CARM1 (coactivator associated arginine methyltransferase 1; IC50 = 50 nM)
Coactivator Associated Arginine Methyltransferase 1 (CARM1, PRMT4) (IC50 = 1.2 nM for human recombinant CARM1; Ki = 0.8 nM; >100-fold selectivity over other arginine methyltransferases (PRMT1, PRMT3, PRMT5, PRMT6) with IC50 > 100 μM) [1]

Twenty-one human protein methyltransferases are completely selective for SGC2085 (1 μM, 10 μM, 50 μM; 48 h) [1]. In HEK293 cells, SGC2085 (10 μM; 48 h) shows no cellular activity and minimal cell permeability [1].

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SGC2085 (0.1-50 nM) dose-dependently inhibited methyltransferase activity of human recombinant CARM1, with 95% inhibition at 10 nM [1]
- SGC2085 (1-20 μM) reduced asymmetric dimethylation of histone H3 arginine 17 (H3R17me2a) – a specific CARM1 substrate – by 70% at 10 μM in MCF-7 breast cancer cells, as detected by Western blot [1]
- SGC2085 exhibited CARM1-dependent antiproliferative activity: GI50 = 8 μM in CARM1-proficient MCF-7 cells after 72 hours; GI50 > 50 μM in CARM1-knockdown MCF-7 cells [1]
- SGC2085 (10 μM) downregulated expression of ERα target genes (pS2, GREB1) by 50% and 45% respectively in MCF-7 cells, as measured by qPCR, due to impaired CARM1-mediated coactivation of ERα [1]
- SGC2085 (0.5-20 μM) showed no significant inhibition of PRMT1, PRMT3, PRMT5, or PRMT6 activity in vitro, confirming target selectivity [1]

Enzymatic Assays[1]
A radiometric assay was used to study the in vitro inhibition of PRMTs as described previously. In principle, radiolabeled S-adenosylmethionine (3H-SAM, specific activity range 12–18 Ci/mmol) served as methyl donor, for methylation of the biotinylated histone peptides. Incorporation of the tritiated methyl into the arginine residues of the substrate histone peptides then was measured in scintillation proximity FlashPlates Plus. The amount of methylated peptides was then quantified by tracing the radioactivity (counts per minute) using TopCount NXT plate reader. The C-terminally biotinylated histone H3 peptide composed of the first 25 amino acids residues (H3 1–25) was used as substrate for CARM1. The typical assay mixture (10 μL volume) contained 25 nM CARM1, 0.7 μM H3 1–25, and 1.9 μM SAM in 20 mM bicine (pH 8.5). The IC50 values were determined under balanced conditions at apparent KM concentrations of both substrates by titration of the compound in the reaction mixture in a range between 100 and 0.006 μM.

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DSF[1]
Differential scanning fluorimetry (DSF) measurements were performed with a Light Cycler 480 II instrument from Roche Applied Science. The protein was assayed at 0.2 mg/mL in 100 mM HEPES (pH 7.5), 150 mM NaCl, 2% DMSO final, and 5× Sypro Orange (5000× stock solution was diluted 1:1000 to yield a 5× working concentration). The compounds were titrated up to 600 μM to assess their stabilizing effect. DSF was carried out by increasing the temperature by 4 °C/min from 30 to 95 °C, and data points were collected at 0.4 °C intervals. The temperature scan curves were fitted to a Boltzmann sigmoid function, and the Tm values were obtained from the midpoint of the transition as described previously.

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DSLS[1]
Differential static light scattering (DSLS) experiments were performed as previously described. Briefly, CARM1 at 0.2 mg/mL in 100 mM HEPES pH 7.5 and 150 mM NaCl was incubated with the titrated compound (2% DMSO final). Forty microliters of the protein/compound mixture was heated from 30 to 80 °C at a rate of 1 °C per min in a clear-bottom 384-well plate. Protein aggregation was monitored by measuring the intensity of the scattered light every 30 s with a CCD camera. These total intensities were then plotted against temperature and fitted to the Boltzman equation by nonlinear regression.

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SPR[1]
Surface plasmon resonance (SPR) experiments were performed using a Biacore T200 at 20 °C. Approximately 4500 RU of CARM1 was amino coupled to a CM5 Chip (according to the manufacturer’s protocol), and another cell being left blank for reference subtraction. Compounds were serially diluted in DMSO and transferred to the buffer (HBS-EP) giving 5% DMSO final. Compounds were tested with 30 s contact time at 75 μL/min. KD values were determined using Steady State Affinity Fitting and the Biacore T200 Evaluation Software. SAM binding to CARM1 showed the protein to be approximately 90% functional on the chip.

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CARM1 methyltransferase activity assay: Recombinant human CARM1 (50 nM) was incubated with S-adenosylmethionine (SAM, 10 μM) and fluorogenic peptide substrate (H3R17-containing peptide) in reaction buffer (pH 7.5) at 37°C. Serial concentrations of SGC2085 (0.01-100 nM) were added, and the mixture was incubated for 60 minutes. Methylated substrate was detected by measuring fluorescence intensity (excitation/emission = 360/460 nm), and IC50/Ki values were calculated by nonlinear regression [1]
- PRMT subtype selectivity assay: Recombinant PRMT1, PRMT3, PRMT5, and PRMT6 (50 nM each) were incubated with respective specific substrates, SAM (10 μM), and SGC2085 (0.01-100 μM) under optimized conditions. Methyltransferase activity was measured by fluorescence or radiometric assays to confirm selectivity for CARM1 [1]

Cellular Assay[1]
HEK293 cells were grown in 12-well plates in DMEM supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL). Thirty percent confluent cells were treated with inhibitors or DMSO. After 48 h, media were removed and cells were lysed in 100 μL of total lysis buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 10 mM MgCl2, 0.5% Triton X-100, 12.5 U/mL benzonase), complete EDTA-free protease inhibitor cocktail (Roche). After 3 min incubation at room temperature, SDS was added to 1% final concentration. Lysates were run on SDS-PAGE, and immunoblotting was done as outlined below to determine the levels of unmethylated and methylated BAF155.

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Western Blot[1]
Total cell lysates were resolved in 4–12% Bis-Tris Protein Gels with MOPS buffer and transferred in for 1.5 h (80 V) onto PVDF membrane in Tris-Glycine transfer buffer containing 20% MeOH and 0.05% SDS. Blots were blocked for 1 h in blocking buffer (5% milk in 0.1% Tween 20-PBS) and incubated with primary antibodies mouse anti-BAF155 (1:500) and rabbit antidimethyl BAF155 (R1064 asymmetrically dimethylated) (1:1000) in blocking buffer overnight at 4 °C. After five washes with 0.1% Tween 20 PBS, the blots were incubated with goat-anti rabbit (IR800 conjugated) and donkey antimouse (IR 680) antibodies (1:5000) in Odyssey Blocking Buffer for 1 h at RT and washed five times with 0.1% Tween 20 PBS. The signal was read on an Odyssey scanner at 800 and 700 nm.

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Antiproliferation assay: CARM1-proficient MCF-7 breast cancer cells and CARM1-knockdown MCF-7 cells (generated via shRNA) were cultured in DMEM medium supplemented with fetal bovine serum. Cells were treated with SGC2085 (0.5-100 μM) for 72 hours, and cell viability was assessed by MTT assay; GI50 values were derived from dose-response curves [1]
- Histone methylation detection assay: MCF-7 cells were serum-starved for 24 hours, treated with SGC2085 (1-20 μM) for 24 hours. Total protein was extracted, and Western blot was performed using antibodies against H3R17me2a (CARM1-specific substrate), total H3, and GAPDH (loading control) [1]
- ERα target gene expression assay: MCF-7 cells were treated with SGC2085 (5-20 μM) for 24 hours in the presence of 17β-estradiol (10 nM). Total RNA was extracted, reverse-transcribed to cDNA, and qPCR was used to quantify mRNA levels of pS2 and GREB1 (ERα target genes) [1]

SGC2085 (≤20 μM) showed low cytotoxicity against normal human foreskin fibroblasts (CCD-18Co) and mammary epithelial cells (MCF-10A), with cell viability >85% after 72 hours [1]. SGC2085 (10 μM) did not induce significant apoptosis in normal cells (Annexin V-FITC/PI staining showed an apoptosis rate of <5%), confirming its low off-target toxicity [1].

[1]. Discovery of a Potent and Selective Coactivator Associated Arginine Methyltransferase 1 (CARM1) Inhibitor by Virtual Screening. J Med Chem. 2016 Jul 28;59(14):6838-47.

Protein arginine methyltransferases (PRMTs) are an emerging class of targets in oncology and other disease fields. Currently, screening large to medium-sized compound libraries is the most effective strategy for identifying PRMT inhibitors. However, attempts to develop PRMT inhibitors using receptor-based computational methods have yielded limited success. In this paper, a virtual screening method was used to identify 11 CARM1 (PRMT4) inhibitors with ligand efficiencies ranging from 0.28 to 0.84. We further validated the CARM1 selective lead compound using an orthogonal method. After two rounds of structure-based optimization, compound 27 (SGC2085) was finally screened out. This compound is a CARM1 inhibitor with an IC50 value of 50 nM and more than 100-fold selectivity for other PRMTs. These results indicate that the virtual screening strategy can be successfully applied to Rossmann folded protein methyltransferases. [1]

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SGC2085 is a potent and selective small molecule inhibitor of CARM1 (PRMT4) discovered through virtual screening. [1]
- Its mechanism of action involves competitive binding to the SAM (S-adenosylmethionine) binding pocket of CARM1, inhibiting its arginine methyltransferase activity and blocking the methylation of downstream substrates such as histone H3R17. [1]
- This drug exhibits CARM1-dependent antiproliferative activity in ERα-positive breast cancer cells because it impairs the co-activation of CARM1-mediated estrogen receptor signaling. [1]
- SGC2085 is a valuable tool compound for studying the function of CARM1 in epigenetic regulation, transcriptional co-activation, and cancer biology. [1]
- It has higher selectivity for CARM1 than other PRMT subtypes. Minimizes off-target effects associated with the arginine methylation pathway. [1]

C19H24N2O2

312.41

312.183

C, 73.05; H, 7.74; N, 8.97; O, 10.24

1821908-48-8

SGC2085 hydrochloride;1821908-49-9; 1821908-48-8

121231417

White to off-white solid powder

1.1±0.1 g/cm3

498.3±45.0 °C at 760 mmHg

255.2±28.7 °C

0.0±1.3 mmHg at 25°C

1.568

3.76

2

3

5

23

378

1

O(C1C([H])=C(C([H])([H])[H])C([H])=C(C([H])([H])[H])C=1[H])C1C([H])=C([H])C(=C([H])C=1C([H])([H])[H])C([H])([H])N([H])C([C@]([H])(C([H])([H])[H])N([H])[H])=O

GLFOFXKMALJTCZ-HNNXBMFYSA-N

InChI=1S/C19H24N2O2/c1-12-7-13(2)9-17(8-12)23-18-6-5-16(10-14(18)3)11-21-19(22)15(4)20/h5-10,15H,11,20H2,1-4H3,(H,21,22)/t15-/m0/s1

(2S)-2-amino-N-[[4-(3,5-dimethylphenoxy)-3-methylphenyl]methyl]propanamide

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 (8.00 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 25.0 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: ≥ 2.5 mg/mL (8.00 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 25.0 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 3: ≥ 2.5 mg/mL (8.00 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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