Protein arginine methyltransferase 5 (PRMT5) [1][2]

尽管 CMP-5（0-100 μM；24-72 小时）会发挥作用，但即使经过较长时间，其对正常静息 B 的毒性也是有限的 [1]。与用 DMSO 处理的组相比，CMP-5（40 μM；24 小时）降低了 60A 细胞中 p-BTK 和 pY (416) SRC 的表达 [1]。当暴露于 CMP-5（0–40 μM；24 小时）时，PRMT5 优先在 Th1 细胞中转录，而不是 Th2 细胞；在人 Th1 细胞和 Th2 细胞中，IC50 值分别为 26.9 μM 和 31.6 μM。 CMP-5（25 μM；24 小时）使小鼠 Th1 细胞增殖降低 91%。应用不同剂量的IL-2，IL-2增强增殖的最大程度在5ng/mL时观察到[1]。

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- CMP5可阻断EB病毒（EBV）驱动的B淋巴细胞转化和存活，对正常B细胞无影响。在40μM浓度下，它占据hPRMT5的催化位点，抑制S2me-H4R3和S2me-H3R8的对称二甲基化，但不影响淋巴母细胞系中H4R3的不对称甲基化。抑制PRMT5会减少PRMT5/p65/HDAC3抑制复合物在miR96启动子上的募集，恢复miR96表达并下调PRMT5。RNA测序和染色质免疫沉淀显示，CMP5可增强抑癌基因PTPRO的表达，导致调节B细胞受体信号的激酶去磷酸化[1]
- CMP5抑制TH1细胞增殖的IC50为3.7μM，抑制TH2细胞增殖的IC50为9.2μM[2]

抑制PRMT5抑制ova诱导的DTH炎症反应[2]
PRMT5抑制剂抑制炎症记忆T细胞反应的有效性表明它们可能对炎症或自身免疫性疾病有益。为了验证这一点，我们使用了OVA诱导的DTH小鼠模型和HLCL65，这是一种更有效和生物可利用的CMP5的衍生物（图1H， 2H，补充图2）。首先，我们分析了CFA免疫OVA后未治疗小鼠脾脏中PRMT5的表达。我们观察到，在免疫后10 d， PRMT5在脾脏中的表达显著上调（图6A），这表明PRMT5的表达与体内DTH免疫应答有关。在DTH模型中（如图6B所示），用CFA免疫OVA诱导OVA特异性T细胞反应，在随后暴露于无佐剂的OVA和记忆性CD4+ T细胞扩增后，引起小鼠足垫炎症。在再挑战期间，HLCL65治疗减少了40%的足底肿胀（炎症的衡量标准）（p < 0.05，图6C）。此外，与载体相比，HLCL65处理降低了36%的ova特异性T细胞增殖（图6D）和70%的IFN-γ产生（图6E）。这些数据表明，我们的新型PRMT5抑制剂HLCL65在体内抑制T细胞介导的反应和炎症。

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小鼠中CMP5处理可抑制回忆性T细胞反应，减轻迟发型超敏反应模型的炎症，并缓解实验性自身免疫性脑脊髓炎的临床症状[2]

蛋白质印迹分析 [1]
细胞类型： 60A 细胞
测试浓度： 40 μM
孵育时间： 24 hrs (hrs (小时))
实验结果： p-BTK 和 pY(416)SRC 蛋白水平的抑制。

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细胞活力测定 [1]
细胞类型：人类 Th1 细胞和 Th2 细胞
测试浓度： 25 μM
孵育持续时间：24小时
实验结果：抑制小鼠Th1细胞增殖，但添加IL-2剂量依赖性地增加细胞增殖。

OVA-induced DTH [2]
CFA and OVA emulsion was prepared at a 1:1 v/v ratio for a final concentration of 1500 μg of OVA/1 ml of PBS. BALB/c mice were injected with 100 μl of emulsion in the dorsal proximal scruff and the base of the tail (150 μg of OVA per mouse). Control groups included nonimmunized mice and immunized mice that were not subsequently challenged with OVA. One week after immunization, aggregated OVA was prepared by suspending in PBS at a concentration of 10 mg/ml in a 15-ml tube. Solution was heated in an 80°C water bath for 60 min. Mice were challenged with 300 μg of aggregated OVA by injecting 30 μl of solution into the left footpad of immunized mice. After an additional week, mice were rechallenged in the same manner (nonimmunized mice were also challenged at this step). Twenty-four hours after the second challenge, mice were euthanized by CO2 asphyxiation and cervical dislocation. Each footpad was measured using calipers for swelling (pre-euthanasia) and weighed for changes in mass. Additionally, spleens were removed and processed for in vitro studies.

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Experimental autoimmune encephalomyelitis [2]
For induced EAE, commercial Hooke Reagent or myelin oligodendrocyte glycoprotein and CFA emulsion were used. CFA/MOG emulsion was prepared in a 1:1 v/v ratio for a final concentration of 1000 μg MOG/1 ml of PBS. C57/B6 mice received 100 μl of emulsion s.c. in the dorsal proximal scruff and the base of the tail. About 2 h after immunization, mice were injected i.p. with 100 μl of 2 ng/μl pertussis toxin. Twenty-four hours later, mice were injected again with 100 μl of 2 ng/μl pertussis toxin. Mice were monitored for disease every day and treated with 25 mg/kg HLCL65 or DMSO vehicle control. At the indicated time points, mice were euthanized by injection with 20 mg/ml ketamine and 4 mg/ml xylazine (120 μl/20 g mouse) and perfused with PBS. Spleens, brains, and spinal cords were collected from representative mice and processed for in vitro studies. To isolate brain and spinal cord mononuclear cells, brains and spinal cords were processed through a 70-μm strainer and separated by a 70–30% isotonic Percoll gradient. [2]
For spontaneous EAE, three MBPAc1–11 TCR-Tg mice that developed EAE spontaneously (scores = 1.5–2) were euthanized by CO2 asphyxiation and cervical dislocation. Splenocytes were isolated and activated with 2 μg/ml MBPAc1–11 for 48 h in the presence of PRMT5 inhibitors or vehicle control. T-bet, IL-17, and RORγt expression was analyzed by intracellular flow cytometry.

[1]. Selective inhibition of protein arginine methyltransferase 5 blocks initiation and maintenance of B-cell transformation.Blood. 2015 Apr 16;125(16):2530-43.

[2]. PRMT5-Selective Inhibitors Suppress Inflammatory T Cell Responses and Experimental Autoimmune Encephalomyelitis. J Immunol. 2017 Feb 15;198(4):1439-1451.

Key epigenetic events driving lymphocyte transformation remain poorly elucidated. Using an Epstein-Barr virus (EBV)-induced B-cell transformation model, we demonstrated the role of protein arginine methyltransferase 5 (PRMT5) in regulating epigenetic repressor markers during lymphomatogenesis. Both EBV-positive lymphomas and transformed cell lines exhibited high PRMT5 expression. PRMT5 is a type II PRMT enzyme that promotes transcriptional silencing of target genes by methylating arginine residues in histone tails. The exclusive expression of PRMT5 in EBV-transformed cells, rather than in resting or activated B lymphocytes, confirms PRMT5 as an ideal therapeutic target. We developed a first-in-class small-molecule PRMT5 inhibitor that blocks EBV-driven B-lymphocyte transformation and survival without affecting normal B cells. PRMT5 inhibition prevents the PRMT5/p65/HDAC3 repressor complex from recruiting to the miR96 promoter, restoring miR96 expression while downregulating PRMT5 expression. RNA sequencing and chromatin immunoprecipitation assays identified several tumor suppressor genes, including the protein tyrosine phosphatase gene PTPROt, which is silenced during EBV-driven B-cell transformation. PTPROt expression was enhanced upon PRMT5 inhibition, leading to dephosphorylation of the kinase that regulates B-cell receptor signaling. We conclude that PRMT5 is crucial for EBV-driven B-cell transformation and maintenance of malignant phenotypes, and that PRMT5 inhibitors hold promise as a novel treatment for B-cell lymphoma. [1]

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In the autoimmune disease multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE), the expansion of pathogenic myelin-specific Th1 cell populations drives disease activity; selective targeting of this process may be a novel treatment approach. Previous studies have suggested that protein arginine methylation plays a role in immune responses, including T-cell-mediated autoimmunity and EAE. However, the role of protein arginine methyltransferases (PRMTs) that catalyze these responses remains unclear. PRMT5 is the major PRMT responsible for the symmetrical dimethylation of histone and other protein arginine residues. PRMT5 drives embryonic development and cancer development, but its role in T cells (if any) has not been studied. This article shows that PRMT5 is an important regulator of CD4+ T cell proliferation. PRMT5 expression is transiently upregulated during peak proliferation of memory Th cells in mice and humans. PRMT5 expression is regulated upstream of the NF-κB pathway and promotes IL-2 production and cell proliferation. Blocking PRMT5 with a novel, highly selective small molecule PRMT5 inhibitor significantly inhibits the proliferation of memory Th cells, and the inhibitory effect on Th1 cells is stronger than that on Th2 cells. In vivo experiments showed that PRMT5 blockade effectively inhibits memory T cell responses and reduces inflammatory responses in delayed-type hypersensitivity and EAE mouse models. These data suggest that PRMT5 is involved in regulating adaptive memory Th cell responses and suggest that PRMT5 inhibitors may be a new strategy for treating T cell-mediated inflammatory diseases. [2]

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- CMP5 is a small molecule PRMT5 inhibitor. EBV infection induces PRMT5 overexpression and epigenetic alterations that are crucial for B-lymphocyte transformation. PRMT5 is expressed in EBV-transformed cells but not in resting/activated B lymphocytes, thus making PRMT5 a therapeutic target for B-cell malignancies [1].

C₂₁H₂₁N₃

315.41

315.173

880813-42-3

CMP-5 hydrochloride;1030021-40-9; 880813-42-3; 2309409-79-6 (2HCl)

4722595

Light yellow to yellow ointment

1.1±0.1 g/cm3

505.1±48.0 °C at 760 mmHg

259.3±29.6 °C

0.0±1.3 mmHg at 25°C

1.637

4.23

29.8

1

2

5

24

399

0

N1C(CNCC2C=C3C4C(N(C3=CC=2)CC)=CC=CC=4)=CC=CC=1

YPJMOVVQKBFRNH-UHFFFAOYSA-N

InChI=1S/C21H21N3/c1-2-24-20-9-4-3-8-18(20)19-13-16(10-11-21(19)24)14-22-15-17-7-5-6-12-23-17/h3-13,22H,2,14-15H2,1H3

1-(9-ethylcarbazol-3-yl)-N-(pyridin-2-ylmethyl)methanamine

CMP5; cmp-5; 880813-42-3; 1-(9-ethyl-9H-carbazol-3-yl)-N-(pyridin-2-ylmethyl)methanamine; 1-(9-ethylcarbazol-3-yl)-N-(pyridin-2-ylmethyl)methanamine; CHEMBL4245087; SCHEMBL21308321; (9-Ethyl-9H-carbazol-3-ylmethyl)-pyridin-2-ylmethyl-amine; CMP 5

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)

DMSO : ~125 mg/mL (~396.31 mM)

配方 1 中的溶解度: ≥ 6.25 mg/mL (19.82 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 62.5 mg/mL澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入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: ≥ 6.25 mg/mL (19.82 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 62.5 mg/mL 澄清 DMSO 储备液加入900 μL 玉米油中，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；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
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6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。