PRMT5 (IC50 = 6.2 nM)

GSK3326595 (10-100 nM, 24-72 h) inhibits the ACE2-RBD interaction, which in turn prevents SARS-CoV-2 spike pseudovirus infection of HEK-293 cells and A549 cells [1]. Peritoneal macrophages are polarized toward the M1 type that is induced by IFN-γ when exposed to GSK3326595 (100 nM, 12 h) [3]. MCL cell death is induced by GSK3326595 (0.15-10 μM, 72 h) [4].

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Type I protein arginine methyltransferases (PRMTs) catalyze asymmetric dimethylation of arginines on proteins. Type I PRMTs and their substrates have been implicated in human cancers, suggesting inhibition of type I PRMTs may offer a therapeutic approach for oncology. The current report describes GSK3368715 (EPZ019997), a potent, reversible type I PRMT inhibitor with anti-tumor effects in human cancer models. Inhibition of PRMT5, the predominant type II PRMT, produces synergistic cancer cell growth inhibition when combined with GSK3368715. Interestingly, deletion of the methylthioadenosine phosphorylase gene (MTAP) results in accumulation of the metabolite 2-methylthioadenosine, an endogenous inhibitor of PRMT5, and correlates with sensitivity to GSK3368715 in cell lines. These data provide rationale to explore MTAP status as a biomarker strategy for patient selection[2].

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Researchers reveal that the PRMT5-specific inhibitor GSK3326595 is able to dramatically reduce ACE2 binding with RBD. Moreover, we discovered that meR671-ACE2 plays an important role in ACE2 binding with Spike1 of the SARS-CoV-2 Omicron, Delta, and Beta variants; and we found that GSK3326595 strongly attenuates ACE2 interaction with Spike1 of the SARS-CoV-2 Omicron, Delta, and Beta variants. Finally, SARS-CoV-2 pseudovirus infection assays uncovered that PRMT5-mediated meR671-ACE2 is essential for SARS-CoV-2 infection in human cells, and pseudovirus infection experiments confirmed that GSK3326595 can strongly suppress SARS-CoV-2 infection of host cells. These findings suggest that as a clinical phase II drug for several kinds of cancers, GSK3326595 is a promising candidate to decrease SARS-CoV-2 infection by inhibiting ACE2 methylation and ACE2-Spike1 interaction[4].

In LDL receptor knockout mice, GSK3326595 (5 mg/kg, Intraperitoneal injection, three times a week for nine weeks) raises liver triglyceride levels without changing atherosclerosis [3]. Enhances resistance to programmed cell death protein 1 (PD-1) immune checkpoint therapy (ICT) in myeloid neoplasia transgene-initiated (MYC-ON) small efficacy in murine hepatocellular carcinoma (HCC) [5]. GSK3326595 is administered orally, once daily, for two weeks.

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Given the important role of inflammation and metabolism in atherosclerotic cardiovascular disease, here we examined the role of PRMT5 in atherosclerosis using the specific PRMT5 inhibitor GSK3326595. Cultured thioglycollate-elicited peritoneal macrophages were exposed to GSK3326595 or DMSO control and stimulated with either 1 ng/mL LPS or 100 ng/mL interferon-gamma for 24 h. Furthermore, male low-density lipoprotein (LDL) receptor knockout mice were fed an atherogenic Western-type diet and injected intraperitoneally 3×/week with a low dose of 5 mg/kg GSK3326595 or solvent control for 9 weeks. In vitro, GSK3326595 primed peritoneal macrophages to interferon-gamma-induced M1 polarization, as evidenced by an increased M1/M2 gene marker ratio. In contrast, no difference was found in the protein expression of iNOS (M1 marker) and ARG1 (M2 marker) in peritoneal macrophages of GSK3326595-treated mice. Also no change in the T cell activation state or the susceptibility to atherosclerosis was detected. However, chronic GSK3326595 treatment did activate genes involved in hepatic fatty acid acquisition, i.e. SREBF1, FASN, and CD36 (+59%, +124%, and +67%, respectively; p < 0.05) and significantly increased hepatic triglyceride levels (+50%; p < 0.05). PRMT5 inhibition by low-dose GSK3326595 treatment does not affect the inflammatory state or atherosclerosis susceptibility of Western-type diet-fed LDL receptor knockout mice, while it induces hepatic triglyceride accumulation. Severe side effects in liver, i.e. development of non-alcoholic fatty liver disease, should thus be taken into account upon chronic treatment with this PRMT5 inhibitor.[3]

High Throughput Screen[2]
Type I PRMT inhibitors were found through screening Epizyme’s proprietary HMT-biased library (Mitchell et al., 2015). In summary, compound was incubated with PRMT1 for 30 minutes at room temperature (384-well plate) and reactions were initated upon the addition of SAM and peptide. Final assay conditions were 0.75 nM PRMT1 (NP_001527.3, GST-PRMT1 amino acids 1-371), 200 nM 3H-SAM (specific activity 80 Ci/mmol), 1.5 μM SAM , and 20 nM peptide (Biotin-Ahx-RLARRGGVKRISGLI-NH2, 21st Century Biochemicals) in 20 mM bincine (pH 7.6), 1mM TCEP, 0.005% bovine skin gelatin, 0.002% Tween-20 and 2% DMSO. Reactions were quenched by the addition of SAM (400 μM final). Terminated reactions were transferred to a Streptavidin-coated Flashplate, incubated for at least 1 hour and then the plate was washed with 0.1% Tween-20 using a Biotek ELx405 plate washer. The quantity of 3H-peptide bound to the Flashplate was measured using a PerkinElmer TopCount plate reader.

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PRMT Biochemical Assays[2]
All assays were performed with compound or DMSO prestamped (49x, 2% final) in 96 well plates. Assays for PRMT1, PRMT3, PRMT6 and PRMT8 used H4 1-21 peptide and a buffer comprised of 50 mM Tris (pH 8), 0.002% Tween-20, 0.5 mM EDTA and 1 mM DTT. Briefly, Flag-his-tev-PRMT8 (61-394) was expressed in a baculovirus expression system and purified using Ni-NTA agarose affinity chromatography and Superdex 200 gel filtration chromatography. For all assays, final Adenosyl-L-Methionine (SAM) concentration listed contains a mixture of unlabeled SAM and 3H-SAM All reactions were quenched upon the addition of SAH (0.5 mM final).[2]
For competition studies, substrate was added to the compound plate followed by the addition of enzyme. For SAM competition studies, final assay concentrations consisted of 2 nM PRMT1, 40 nM peptide and titrating SAM (50-8000 nM). For peptide competition studies, final assay concentrations consisted of 2 nM PRMT1, 1000 nM and titrating peptide (1.6-1000 nM). Reactions were incubated at room temperature for 18 minutes prior to quench.[2]
For time dependence studies, enzyme/SAM mix was added to the compound plate and incubated for 3-60 minutes prior to addition of the peptide. For no preincubation assay, peptide was added to the compound plate followed by enzyme/SAM mix to initiate the reaction. Final PRMT1 assay concentrations were 0.5 nM PRMT1, 40 nM peptide and 1100 nM SAM. Reactions were incubated at room temperature for 20 minutes prior to quench.

Western Blot Analysis[4]
Cell Types: HEK-293T cells, A549 cells
Tested Concentrations: 10 nM, 25 nM, 50 nM, 100 nM
Incubation Duration: 48 h
Experimental Results: Strongly inhibited ACE2-RBD interaction at low concentration. Inhibited SARS-CoV -2 Omicron and other variants Spike1 binding with ACE2. Inhibits SARS-CoV-2 spike pseudovirus infection host cells.

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Cell Cytotoxicity Assay[1]
Cell Types: MCL cells
Tested Concentrations: 0.15 μM, 0.3 μM, 0.6 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM
Incubation Duration: 72 h
Experimental Results: Resulted in modest growth inhibition in MCL cells.

Animal/Disease Models: LDL receptor knockout mice[3]
Doses: 5 mg/kg
Route of Administration: intraperitoneal (ip)injection
Experimental Results: Did not alter atherosclerosis susceptibility. Increased hepatic triglyceride levels without changing the hyperlipidemia extent. Activated genes involved in fatty acid acquisition.

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Animal/Disease Models: myelocytomatosis transgene turned on mice[5]
Doses: 25 mg/kg, 50 mg/kg
Route of Administration: Oral
Experimental Results: Dramatically suppressed tumor growth at 50 mg/kg. demonstrated better therapeutic efficacy at 25 mg/kg.

[1]. Exploiting PRMT5 as a target for combination therapy in mantle cell lymphoma characterized by frequent ATM and TP53 mutations. Blood cancer journal, 2023, 13(1): 27.

[2]. Anti-tumor activity of the type I PRMT inhibitor, GSK3368715, synergizes with PRMT5 inhibition through MTAP loss. Cancer cell, 2019, 36(1): 100-114. e25.

[3]. PRMT5 inhibition induces pro‐inflammatory macrophage polarization and increased hepatic triglyceride levels without affecting atherosclerosis in mice. Journal of Cellular and Molecular Medicine, 2023, 27(8): 1056-1068.

[4]. GSK3326595 is a promising drug to prevent SARS-CoV-2 Omicron and other variants infection by inhibiting ACE2-R671 di-methylation. Journal of Medical Virology, 2023, 95(1): e28158.

[5]. Myelocytomatosis-protein arginine N-methyltransferase 5 Axis defines the tumorigenesis and immune response in hepatocellular carcinoma. Hepatology, 2021, 74(4): 1932-1951.

Pemrametostat is an orally available, selective small molecule inhibitor of protein arginine methyltransferase 5 (PRMT5), with potential antiproliferative and antineoplastic activities. Although the mechanism of action has not been completely determined, pemrametostat binds to the substrate recognition site of PRMT5 following oral administration and inhibits its methyltransferase activity, which decreases the levels of both monomethylated and dimethylated arginine residues in histones H2A, H3 and H4 and modulates the expression of genes involved in several cellular processes, including cell proliferation. Therefore, this agent may increase the expression of antiproliferative genes and/or decrease the expression of genes that promote cell proliferation and may lead to decreased growth of rapidly proliferating cells, including cancer cells. PRTM5, an arginine methyltransferase that can catalyze the formation of both omega-N monomethylarginine (MMA) and symmetrical dimethylarginine (sDMA) on histones and a variety of other protein substrates, is overexpressed in several neoplasms.

C24H32N6O3

452.55

452.253

C, 63.70; H, 7.13; N, 18.57; O, 10.61

1616392-22-3

1848944-46-6 (succinate); 1616392-22-3

90241742

Typically exists as white to light yellow solids at room temperature

1.3±0.1 g/cm3

760.3±60.0 °C at 760 mmHg

413.6±32.9 °C

0.0±2.7 mmHg at 25°C

1.626

2.88

3

7

7

33

656

1

O([H])[C@@]([H])(C([H])([H])N([H])C(C1=C([H])C(=NC([H])=N1)N([H])C1([H])C([H])([H])C([H])([H])N(C(C([H])([H])[H])=O)C([H])([H])C1([H])[H])=O)C([H])([H])N1C([H])([H])C2=C([H])C([H])=C([H])C([H])=C2C([H])([H])C1([H])[H]

JLCCNYVTIWRPIZ-NRFANRHFSA-N

InChI=1S/C24H32N6O3/c1-17(31)30-10-7-20(8-11-30)28-23-12-22(26-16-27-23)24(33)25-13-21(32)15-29-9-6-18-4-2-3-5-19(18)14-29/h2-5,12,16,20-21,32H,6-11,13-15H2,1H3,(H,25,33)(H,26,27,28)/t21-/m0/s1

(S)-6-((1-acetylpiperidin-4-yl)amino)-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)pyrimidine-4-carboxamide

EPZ-015938; EPZ 015938; EPZ015938; GSK3326595; 1616392-22-3; GSK-3326595; Pemrametostat; 6-[(1-acetylpiperidin-4-yl)amino]-N-[(2S)-2-hydroxy-3-(1,2,3,4-tetrahydroisoquinolin-2-yl)propyl]pyrimidine-4-carboxamide; EPZ015938; (S)-6-((1-acetylpiperidin-4-yl)amino)-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)pyrimidine-4-carboxamide; GSK-3326595A; Pemrametostat; GSK3326595; GSK 3326595; GSK-3326595;

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.52 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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: ≥ 2.5 mg/mL (5.52 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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.

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Solubility in Formulation 3: 2.08 mg/mL (4.60 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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 4: 2.08 mg/mL (4.60 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 5: ≥ 2.08 mg/mL (4.60 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 6: 2.2mg/mL in10% DMSO : 40% PEG300 : 5% Tween80 + : 45% saline

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