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]. Keystone Symposia 2019 - Epigenetics and Human Disease.

[2]. The DNMT1 inhibitor GSK-3484862 mediates global demethylation in murine embryonic stem cells. [Epigenetics Chromatin. 2021 Dec 15;14(1):56. https://pubmed.ncbi.nlm.nih.gov/34906184/ ]

From a research perspective, GSK-3484862 shows a great deal of promise. 5-Azanucleosides have substantial non-specific toxicity and work within a narrow concentration band. We observed reactivation of some methylated genes after treatment with 0.3 µM 5-azacytidine, but very few cells survived at this concentration, while 0.1 µM 5-azacytidine was inadequate to reactivate methylated genes. Furthermore, we had to allow two days for colonies of surviving cells to emerge in culture. By contrast, 2 µM or 10 µM GSK-3484862 reactivated methylated gene expression and produced only modest reduction in growth, most noticeable between days six and 10 of treatment. This reduced growth may reflect specific activity of the compound. When mESCs undergo demethylation mediated by treatment with MEK inhibitor, Glycogen Synthase Kinase 3 inhibitor and high concentration of ascorbic acid, they have a burst of transposon expression and undergo reconfiguration of heterochromatin state approximately during this interval [2].
DNA methylation of the GSK-3484862-treated mESCs never fell below 16% regardless of the dosage used or time of treatment, and methylated genes were not reactivated to the extent observed in the TKO cells. This likely reflects the high level of DNMT3A and DNMT3B activity in mESCs, as evidenced by the similar level of DNA methylation in published Dnmt1 KO mESCs. The Dnmt1 deficient or inhibited cells potentially reach an equilibrium in which methylation is constantly added by DNMT3A and DNMT3B and lost through replication and Tet-protein activity. Other cell types may respond to Dnmt1 inhibition differently. Most somatic and cancer cells do not express such high levels of the de novo DNA methyltransferases and may not be able to maintain such high levels of DNA methylation in the absence of Dnmt1 activity. At the same time, somatic or cancer cells may not survive dramatic DNA methylation loss. Dnmt1 deficient and Dnmt1/3a/3b TKO mESCs are not viable upon differentiation, and DNMT1 becomes essential after uterine implantation. This shift may reflect the fact that mESCs depend heavily on TRIM28 to silence transposons, but upon differentiation of mESCs or uterine implantation of embryos, DNA methylation gains importance for transposon repression. Thus, researchers working with other cell types may well observe specific toxicity at lower doses, and indeed GSK-3484862 and related compounds show a striking effect in leukemias. We also cannot rule out that non-specific toxicity may occur in some cell types, a result suggested by the GSK-3484862’s ability to halt mouse development at relatively low concentrations. Nonetheless, this novel DNA methyltransferase inhibitor appears to be a substantial improvement over 5-azanucleosides and a promising research tool.
Conclusions GSK-3484862 mediates dramatic demethylation in murine embryonic stem cells. With regard to both activation of methylated genes and non-specific toxicity, GSK-3484862 performs far better than 5-azanucleosides.

C19H19N5OS

365.452061891556

365.131

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

2170136-65-7

(Rac)-GSK-3484862;2170136-02-2

132233666

Light yellow to yellow solid powder

3

1

6

6

26

585

1

S(C1=C(C#N)C(=C(C#N)C(=N1)N(C)C)CC)[C@@H](C(N)=O)C1C=CC=CC=1

KIEQQZZDWUNUQK-MRXNPFEDSA-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)/t16-/m1/s1

(R)-2-((3,5-dicyano-6-(dimethylamino)-4-ethylpyridin-2-yl)thio)-2-phenylacetamide

GSK3484862; GSK-3484862; 2170136-65-7; GSKMI-714; (2R)-2-[3,5-dicyano-6-(dimethylamino)-4-ethylpyridin-2-yl]sulfanyl-2-phenylacetamide; SCHEMBL19717424; BDBM491120; US10975056, Example 64; NSC825088; GSK 3484862

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)

DMSO : ~20.83 mg/mL (~57.00 mM)

Solubility in Formulation 1: 2.08 mg/mL (5.69 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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: 2.08 mg/mL (5.69 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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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 (Please use freshly prepared in vivo formulations for optimal results.)