PRMT5

即使在延长孵育后，CMP-5（0-100 μM；24-72 小时）对健康的静息 B 淋巴细胞也只有一定程度的危害，但对淋巴瘤细胞的危害尤其大 [1]。与 DMSO 处理组相比，CMP-5（40 μM；24 小时）可以降低 60A 细胞中 p-BTK 和 pY(416)SRC 的表达 [1]。人类 Th1 细胞比 Th2 细胞更容易被 CMP-5（0–40 μM；24 小时）抑制（分别抑制 43% 和 9%）。人Th1和Th2细胞的IC50值分别为26.9 μM和31.6 μM，并且Th1细胞对PRMT5抑制的敏感性低于Th2细胞[1]。 CMP-5 本身（25 μM；24 小时）可使小鼠 Th1 细胞的生长减少 91%。当给予不同剂量的 IL-2 时，它会增加增殖，达到 5 ng/ml 的峰值[1]。

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。

细胞增殖测定 [1]
细胞类型：人类 Th1 细胞和 Th2 细胞
测试浓度： 25 μM
孵育时间：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。

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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小时
实验结果：p-BTK和pY(416)SRC蛋白水平受到抑制。

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.

Epigenetic events that are essential drivers of lymphocyte transformation remain incompletely characterized. We used models of Epstein-Barr virus (EBV)-induced B-cell transformation to document the relevance of protein arginine methyltransferase 5 (PRMT5) to regulation of epigenetic-repressive marks during lymphomagenesis. EBV(+) lymphomas and transformed cell lines exhibited abundant expression of PRMT5, a type II PRMT enzyme that promotes transcriptional silencing of target genes by methylating arginine residues on histone tails. PRMT5 expression was limited to EBV-transformed cells, not resting or activated B lymphocytes, validating it as an ideal therapeutic target. We developed a first-in-class, small-molecule PRMT5 inhibitor that blocked EBV-driven B-lymphocyte transformation and survival while leaving normal B cells unaffected. Inhibition of PRMT5 led to lost recruitment of a PRMT5/p65/HDAC3-repressive complex on the miR96 promoter, restored miR96 expression, and PRMT5 downregulation. RNA-sequencing and chromatin immunoprecipitation experiments identified several tumor suppressor genes, including the protein tyrosine phosphatase gene PTPROt, which became silenced during EBV-driven B-cell transformation. Enhanced PTPROt expression following PRMT5 inhibition led to dephosphorylation of kinases that regulate B-cell receptor signaling. We conclude that PRMT5 is critical to EBV-driven B-cell transformation and maintenance of the malignant phenotype, and that PRMT5 inhibition shows promise as a novel therapeutic approach for B-cell lymphomas.[1]

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In the autoimmune disease multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), expansion of pathogenic, myelin-specific Th1 cell populations drives active disease; selectively targeting this process may be the basis for a new therapeutic approach. Previous studies have hinted at a role for protein arginine methylation in immune responses, including T cell-mediated autoimmunity and EAE. However, a conclusive role for the protein arginine methyltransferase (PRMT) enzymes that catalyze these reactions has been lacking. PRMT5 is the main PRMT responsible for symmetric dimethylation of arginine residues of histones and other proteins. PRMT5 drives embryonic development and cancer, but its role in T cells, if any, has not been investigated. In this article, we show that PRMT5 is an important modulator of CD4+ T cell expansion. PRMT5 was transiently upregulated during maximal proliferation of mouse and human memory Th cells. PRMT5 expression was regulated upstream by the NF-κB pathway, and it promoted IL-2 production and proliferation. Blocking PRMT5 with novel, highly selective small molecule PRMT5 inhibitors severely blunted memory Th expansion, with preferential suppression of Th1 cells over Th2 cells. In vivo, PRMT5 blockade efficiently suppressed recall T cell responses and reduced inflammation in delayed-type hypersensitivity and clinical disease in EAE mouse models. These data implicate PRMT5 in the regulation of adaptive memory Th cell responses and suggest that PRMT5 inhibitors may be a novel therapeutic approach for T cell-mediated inflammatory disease.[2]

C21H22CLN3

351.872483730316

351.15

C, 71.68; H, 6.30; Cl, 10.07; N, 11.94

1030021-40-9

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

6462334

Typically exists as solid at room temperature

29.8

2

2

5

25

399

0

C(N1C2=CC=CC=C2C2=CC(CNCC3N=CC=CC=3)=CC=C12)C.Cl

LTMOFUKBECEABM-UHFFFAOYSA-N

InChI=1S/C21H21N3.ClH/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;1H

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

1030021-40-9; CMP5; CMP-5 hydrochloride; CMP-5 (hydrochloride); CMP-5 HCl; 1-(9-ethylcarbazol-3-yl)-N-(pyridin-2-ylmethyl)methanamine;hydrochloride; 1-(9-Ethyl-9H-carbazol-3-yl)-N-(pyridin-2-ylmethyl)methanamine hydrochloride; 1030021-40-9 (HCl);

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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;
5、为保证最佳实验结果，工作液请现配现用！
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。