DNMT1/DNA methyltransferase

Significant reduction of DNA methylation is caused by GSK-3484862 (0-10 μM, 6 or 14 days) [2]. GSK-3484862 (0-10 μM, 4 days) results in a slight reduction in the levels of DNMT1 protein [2].

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In this study, researchers determined the cytotoxicity and optimal concentration of GSK-3484862 by treating wild-type (WT) or Dnmt1/3a/3b triple knockout (TKO) mESC with different concentrations of the compound, which was obtained from two commercial sources. Concentrations of 10 µM or below were readily tolerated for 14 days of culture. Known DNA methylation targets such as germline genes and GLN-family transposons were upregulated within 2 days of the start of GSK-3484862 treatment. By contrast, 5-azacytidine and decitabine induced weaker upregulation of methylated genes and extensive cell death. Whole-genome bisulfite sequencing showed that treatment with GSK-3484862 induced dramatic DNA methylation loss, with global CpG methylation levels falling from near 70% in WT mESC to less than 18% after 6 days of treatment with GSK-3484862. The treated cells showed a methylation level and pattern similar to that observed in Dnmt1-deficient mESCs. Conclusions: GSK-3484862 mediates striking demethylation in mESCs with minimal non-specific toxicity.[2]

Determining GSK-3484862 toxicity[1] An assay to determine the optimal concentration and toxicity of GSK-3484862 was performed using J1 WT and DNMT TKO mESCs. 30,000 cells were seeded in 24-well plates pre-coated with 0.1% gelatin. The next day, medium was changed to fresh mESC medium or medium containing DMSO (0.1% or 1%) for the following concentrations of GSK-3484862: 2 pM, 20 pM, 200 pM, 2 nM, 20 nM, 200 nM, 2 µM, 20 µM (in 0.1% DMSO) and 200 µM (in 1% DMSO). The medium was refreshed every day for the next six days, after which cell morphology was assessed, followed by cell dissociation with 0.05% Trypsin–EDTA for cell counting.[1] Next, the demethylation efficacy and long-term cytotoxicity of GSK-3484862 was evaluated in duplicate experiments. To improve solubility, after resuspension in DMSO, GSK-3484862 was subjected to ultrasonication. GSK-3484862 from both companies were sonicated for 6 min at 42 kHz in an ultrasonic water bath (Sper Scientific). Still, drug precipitation was observed for concentrations at or above 20 µM in media, and therefore an upper concentration of 10 µM was chosen. WT and DNMT TKO cells were seeded in 12-well plates pre-coated with 0.1% gelatin and had 0.1% DMSO, 2 µM or 10 µM GSK3484862 added in medium from day zero. The medium was refreshed every day and cells were counted using the Countess II FL instrument and passaged every 2–3 days for the next 14 days.

Cell Viability Assay[2]
Cell Types: Mouse Embryonic Stem Cells (mESC, Wild Type (WT) or Dnmt1/3a/3b Triple Knockout (TKO))
Tested Concentrations: 2 µM and 10 µM
Incubation Duration: 6 or 14 days
Experimental Results: Result There was a dramatic loss of DNA methylation, with overall CpG methylation levels in WT mESCs falling from nearly 70% to less than 18% after 6 days.

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Western Blot Analysis[2]
Cell Types: Mouse Embryonic Stem Cells (mESC, Wild Type (WT) or Dnmt1/3a/3b Triple Knockout (TKO))
Tested Concentrations: 2 µM and 10 µM
Incubation Duration: 4 days
Experimental Results: Production of DNMT1 Protein levels are moderately diminished.

[1]. The DNMT1 inhibitor GSK-3484862 mediates global demethylation in murine embryonic stem cells.

[2]. Keystone Symposia 2019 - Epigenetics and Human Disease.

From a research perspective, GSK-3484862 shows great potential. 5-azanucleotides have significant non-specific toxicity and a narrow concentration range. We observed that treatment with 0.3 µM 5-azacytidine reactivated some methylated genes, but very few cells survived at this concentration; while 0.1 µM 5-azacytidine was insufficient to reactivate methylated genes. In addition, we needed to culture for two days to observe the formation of clonal cells. In contrast, 2 µM or 10 µM GSK-3484862 could reactivate the expression of methylated genes and only caused slight inhibition of cell growth, which was most pronounced from day 6 to day 10 of treatment. This growth inhibition may reflect the specific activity of the compound. When mouse embryonic stem cells (mESCs) are demethylated after treatment with MEK inhibitors, glycogen synthase kinase 3 inhibitors and high concentrations of ascorbic acid, transposon expression increases explosively and heterochromatin state is remodeled during this period [2]. Regardless of the dosage or treatment duration, the DNA methylation level of mESCs treated with GSK-3484862 never fell below 16%, and the reactivation of methylated genes did not reach the levels observed in TKO cells. This may reflect the high activity of DNMT3A and DNMT3B in mESCs, as confirmed by similar DNA methylation levels in published Dnmt1 KO mESCs. Dnmt1-deficient or suppressed cells may reach an equilibrium where methylation is continuously added by DNMT3A and DNMT3B and lost through replication and Tet protein activity. Other cell types may respond differently to Dnmt1 inhibition. Most somatic and cancer cells do not express such high levels of de novo DNA methyltransferases and therefore may not be able to maintain such high DNA methylation levels in the absence of Dnmt1 activity. Simultaneously, somatic or cancer cells may not survive in the presence of significantly reduced DNA methylation levels. Dnmt1-deficient and Dnmt1/3a/3b triple knockout mouse embryonic stem cells (mESCs) fail to survive post-differentiation, while DNMT1 becomes crucial after uterine implantation. This shift may reflect the high dependence of mESCs on TRIM28 to silence transposons, but the crucial role of DNA methylation in transposon inhibition after mESC differentiation or embryonic implantation. Therefore, researchers studying other cell types are likely to observe specific toxicities at lower doses; indeed, GSK-3484862 and related compounds have shown significant efficacy in leukemia. We also cannot rule out the possibility of non-specific toxicity in certain cell types, as GSK-3484862 inhibits mouse development at relatively low concentrations, suggesting this possibility. Nevertheless, this novel DNA methyltransferase inhibitor appears to be a significant improvement over 5-azanucleoside analogs and is a promising research tool. Conclusion: GSK-3484862 significantly reduces methylation levels in mouse embryonic stem cells. In terms of methylation gene activation and non-specific toxicity, GSK-3484862 outperforms 5-azanucleoside analogs.

C19H19N5OS

365.452061891556

365.131

C, 62.45; H, 5.24; N, 19.16; O, 4.38; S, 8.77

2170136-02-2

GSK-3484862;2170136-65-7

132233424

Brown to orange solid powder

3

1

6

6

26

585

0

KIEQQZZDWUNUQK-UHFFFAOYSA-N

InChI=1S/C19H19N5OS/c1-4-13-14(10-20)18(24(2)3)23-19(15(13)11-21)26-16(17(22)25)12-8-6-5-7-9-12/h5-9,16H,4H2,1-3H3,(H2,22,25)

2-[3,5-dicyano-6-(dimethylamino)-4-ethylpyridin-2-yl]sulfanyl-2-phenylacetamide

(Rac)-GSK-3484862; 2170136-02-2; 2-{[3,5-dicyano-6-(dimethylamino)-4-ethylpyridin-2-yl]sulfanyl}-2-phenylacetamide; SCHEMBL19717177; US10975056, Example 3; BDBM491060;

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.)