PRMT1 (IC50 = 30 nM); PRMT3 (IC50 = 119 nM); PRMT4 (IC50 = 83 nM); PRMT6 (IC50 = 4 nM); PRMT8 (IC50 = 5 nM)

PRMT1 methyltransferase activity in MCF7 cells is inhibited by MS023 dihydrochloride (1-1000 nM; 48 hours) [1]. PRMT6 methyltransferase activity in HEK293 cells is inhibited by MS023 dihydrochloride (1-1000 nM; 20 hours) [1].

MS023 dihydrochloride (160 mg/kg, ip) coupled with PKC412 (100 mg/kg, ig) prevents MLL-r timely cycling (ALL) by reducing the support of functional MLL-r ALL starting cells. )

PRMT biochemical assays[1]
A scintillation proximity assay (SPA) was used for assessing the effect of test compounds on inhibiting the methyl transfer reaction catalyzed by PRMTs as described previously.27 In brief, the tritiated S-adenosyl-L-methionine was used as the donor of methyl group. The (3H) methylated biotin labelled peptide was captured in streptavidin/scintillant-coated microplate which brings the incorporated 3H-methyl and the scintillant to close proximity resulting in light emission that is quantified by tracing the radioactivity signal (counts per minute) as measured by a TopCount NXT™ Microplate Scintillation and Luminescence Counter. When necessary, non-tritiated SAM was used to supplement the reactions. The IC50 values were determined under balanced conditions at Km concentrations of both substrate and cofactor by titration of test compounds in the reaction mixture.

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 Cellular PRMT1 assay[1]
MCF7 cells were grown in 12-well plates in DMEM supplemented with 10% FBS, penicillin (100 units mL−1) and streptomycin (100 μg mL−1). 40% confluent cells were treated with different concentrations of MS023 and compounds 4 – 6 at indicated concentrations or DMSO control for 48 h. Cells were lysed in 100 μL of total lysis buffer (20 mM Tris-HCl pH 8, 150 mM NaCl, 1 mM EDTA, 10 mM MgCl2, 0.5% TritonX-100, 12.5 U mL−1 benzonase, complete EDTA-free protease inhibitor cocktail). After 3 min incubation at RT, SDS was added to final 1% concentration. Lysates were run on SDS-PAGE and immunoblotting was done as outlined below to determine H4R3me2a, arginine asymmetric dimethylation, arginine symmetric dimethylation and arginine monomethylation in western blot.

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 Cellular PRMT6 assay[1]
HEK293 cells were grown in 12-well plates in DMEM supplemented with 10% FBS, penicillin (100 U mL−1) and streptomycin (100 μg mL−1). 50 % confluent cells were transfected with FLAG-tagged PRMT6 or mutant V86K/D88A PRMT6 (1 μg of DNA per well) using jetPRIME® transfection reagent (Polyplus-Transfection), following manufacturer instructions. After 4 h media were removed and cells were treated with MS023 at indicated concentrations or DMSO control. After 20 h, media was removed and cells were lysed in 100 μL of total lysis buffer.

Western Blot Analysis [1]
Cell Types: MCF7 and HEK293 cells
Tested Concentrations: 1.4, 4, 12, 37, 111, 333 and 1000 nM
Incubation Duration: 48 hrs (hours) for MCF7 cells; 20 hrs (hours) for HEK293 cells
Experimental Results:: Treatment potently and concentration-dependently diminished cellular levels of H4R3me2a (IC50=9±0.2 nM). Treatment concentration-dependently diminished the H3R2me2a mark (IC50=56±7 nM).

Animal/Disease Models: NOD-scid IL2Rgnull (NSG) mice carrying primary MLL-r ALL cells [2]
Doses: 160 mg/kg
Route of Administration: intraperitoneal (ip) injection; spread of [2]. Results of 4 weeks of PKC412 (100 mg/kg, ig), MS023 (160 mg/kg, ip) or combination treatment: Combination treatment prolonged the survival of leukemia mice relative to single treatment.

[1]. A Potent, Selective, and Cell-Active Inhibitor of Human Type I Protein Arginine Methyltransferases. ACS Chem Biol. 2016 Mar 18;11(3):772-81.

[2]. Targeting PRMT1-mediated FLT3 methylation disrupts maintenance of MLL-rearranged acute lymphoblastic leukemia. Blood. 2019 Oct 10;134(15):1257-1268.

Protein arginine methyltransferases (PRMTs) play crucial roles in various biological processes. Overexpression of PRMTs is closely associated with a range of human diseases, including cancer. Therefore, both academia and the pharmaceutical industry are actively developing selective small-molecule inhibitors of PRMTs as chemical tools to validate biological and therapeutic hypotheses. PRMTs are classified into three classes: Type I PRMTs catalyze monomethylation and asymmetric dimethylation of arginine residues; Type II PRMTs catalyze monomethylation and symmetric dimethylation of arginine residues; and Type III PRMTs catalyze only monomethylation of arginine residues. This paper reports the discovery of MS023, a highly efficient, selective, and cellularly active Type I PRMT inhibitor, and characterizes it through a series of biochemical, biophysical, and cellular experiments. MS023 exhibits highly efficient inhibitory activity against Type I PRMTs (including PRMT1, -3, -4, -6, and -8), but is completely ineffective against Type II and III PRMTs, protein lysine methyltransferases, and DNA methyltransferases. Crystal structure of PRMT6 complex with MS023 shows that MS023 can bind to substrate binding sites. MS023 significantly reduces the level of intracellular histone arginine asymmetric dimethylation. It also reduces the overall level of intracellular arginine asymmetric dimethylation while increasing the levels of arginine monomethylation and symmetric dimethylation. We also developed MS094, an analog of MS023, which is inactive in both biochemical and cellular experiments and can be used as a negative control for chemical biology studies. MS023 and MS094 are effective chemical tools for studying the role of type I PRMT in health and disease. [1] Relapse remains the main cause of treatment failure in MLL rearrangement (MLL-r) acute lymphoblastic leukemia (ALL) due to the persistence of drug-resistant clones after conventional chemotherapy or targeted therapy. Therefore, elucidating the mechanisms of MLL-r ALL maintenance is crucial for developing effective treatments. PRMT1 can deposit asymmetric dimethylarginine tags on histones/non-histones and has been reported to be overexpressed in a variety of cancers. In this study, we demonstrated elevated PRMT1 levels in MLL-rALL cells and showed that inhibiting PRMT1 significantly suppressed the growth and survival of leukemia cells. Mechanistically, we found that PRMT1 methylates arginine (R) residues 972 and 973 (R972/973) of the Fms-like receptor tyrosine kinase 3 (FLT3), and its oncogenic function in MLL-relapsed acute lymphoblastic leukemia (MLL-rALL) cells depends on FLT3 methylation. Biochemical and computational analyses showed that R972/973 methylation can promote adaptor protein recruitment to FLT3 in a manner dependent on or independent of phosphorylated tyrosine (Y) residue 969 (Y969). Cells expressing R972/973 methylation-deficient FLT3 exhibited stronger apoptosis and growth inhibition than transduced cells expressing Y969 phosphorylated-deficient FLT3. We also found that the ability of the type I PRMT inhibitor MS023 to inhibit leukemia cell viability was positively correlated with baseline FLT3 R972/973 methylation levels. Finally, in a patient-derived mouse xenograft model, treatment with the FLT3 tyrosine kinase inhibitor PKC412 in combination with MS023 was more effective in clearing MLL-rALL cells than treatment with PKC412 alone. These results suggest that eliminating FLT3 arginine methylation by inhibiting PRMT1 is a promising strategy for targeting MLL-rALL cells. [2]

C17H28CL3N3O

287.199

C, 51.46; H, 7.11; Cl, 26.80; N, 10.59; O, 4.03

2108631-19-0

MS023;1831110-54-3;MS023 dihydrochloride;1992047-64-9

129626591

Typically exists as solid at room temperature

1.1±0.1 g/cm3

437.8±45.0 °C at 760 mmHg

218.6±28.7 °C

0.0±1.1 mmHg at 25°C

1.567

2.3

5

3

7

24

290

0

VEUUSCXROKBMMJ-UHFFFAOYSA-N

InChI=1S/C17H25N3O.3ClH/c1-13(2)21-16-6-4-14(5-7-16)17-11-19-10-15(17)12-20(3)9-8-18;;;/h4-7,10-11,13,19H,8-9,12,18H2,1-3H3;3*1H

N1-((4-(4-isopropoxyphenyl)-1H-pyrrol-3-yl)methyl)-N1-methylethane-1,2-diamine trihydrochloride

MS023 trihydrochloride); MS023 triHCl

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.)