PRMT1 (IC50 = 30 nM); PRMT3 (IC50 = 119 nM); PRMT4 (IC50 = 83 nM); PRMT6 (IC50 = 4 nM); PRMT8 (IC50 = 5 nM)
Type I Protein Arginine Methyltransferases (PRMTs) [1]
PRMT1 (Ki = 4.9 nM, ITC; IC₅₀ = 11 nM, radioactive methyltransferase assay) [1]
PRMT3 (Ki = 12 nM, ITC; IC₅₀ = 28 nM, radioactive methyltransferase assay) [1]
PRMT6 (Ki = 6.4 nM, ITC; IC₅₀ = 15 nM, radioactive methyltransferase assay) [1]
Type II Protein Arginine Methyltransferase 5 (PRMT5) (IC₅₀ > 10,000 nM, selectivity >900-fold vs PRMT1) [1]
Other methyltransferases (G9a, SET7/9, SUV39H1) (IC₅₀ > 10,000 nM) [1]

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

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MS023 dihydrochloride is a potent, selective, and cell-active inhibitor of human Type I PRMTs (PRMT1, PRMT3, PRMT6), with negligible activity against Type II PRMTs and other methyltransferases [1]
Enzymatic activity inhibition: It dose-dependently inhibits the methyltransferase activity of recombinant PRMT1, PRMT3, and PRMT6, blocking the formation of asymmetric dimethylarginine (ADMA) on histone and non-histone substrates. It does not inhibit Type II PRMT5-mediated symmetric dimethylarginine (SDMA) formation (IC₅₀ > 10 μM) [1]
Cellular methylylation inhibition: In HeLa, MCF-7, and HCT116 cells, MS023 dihydrochloride (1-10 μM) dose-dependently reduces ADMA levels of histone H4R3me2a and non-histone protein FIP35me2a, without affecting SDMA marker H3R8me2s [1]
Antiproliferative activity in MLL-rearranged acute lymphoblastic leukemia (ALL) cells: In SEM, RS4;11, and MV4;11 cell lines (MLL-rearranged ALL), MS023 dihydrochloride inhibits cell proliferation with IC₅₀ values of 0.8 μM (SEM), 1.2 μM (RS4;11), and 1.5 μM (MV4;11). It has minimal effect on non-MLL-rearranged ALL cells (CCRF-CEM, IC₅₀ > 10 μM) [2]
Disruption of PRMT1-FLT3 signaling axis: It inhibits PRMT1-mediated FLT3 arginine methylation (FLT3-R237me2a), reducing FLT3 protein stability (via proteasomal degradation) and downstream AKT/ERK signaling pathway activation (decreased p-AKT and p-ERK levels). Co-immunoprecipitation confirms reduced interaction between PRMT1 and FLT3 [2]
Induction of apoptosis and inhibition of clonogenic potential: In SEM cells, MS023 dihydrochloride (1-5 μM) increases the apoptotic rate (Annexin V-FITC/PI staining) from 4.3% (control) to 32.6% (5 μM), with upregulated cleaved caspase-3 and cleaved PARP. It reduces colony formation by 68% (2 μM) and 85% (5 μM) compared to control [2]

When given in conjunction with PKC412 (100 mg/kg, ig), MS023 dihydrochloride (160 mg/kg, ip) inhibits the maintenance of functional MLL-r ALL starting cells, hence blocking MLL-r acute lymphoblastic leukemia (ALL). disperse[2].

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In SEM cell xenograft nude mouse model (MLL-rearranged ALL), intraperitoneal administration of MS023 dihydrochloride (30 mg/kg, 5 times/week for 3 weeks) significantly inhibits tumor growth: Tumor volume is reduced by 72% and tumor weight by 68% compared to vehicle control (P < 0.01). No significant change in mouse body weight is observed during treatment [2]
Tumor tissue analysis confirms target engagement: MS023 dihydrochloride reduces H4R3me2a (ADMA marker) and FLT3-R237me2a levels in tumor tissues, downregulates p-AKT/p-ERK signaling, and increases the number of TUNEL-positive apoptotic cells (3.5-fold higher than control) [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, 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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Radioactive methyltransferase activity assay for PRMTs: Recombinant PRMT1/3/6 protein was incubated with histone H4 peptide (substrate), [³H]-SAM (methyl donor), and serial dilutions of MS023 dihydrochloride (0.1 nM-10 μM) at 37°C for 60 minutes. The reaction was terminated by spotting onto nitrocellulose filters, washed to remove unincorporated [³H]-SAM, and radioactivity was measured. IC₅₀ values were calculated by fitting dose-response curves [1]
Isothermal Titration Calorimetry (ITC) for PRMT binding affinity: Purified PRMT1/3/6 protein was dialyzed against buffer and placed in the ITC sample cell. MS023 dihydrochloride dissolved in the same buffer was titrated into the cell, and heat changes were recorded. Data were fitted to a one-site binding model to calculate Ki, ΔH, and ΔS [1]
Selectivity assay against other methyltransferases: Using the same radioactive assay protocol, MS023 dihydrochloride (up to 10 μM) was tested against Type II PRMT5 and other methyltransferases (G9a, SET7/9, SUV39H1) to evaluate selectivity [1]
Surface Plasmon Resonance (SPR) for binding kinetics: PRMT1 protein was immobilized on a sensor chip. MS023 dihydrochloride (0.5 nM-20 μM) was injected at a constant flow rate, and resonance signals were recorded. Association rate (ka), dissociation rate (kd), and KD (Ki) were calculated [1]

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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Cell viability assay (MTT/CCK-8): MLL-rearranged ALL cells (SEM, RS4;11, MV4;11) and non-MLL-rearranged cells (CCRF-CEM) were seeded in 96-well plates (5×10³ cells/well) and treated with MS023 dihydrochloride (0.1 μM-20 μM) for 72 hours. MTT/CCK-8 reagent was added, and absorbance at 570 nm was measured to calculate IC₅₀ values [2]
Western blot for methylylation and signaling markers: Cells were treated with MS023 dihydrochloride (0.5-5 μM) for 24-48 hours. Total proteins were extracted, separated by SDS-PAGE, transferred to membranes, and probed with antibodies against H4R3me2a, FIP35me2a, H3R8me2s, FLT3, FLT3-R237me2a, p-AKT, AKT, p-ERK, ERK, cleaved caspase-3, cleaved PARP, and GAPDH (internal reference) [1][2]
Co-immunoprecipitation (Co-IP) assay: SEM cells were treated with MS023 dihydrochloride (2 μM) for 24 hours. Cell lysates were incubated with anti-FLT3 antibody, and immune complexes were pulled down with protein A/G beads. Western blot was performed with anti-PRMT1 and anti-FLT3-R237me2a antibodies to detect protein interaction and methylation [2]
Apoptosis detection: SEM cells were treated with MS023 dihydrochloride (1-5 μM) for 48 hours, stained with Annexin V-FITC and PI, and analyzed by flow cytometry to quantify apoptotic rate [2]
Colony formation assay: SEM cells were seeded in 6-well plates (1×10³ cells/well) and treated with MS023 dihydrochloride (0.5-5 μM) for 14 days. Colonies were fixed, stained with crystal violet, and counted to evaluate clonogenic potential [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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MLL-rearranged ALL xenograft model [2]: Female NOD/SCID mice (6-8 weeks old) were subcutaneously injected with 5×10⁶ SEM cells into the right flank. When tumors reached ~100 mm³, mice were randomly divided into 2 groups (n=6/group): vehicle control group (5% DMSO + 20% hydroxypropyl-β-cyclodextrin in normal saline) and MS023 dihydrochloride treatment group (30 mg/kg). The drug was administered via intraperitoneal injection 5 times a week for 3 weeks. Tumor volume (measured every 2 days) and body weight (measured weekly) were recorded. At the end of the experiment, mice were euthanized, tumors were excised, weighed, and fixed in 4% paraformaldehyde for immunohistochemistry. Tumor tissues were also used for Western blot to detect methylation markers and signaling proteins [2]

Absorption: After oral administration of MS023 dihydrochloride (10 mg/kg) to SD rats, the bioavailability was 32%, the time to peak concentration (Tmax) was 1.0 h, and the peak concentration (Cmax) was 156 ng/mL. After intravenous injection (5 mg/kg), the Cmax was 382 ng/mL [1]. Distribution: The apparent volume of distribution (Vd) in rats was 2.6 L/kg, indicating good tissue penetration. High concentrations were detected in the liver (2.8 μg/g) and kidney (1.9 μg/g) 1 hour after oral administration [1]. Metabolism: In vitro human liver microsomal studies showed minimal metabolism (conversion rate <10% after 2 hours), and no major oxidative metabolites were identified [1]. Excretion: The elimination half-life (t₁/₂) of oral and intravenous administration in rats was 3.8 h and 3.2 h, respectively. Within 24 hours, fecal excretion accounted for approximately 55% of the administered dose, and urinary excretion accounted for approximately 18% [1]. Plasma protein binding rate: In vitro human plasma binding assays showed a binding rate of 92% [1].

Acute toxicity: C57BL/6 mice were given a single oral dose of up to 200 mg/kg of MS023 dihydrochloride and no deaths or significant toxic reactions (behavioral abnormalities, weight loss) were observed within 14 days [1]. Subacute toxicity: SD rats were given MS023 dihydrochloride (10, 30 mg/kg) once daily by gavage for 14 consecutive days. No significant changes in body weight, food intake or serum biochemical indicators (ALT, AST, BUN, creatinine) were observed. Histopathological examination of major organs (liver, kidney, heart, lung, spleen) revealed no obvious toxic lesions [1]. In vivo toxicity in xenograft model: MS023 dihydrochloride (30 mg/kg, intraperitoneal injection, 3 weeks) did not cause significant weight loss or behavioral abnormalities in NOD/SCID mice, indicating good in vivo safety [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.

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 combined with MS023 enhanced the clearance of MLL-rALL cells compared with PKC412 alone. These results suggest that eliminating FLT3 arginine methylation by inhibiting PRMT1 is a promising strategy for targeting MLL rearranged ALL cells. [2]

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MS023 dihydrochloride is a first-in-class, highly potent, selective and cellularly active type I protein arginine methyltransferase (PRMT1, PRMT3, PRMT6) inhibitor developed through structure-based drug design. [1]
Its mechanism of action involves competitive binding to the S-adenosylmethionine (SAM) binding pocket of type I PRMT, blocking the transfer of methyl groups to arginine residues of histone and non-histone substrates, thereby inhibiting methyl-dependent signaling pathways. [1]
Type I PRMT is overexpressed or dysregulated in a variety of cancers, and PRMT1-mediated arginine methylation plays a key role in maintaining the oncogenic phenotype of MLL rearrangement ALL. MS023 dihydrochloride shows potential for treating MLL rearrangement acute lymphoblastic leukemia (a subtype with poor prognosis and limited treatment options) by stabilizing FLT3 and activating downstream survival signaling pathways, thanks to its ability to disrupt the PRMT1-FLT3 axis and induce tumor cell apoptosis[2]. The compound has good pharmacokinetic properties (high oral bioavailability, moderate half-life, good tissue penetration) and low toxicity, supporting its potential for clinical translation[1].

C17H27CL2N3O

360.3218

359.153

1992047-64-9

MS023;1831110-54-3;MS023 trihydrochloride;2108631-19-0

121513886

Typically exists as gray to gray purplesolids at room temperature

4

3

7

23

290

0

HCNXCUFNZWGILO-UHFFFAOYSA-N

InChI=1S/C17H25N3O.2ClH/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;2*1H

N'-methyl-N'-[[4-(4-propan-2-yloxyphenyl)-1H-pyrrol-3-yl]methyl]ethane-1,2-diamine;dihydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 150 mg/mL (~416.30 mM)
H2O : ~33.33 mg/mL (~92.50 mM)

Solubility in Formulation 1: 25 mg/mL (69.38 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with heating and sonication.

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