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

Although CMP-5 (0-100 μM; 24-72 hours) exerts effects, even after prolonged times, its toxicity to normal resting B is limited [1]. In comparison to the group treated with DMSO, CMP-5 (40 μM; 24 hours) decreased the expression of p-BTK and pY (416) SRC in 60A cells [1]. PRMT5 is preferentially transcribed in Th1 cells as opposed to Th2 cells when exposed to CMP-5 (0–40 μM; 24 hours); in human Th1 cells and Th2 cells, the IC50 values are 26.9 μM and 31.6 μM, respectively. CMP-5 (25 μM; 24 hours) decreased mouse Th1 cell proliferation by 91%. Different IL-2 dosages were applied, and the maximum degree of IL-2-enhanced proliferation was seen at 5 ng/mL [1].

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- CMP5 blocks EBV-driven B-lymphocyte transformation and survival without affecting normal B cells. At 40 μM, it occupies the catalytic site of hPRMT5, inhibiting symmetric dimethylation of S2me-H4R3 and S2me-H3R8, while having no effect on asymmetric methylation of H4R3 in lymphoblastoid cell lines. Inhibition of PRMT5 reduces recruitment of the PRMT5/p65/HDAC3 repressive complex on the miR96 promoter, restoring miR96 expression and downregulating PRMT5. RNA sequencing and chromatin immunoprecipitation show CMP5 enhances tumor suppressor gene PTPRO expression, leading to dephosphorylation of kinases regulating B-cell receptor signaling [1]
- CMP5 inhibits TH1 cell proliferation with an IC50 of 3.7 μM and TH2 cell proliferation with an IC50 of 9.2 μM [2]
- In Vivo： - CMP5 treatment in mice suppresses recall T-cell responses, reduces inflammation in delayed-type hypersensitivity models, and alleviates clinical symptoms of experimental autoimmune encephalomyelitis [2]

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.

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- CMP5 treatment in mice suppresses recall T-cell responses, reduces inflammation in delayed-type hypersensitivity models, and alleviates clinical symptoms of experimental autoimmune encephalomyelitis [2]

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

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Cell viability 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.

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

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)

Solubility in Formulation 1: ≥ 6.25 mg/mL (19.82 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 62.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 6.25 mg/mL (19.82 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 62.5 mg/mL clear DMSO stock solution to 900 μL corn oil and mix evenly.

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