PRMT5

Even after extended incubation, CMP-5 (0-100 μM; 24-72 hours) is only somewhat harmful to healthy resting B lymphocytes, but it is specifically hazardous to lymphoma cells [1]. When compared to the DMSO-treated group, CMP-5 (40 μM; 24 hours) can lower the expression of p-BTK and pY(416)SRC in 60A cells [1]. Human Th1 cells are more preferentially inhibited by CMP-5 (0–40 μM; 24 h) than Th2 cells (43% and 9% inhibition, respectively). The IC50 values of human Th1 and Th2 cells are 26.9 μM and 31.6 μM, respectively, and Th1 cells are less sensitive to PRMT5 suppression than Th2 cells [1]. CMP-5 by itself (25 μM; 24 hours) reduced the growth of mouse Th1 cells by 91%. When IL-2 was given in varying dosages, it increased proliferation, peaking at 5 ng/ml[1].

PRMT5 inhibition suppresses in vivo OVA-induced DTH inflammatory responses [2]
The effectiveness of PRMT5 inhibitors at suppressing inflammatory memory T cell responses suggested that they may be beneficial in inflammatory or autoimmune disease. To test this, we used the OVA-induced DTH mouse model and HLCL65, a more potent and bioavailable derivative of CMP5 (Figs. 1H, 2H, Supplemental Fig. 2). First, we analyzed PRMT5 expression in the spleen of untreated mice after OVA immunization with CFA. We observed that, at 10 d after immunization, PRMT5 expression was upregulated significantly in the spleen (Fig. 6A), suggesting that PRMT5 expression is relevant to in vivo DTH immune responses. In the DTH model (outlined in Fig. 6B), OVA immunization with CFA induces an OVA-specific T cell response that causes footpad inflammation in mice upon subsequent exposure to adjuvant-free OVA and memory CD4+ T cell expansion. HLCL65 treatment during the rechallenge period reduced footpad swelling, a measure of inflammation, by 40% (p < 0.05, Fig. 6C). In addition, compared with vehicle, HLCL65 treatment reduced OVA-specific T cell proliferation by 36% (Fig. 6D) and IFN-γ production by 70% (Fig. 6E). These data indicate that our novel PRMT5 inhibitor HLCL65 suppresses T cell–mediated responses and inflammation in vivo.

Cell proliferation assay [1]
Cell Types: human Th1 cells and Th2 cells
Tested Concentrations: 25 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Inhibited mouse Th1 cell proliferation, but adding IL-2 dose-dependently increased cell proliferation.

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Western Blot Analysis[1]
Cell Types: 60A Cell
Tested Concentrations: 40 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: p-BTK and pY(416)SRC protein levels were inhibited.

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

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

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)