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 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

2170136-65-7

(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.)