PRMT1 (IC50 = 30 nM); PRMT3 (IC50 = 119 nM); PRMT4 (IC50 = 83 nM); PRMT6 (IC50 = 4 nM); PRMT8 (IC50 = 5 nM)
Human type I Protein Arginine Methyltransferases (PRMTs): MS023 exhibits high potency against PRMT1, PRMT3, PRMT4, PRMT6, and PRMT8; for PRMT6, the dissociation constant (K_d) is 6 nM (measured by ITC). It is completely inactive against type II PRMTs, type III PRMTs, protein lysine methyltransferases (PKMTs), and DNA methyltransferases (DNMTs) [1]
- PRMT1 (a member of type I PRMTs): MS023 inhibits PRMT1-mediated methylation of Fms-like receptor tyrosine kinase 3 (FLT3) at arginine residues 972 and 973 (R972/973) in MLL-rearranged (MLL-r) acute lymphoblastic leukemia (ALL) cells, with the inhibitory effect paralleling baseline FLT3 R972/973 methylation levels [2]

In MCF7 cells, PRMT1 methyltransferase activity is inhibited by MS023 (1-1000 nM; 48 h) [1]. In HEK293 cells, MS023 (1-1000 nM; 20 h) suppresses PRMT6 methyltransferase activity [1].

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Type I PRMT Inhibition Assays: MS023 potently inhibits the activity of type I PRMTs (PRMT1, -3, -4, -6, -8) in biochemical assays. Differential scanning fluorimetry (DSF) shows that 200 μM MS023 increases the melting temperature (T_m) of PRMT6 by 20 °C (vs. T_m = 52 °C for PRMT6 alone), confirming binding. When combined with 100 μM S-adenosylmethionine (SAM), the ΔT_m reaches 22 °C. Selectivity profiling against 25 PKMTs, 3 DNMTs, and 3 histone lysine demethylases at 1 μM and 10 μM shows no inhibitory activity. Kinetic analysis reveals noncompetitive inhibition of PRMT6, as IC₅₀ values remain unchanged with varying peptide or SAM concentrations [1]
- Cellular Methylation Modulation: In MCF7 cells treated with MS023 for 48 h, Western blot analysis shows concentration-dependent reduction of histone H4 arginine 3 asymmetric dimethylation (H4R3me2a, a marker of PRMT1 activity). In HEK293 cells transfected with FLAG-tagged PRMT6, MS023 (20 h treatment) concentration-dependently decreases histone H3 arginine 2 asymmetric dimethylation (H3R2me2a, a marker of PRMT6 activity). Additionally, MS023 reduces global cellular arginine asymmetric dimethylation (Rme2a) while increasing arginine monomethylation (Rme1) and symmetric dimethylation (Rme2s) in MCF7 cells [1]
- MLL-r ALL Cell Effects: In MLL-r ALL cell lines (KOCL45, KOCL50, KOCL69, RS4;11, SEM) and primary blasts, MS023 (5 μM) reduces FLT3 R972/973 asymmetric dimethylation (detected by Western blot). The IC₅₀ of MS023 for inhibiting MLL-r ALL cell viability correlates with baseline FLT3 R972/973 methylation levels. Combined with FLT3 tyrosine kinase inhibitor (TKI) PKC412, MS023 enhances inhibition of primary MLL-r ALL cell viability and induces higher apoptosis rates compared to PKC412 alone. In SEM cells with PRMT1 knockdown, MS023 further sensitizes cells to PKC412-induced apoptosis [2]
- FLT3 Methylation-Dependent Effects: In 293T cells expressing FLT3 wild-type (WT) or R972/973 mutant (R972K/R973K), MS023 reduces PRMT1-mediated FLT3 R972/973 methylation in WT cells but has no effect on mutant cells. In BaF3 cells transduced with FLT3-WT or FLT3-R972/973K, MS023 inhibits proliferation and induces apoptosis in FLT3-WT cells, with weaker effects in FLT3-R972/973K cells. It also blocks PRMT1-FLT3 interaction (detected by co-immunoprecipitation) and reduces downstream signaling (e.g., phospho-FLT3, phospho-ERK) in MLL-r ALL cells [2]

When combined with PKC412 (100 mg/kg, ig), MS023 (160 mg/kg, ip) prevents the growth of MLL-r acute lymphoblastic leukemia (ALL) by preventing the maintenance of functional MLL-r ALL initiating cells. disperse[2].

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Patient-Derived Xenograft (PDX) Models of MLL-r ALL: NOD scid gamma (NSG) mice were transplanted with primary MLL-r ALL cells (patient #5 and #6). Once human cell engraftment reached 1%, mice were treated with: vehicle, MS023 (dose not specified), PKC412 (dose not specified), or MS023 + PKC412 for 4 weeks. The combination treatment significantly reduced the percentage of human CD45⁺/CD19⁺ cells in bone marrow (BM), spleen (SP), and peripheral blood (PB) compared to single-agent or vehicle treatment. In secondary transplantation (using BM cells from primary treated mice), the combination group maintained lower engraftment levels. Survival analysis showed that mice in the MS023 + PKC412 group had significantly longer survival than other groups [2]

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.

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ITC Assay for PRMT6 Binding: Purified PRMT6 protein and MS023 were prepared in appropriate buffers. MS023 was titrated into the PRMT6 solution at a constant temperature, and heat changes during binding were recorded. Data analysis revealed a K_d value of 6 nM, indicating high-affinity binding between MS023 and PRMT6 [1]
- DSF Assay for PRMT6 Stabilization: PRMT6 protein was mixed with different concentrations of MS023 (with or without SAM). The mixture was heated gradually, and changes in fluorescence (due to protein unfolding) were measured. The T_m was determined as the inflection point of the fluorescence-temperature curve. PRMT6 alone had a T_m of 52 °C; 200 μM MS023 increased T_m by 20 °C, and 100 μM SAM + 200 μM MS023 increased T_m by 22 °C, confirming specific binding [1]
- Type I PRMT Enzymatic Activity Assay: Recombinant type I PRMTs (PRMT1, -3, -4, -6, -8) were incubated with peptide substrates, SAM (methyl donor), and varying concentrations of MS023. The formation of methylated peptides was quantified using a detection system (details not specified). IC₅₀ values were calculated by fitting dose-response curves, showing potent inhibition of type I PRMTs. For PRMT6, IC₅₀ was measured under different peptide (0.1–10 μM) and SAM (1–100 μM) concentrations, with no significant changes, indicating noncompetitive inhibition [1]
- FLT3 Methylation Assay: In vitro: GST-tagged FLT3 peptides (aa841–aa993) were incubated with PRMT1 enzyme, SAM, and MS023. Methylation of FLT3 R972/973 was detected by mass spectrometry (MS) and Western blot (using anti-asymmetric dimethylarginine antibody). In cells: 293T cells expressing FLT3-WT were treated with MS023, then FLT3 was immunoprecipitated, and R972/973 methylation was analyzed by Western blot [2]

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 decreased cellular levels of H4R3me2a (IC50=9±0.2 nM). Treatment concentration-dependently decreased the H3R2me2a mark (IC50=56±7 nM).

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Histone Methylation Detection in MCF7/HEK293 Cells: MCF7 cells were seeded and treated with MS023 at different concentrations (0–10 μM) for 48 h. Cells were lysed, and histones were extracted. Western blot was performed using anti-H4R3me2a (PRMT1 marker) and anti-total H4 antibodies. Band intensities were quantified, and nonlinear fits were generated to assess concentration-dependent inhibition. For HEK293 cells: cells were transfected with FLAG-tagged PRMT6 or catalytically inactive PRMT6 (V86K/D88A) for 24 h, then treated with MS023 (0–10 μM) for 20 h. Histones were extracted, and Western blot was done with anti-H3R2me2a and anti-total H3 antibodies [1]
- Global Arginine Methylation Analysis: MCF7 cells were treated with MS023 (0–10 μM) for 48 h. Total cell lysates were prepared, and Western blot was performed using pan-antibodies against Rme2a, Rme1, and Rme2s. β-actin was used as a loading control. Band intensities were quantified to compare methylation level changes [1]
- MLL-r ALL Cell Viability and Apoptosis Assays: Primary MLL-r ALL blasts or cell lines (SEM, RS4;11) were seeded in 96-well plates and treated with MS023 (0–20 μM) alone or with PKC412 (0–5 μM) for 48–72 h. Cell viability was measured using a cell counting kit (details not specified), and IC₅₀ values were calculated. For apoptosis: cells were stained with annexin V-Cy5 and 4',6-diamidino-2-phenylindole (DAPI), then analyzed by flow cytometry to determine the percentage of apoptotic cells [2]
- Co-Immunoprecipitation (Co-IP) for PRMT1-FLT3 Interaction: SEM cells were lysed in lysis buffer, and cell lysates were incubated with anti-FLT3 antibody or IgG (control) overnight at 4 °C. Protein A/G beads were added, and the mixture was incubated for 2 h. Beads were washed, and bound proteins were eluted. Western blot was performed with anti-PRMT1 and anti-FLT3 antibodies to detect their interaction [2]
- Proximity Ligation Assay (PLA) for PRMT1-FLT3 Interaction: Primary MLL-r ALL cells were fixed and permeabilized, then incubated with anti-PRMT1 and anti-FLT3 primary antibodies. PLA probes (complementary oligonucleotides conjugated to secondary antibodies) were added, followed by ligation and amplification. Red fluorescent spots (indicating PRMT1-FLT3 interaction) were visualized by fluorescence microscopy, and the number of spots per cell was counted [2]

Animal/Disease Models: NOD-scid IL2Rgnull (NSG) mice bearing primary MLL-r ALL cells[2]
Doses: 160 mg/kg
Route of Administration: intraperitoneal (ip)injection; PKC412 (100 mg/kg, ig), MS023 (160 mg/kg, ip ), or a combination for 4 weeks
Experimental Results: Combinatorial treatment extended survival of leukemic mice relative to single treatments.

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In vivo treatment of MLL-r ALL-engrafted mouse model[1]
For studying inducible short hairpin PRMT1 (shPRMT1), SEM cells stably expressing either doxycycline (DOX)-inducible short hairpin control (shCtrl) or PRMT1 short hairpin RNA (shRNA) (shPRMT1) were transplanted into irradiated immunodeficient NOD-scid IL2Rgnull (NSG) mice (1 × 106 cells per mouse). Each group was administered DOX treatment (10 mg/kg) orally for 3 weeks after engraftment. To assess R972/973 function in leukemogeneis, KOCL45 cells transduced with constructs expressing an FLT3 variant were sorted by using red fluorescent protein and injected into mice (1 × 105 cells per mouse), and mouse survival was monitored daily. To assess MS023 effects in vivo, we transplanted primary MLL-r ALL cells into NSG mice (0.5 × 106 cells per mouse). After engraftment, grouped mice were treated with vehicle, PKC412 (100 mg/kg, intragastrically), MS023 (160 mg/kg, intraperitoneally), or a combination for 4 weeks. After treatment, engrafted human cells were identified. Secondary transplantations of whole bone marrow (BM) cells from treated or control mice were then performed. Animal procedures were performed in accordance with federal and state government guidelines and established institutional guidelines and protocols approved by the Institutional Animal Care and Use Committee at City of Hope.

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PDX Model of MLL-r ALL and Drug Treatment: Primary MLL-r ALL cells (from patients #5 and #6) were intravenously injected into NSG mice. Peripheral blood was monitored weekly to detect human CD45⁺/CD19⁺ cell engraftment. When engraftment reached 1%, mice were randomized into 4 groups (n = 5–6 per group): (1) Vehicle (formulation not specified), (2) MS023 , (3) PKC412 , (4) MS023 + PKC412. Treatment lasted for 4 weeks. After treatment, mice were euthanized, and BM, SP, and PB were collected. The percentage of human CD45⁺/CD19⁺ cells was analyzed by flow cytometry. For secondary transplantation: BM cells (1×10⁶) from primary treated mice were injected into new NSG mice, and engraftment was monitored for 5 weeks. Survival was recorded for all groups [2]

[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.

MS023 is a type I PRMTs inhibitor.

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MS023 is a highly potent, selective, and cellularly active type I human PRMTs inhibitor. The crystal structure of its complex with PRMT6 and S-adenosylhomocysteine (SAH) shows that it binds to the substrate binding site of PRMT6, forming key hydrogen bonds and hydrophobic interactions [1]
-MS094 is a close analogue of MS023 that is inactive against type I PRMTs in biochemical and cellular experiments (e.g., does not inhibit H4R3me2a in MCF7 cells), and is therefore used as a negative control for MS023 in chemical biology studies [1]
-PRMT1 expression is higher in MLL severe acute lymphoblastic leukemia (MLL-r ALL) than in normal CD34⁺ cells. PRMT1-mediated FLT3 R972/973 methylation promotes the recruitment of adaptor proteins (such as GRB2) to FLT3, activating downstream oncogenic signaling pathways. MS023 can block this methylation, thereby disrupting the maintenance of leukemia cells. Combining MS023 with the FLT3 TKI PKC412 to overcome TKI resistance by simultaneously targeting FLT3 phosphorylation and methylation represents a promising MLL-rALL treatment strategy [2].

C17H25N3O

287.4

287.199

C, 71.04; H, 8.77; N, 14.62; O, 5.57

1831110-54-3

MS023 dihydrochloride;1992047-64-9;MS023 trihydrochloride;2108631-19-0

92136227

Typically exists as white to light yellow solids 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

2.28

2

3

7

21

290

0

O(C(C)C)C1C=CC(=CC=1)C1=CNC=C1CN(C)CCN

FMTVWAGUJRUAKE-UHFFFAOYSA-N

InChI=1S/C17H25N3O/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

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

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: 53.33 mg/mL (185.56 mM) in 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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 (Please use freshly prepared in vivo formulations for optimal results.)