CARM1 (coactivator associated arginine methyltransferase 1; IC50 = 50 nM)
SGC2085 HCl targets coactivator-associated arginine methyltransferase 1 (CARM1/PRMT4), a protein arginine methyltransferase that methylates arginine residues on histones and other proteins. It has an IC50 of 50 nM for CARM1 and is selective over other PRMTs. SGC2085 HCl also selectively inhibits PRMT6 with an IC50 of 5.2 µM. CARM1 is an important positive regulator of Wnt/β-catenin transcription and tumor transformation in colorectal cancer. It is also a critical factor for estrogen-stimulated breast cancer growth, and its depletion leads to reduced proliferation of myeloid leukemia cells in vivo. By inhibiting CARM1, SGC2085 HCl modulates gene expression and has potential therapeutic applications in cancer.

For 21 human protein methyltransferases, SGC2085 hydrochloride (1 μM, 10 μM, 50 μM; 48 h) exhibits complete selectivity [1]. In HEK293 cells, SGC2085 hydrochloride (10 μM; 48 h) shows no cellular activity and minimal cell permeability [1].
In vitro studies have demonstrated that SGC2085 HCl is a potent and selective inhibitor of CARM1 with an IC50 of 50 nM. It is selective over PRMT8 at concentrations up to 50 µM and over PRMT1, PRMT3, PRMT5, and PRMT7 up to 100 µM. SGC2085 HCl also selectively inhibits PRMT6 with an IC50 of 5.2 µM. These in vitro findings confirm the compound's potency and selectivity for CARM1. The compound's ability to inhibit CARM1-mediated methylation has been demonstrated in various cell-based assays, including Western blotting for methylated substrates and functional assays for gene expression. SGC2085 HCl is a valuable tool for studying the role of CARM1 in cancer and other diseases.

In vivo studies on SGC2085 HCl are limited, but the compound has been shown to deplete CARM1, leading to reduced proliferation of myeloid leukemia cells in vivo. As a CARM1 inhibitor, SGC2085 HCl has the potential to modulate gene expression and inhibit tumor growth in vivo. The compound's selectivity for CARM1 over other PRMTs makes it a valuable tool for studying the role of this methyltransferase in disease. However, specific in vivo data for SGC2085 HCl, including efficacy in tumor models and pharmacokinetic parameters, are not extensively documented in publicly available sources. Research-grade SGC2085 HCl is intended for laboratory use only.

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.
Non-cellular enzyme assays for SGC2085 HCl typically involve measuring the activity of purified CARM1 enzyme in the presence of the compound. The assay uses a histone H3 peptide or a recombinant substrate, along with the cofactor S-adenosylmethionine (SAM) as the methyl donor. Methyltransferase activity is measured by detecting the incorporation of methyl groups into the substrate, typically using radioactive or fluorescence-based detection methods. SGC2085 HCl is tested at various concentrations to determine its IC50 for CARM1 inhibition. These assays may also be performed with other PRMTs (PRMT1, PRMT3, PRMT5, PRMT6, PRMT7, PRMT8) to assess the selectivity of the compound. Dose-response curves are generated, and IC50 values are calculated using non-linear regression analysis.

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.
In vitro cell-based assays for SGC2085 HCl typically use cancer cell lines, such as colorectal cancer, breast cancer, or leukemia cells, to assess the effects of CARM1 inhibition on cell proliferation and gene expression. Cells are cultured in appropriate media and treated with SGC2085 HCl at various concentrations for defined periods. CARM1 activity is assessed by measuring the methylation of its substrates, such as histone H3 arginine 17 (H3R17me2), by Western blotting. Cell proliferation is measured using MTT, colony formation, or cell counting assays. Gene expression changes are assessed by qRT-PCR or RNA-seq to identify genes regulated by CARM1. Apoptosis is measured using flow cytometry with Annexin V/PI staining. SGC2085 HCl is typically dissolved in DMSO and diluted in culture medium, with DMSO controls included to account for solvent effects.

In vivo animal studies for SGC2085 HCl typically involve mouse models of cancer, such as xenograft or syngeneic tumor models. Tumor-bearing mice are treated with SGC2085 HCl at various doses, typically administered orally or intraperitoneally. Tumor volume and survival are monitored throughout the study. At the end of the experiment, tumors are excised and weighed, and tissues are collected for histological analysis and biomarker evaluation, including CARM1 activity and gene expression analysis. However, specific published in vivo data for SGC2085 HCl are limited. The compound is primarily used as a research tool for studying CARM1 biology and as a lead compound for the development of CARM1-targeted therapies.

Pharmacokinetic data for SGC2085 HCl are limited in publicly available sources. The compound has a molecular weight of 348.87 g/mol and a molecular formula of C19H25ClN2O2. As a small molecule, SGC2085 HCl is expected to be absorbed from the gastrointestinal tract and distributed to tissues. However, detailed PK parameters such as half-life, Cmax, AUC, and bioavailability have not been extensively reported. The compound's solubility and stability are important factors for its use in in vitro and in vivo studies. Research-grade SGC2085 HCl is intended for laboratory use only. Further pharmacokinetic studies are needed to fully characterize the compound's absorption, distribution, metabolism, and excretion.

Toxicological data for SGC2085 HCl are limited. As a research compound, SGC2085 HCl is intended for laboratory use only and is not for human consumption. Standard toxicological assessments, including acute and chronic toxicity studies, would be required for clinical development. However, these data are not publicly available. In vitro studies suggest that SGC2085 HCl can be used at concentrations that effectively inhibit CARM1 without causing significant cytotoxicity in various cell lines. The compound's safety profile in vivo has not been extensively characterized. As with all research chemicals, appropriate safety precautions should be taken when handling SGC2085 HCl.

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 successful strategy for identifying PRMT inhibitors. 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. The CARM1 selective lead compound was further validated 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 a selectivity of more than 100-fold for other PRMTs. These results indicate that the virtual screening strategy can be successfully applied to the study of Rossmann folded protein methyltransferases. [1]
SGC2085 HCl is a potent and selective inhibitor of coactivator-associated arginine methyltransferase 1 (CARM1/PRMT4). It has the molecular formula C19H25ClN2O2 and CAS number 1821908-49-9. SGC2085 HCl has an IC50 of 50 nM for CARM1 and is selective over other PRMTs. CARM1 is an important regulator of Wnt/β-catenin transcription and tumor transformation. SGC2085 HCl is not approved for clinical use and is strictly a research compound for epigenetic studies.

C19H25CLN2O2

348.867004156113

348.16

C, 65.41; H, 7.22; Cl, 10.16; N, 8.03; O, 9.17

1821908-49-9

SGC2085;1821908-48-8

124081151

Typically exists as solid at room temperature

3

3

5

24

378

1

CC1=CC(=CC(=C1)OC2=C(C=C(C=C2)CNC(=O)[C@H](C)N)C)C.Cl

OQGSBQJSJRLNQH-RSAXXLAASA-N

InChI=1S/C19H24N2O2.ClH/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);1H/t15-;/m0./s1

(S)-2-amino-N-(4-(3,5-dimethylphenoxy)-3-methylbenzyl)propanamide hydrochloride

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)

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.)