as a negative control for chemical biology studies. MS023 and MS094 are useful chemical tools for investigating the role of type I PRMTs in health and disease.[1]

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Relapse remains the main cause of MLL-rearranged (MLL-r) acute lymphoblastic leukemia (ALL) treatment failure resulting from persistence of drug-resistant clones after conventional chemotherapy treatment or targeted therapy. Thus, defining mechanisms underlying MLL-r ALL maintenance is critical for developing effective therapy. PRMT1, which deposits an asymmetric dimethylarginine mark on histone/non-histone proteins, is reportedly overexpressed in various cancers. Here, we demonstrate elevated PRMT1 levels in MLL-r ALL cells and show that inhibition of PRMT1 significantly suppresses leukemic cell growth and survival. Mechanistically, we reveal that PRMT1 methylates Fms-like receptor tyrosine kinase 3 (FLT3) at arginine (R) residues 972 and 973 (R972/973), and its oncogenic function in MLL-r ALL cells is FLT3 methylation dependent. Both biochemistry and computational analysis demonstrate that R972/973 methylation could facilitate recruitment of adaptor proteins to FLT3 in a phospho-tyrosine (Y) residue 969 (Y969) dependent or independent manner. Cells expressing R972/973 methylation-deficient FLT3 exhibited more robust apoptosis and growth inhibition than did Y969 phosphorylation-deficient FLT3-transduced cells. We also show that the capacity of the type I PRMT inhibitor MS023 to inhibit leukemia cell viability parallels baseline FLT3 R972/973 methylation levels. Finally, combining FLT3 tyrosine kinase inhibitor PKC412 with MS023 treatment enhanced elimination of MLL-r ALL cells relative to PKC412 treatment alone in patient-derived mouse xenografts. These results indicate that abolishing FLT3 arginine methylation through PRMT1 inhibition represents a promising strategy to target MLL-r ALL cells.[2]

C17H27CL2N3O

360.3218

359.1531

1992047-64-9

MS023;1831110-54-3;MS023 trihydrochloride;2108631-19-0

121513886

Typically exists as gray to gray purplesolids at room temperature

54.3Ų

Cl[H].Cl[H].O(C([H])(C([H])([H])[H])C([H])([H])[H])C1C([H])=C([H])C(=C([H])C=1[H])C1=C([H])N([H])C([H])=C1C([H])([H])N(C([H])([H])[H])C([H])([H])C([H])([H])N([H])[H]

HCNXCUFNZWGILO-UHFFFAOYSA-N

InChI=1S/C17H25N3O.2ClH/c1-13(2)21-16-6-4-14(5-7-16)17-11-19-10-15(17)12-20(3)9-8-18;;/h4-7,10-11,13,19H,8-9,12,18H2,1-3H3;2*1H

N'-methyl-N'-[[4-(4-propan-2-yloxyphenyl)-1H-pyrrol-3-yl]methyl]ethane-1,2-diamine;dihydrochloride

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 (e.g. under nitrogen), 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 : ≥ 150 mg/mL (~416.30 mM)
H2O : ~33.33 mg/mL (~92.50 mM)

配方 1 中的溶解度: 25 mg/mL (69.38 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 通过加热和超声助溶。

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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));
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10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
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