DNA methyltransferase: DNMT3B (IC50 = 7.5 μM); DNMT3A (IC50 = 8 μM); DNMT1 (IC50 = 12.5 μM)

SGI-1027 is a DNMT inhibitor with IC50s of 7.5 μM, 8 μM and 12.5 μM for DNMT3B, DNMT3A and DNMT1 correspondingly when using poly(dI-dC) as substrate. The IC50 of SGI-1027 against DNMT1 (hemimethylated DNA) is 6 μM. SGI-1027 (1, 2.5 or 5 μM) promotes preferential degradation of DNMT1 in various human cancer cell lines but has little or no cytotoxic effect on rat hepatoma cells and does not trigger apoptosis in rat hepatoma cells [1]. SGI-1027 has an EC50 of 0.9 μM for hDNMT3A and is cytotoxic to KG-1 cells with an EC50 of 4.4 μM [2].

Intravitreous administration of the DNA-methyltransferase inhibitor SGI-1027 induced Oct4 expression at 24 hpi in MG. DNA methylation blockage maintains Oct4 expression at 24 hpi.
To demonstrate a causal relationship between DNA methylation and Oct4 silencing, we intravitreally administered SGI-1027, a DNA-methyltransferase inhibitor which has been shown to block and degrade DNMT1, DNMT3a, and DNMT3b (Yoo et al., 2013; Gros et al., 2015), to a group of mice (n = 10, 5 per condition). We evaluated the expression of Oct4 in GLAST-positive and negative fractions of retinas injured in the presence and absence of SGI-1027. The retinas were extracted 24 h after NMDA injection, since the aforementioned pluripotency-associated marker was silenced at this time in previous experiments. Our results show that SGI-1027 allows the sustained expression of Oct4 after retinal injury, only in the GLAST-positive fraction of retinas (Figure ​(Figure6A).6A). This increase was revealed to be statistically significant (Student's t-test) by qPCR analysis (p < 0.001, when compared to control; p < 0.001 when compared to damaged retinas at 24 hpi without SGI-1027 treatment; Figure ​Figure6B).6B). These results suggest that DNA methylation could be involved in Oct4 silencing at 24 hpi in vivo, and restrict this response to MG. Front Neurosci . 2016 Nov 15:10:523. https://pubmed.ncbi.nlm.nih.gov/27895551/

DNMT3A assay: [2]
DNMT3A enzyme inhibition was adapted from the restriction-based fluorescence assay protocol described by Ceccaldi et al. Briefly, a 5′-labelled biotin oligonucleotide was hybridized to its complementary strand labelled with 6-carboxyfluorescein at the 3′-end and transferred into a 384-well microplate (black Optiplates; PerkinElmer) pre-coated with avidin. The duplex contains a unique CpG site overlapping with a restriction site of a methylation-sensitive restriction enzyme. The human C-terminal DNMT3A (a.a. 623-908), produced as described in Ref., was added to each well (200 ng/well) and mixed with the chemical compounds at the desired concentration and freshly prepared AdoMet (20 μm final concentration) to start the reaction in a total volume of 50 μL. After 1 h incubation at 37 °C, each well was washed three times with phosphate-buffered saline (PBS) containing 0.05 % Tween-20 and NaCl (500 mm) and three more times with phosphate-buffered saline Tween-20 (PBST). Specific fluorescence signals were detected with the methylation-sensitive restriction enzyme HpyCH4IV (New England Biolabs, Ipswich, MA, USA) as described, and measured on a PerkinElmer Envision detector. The percent inhibition was calculated according to Equation (1), where X is the signal determined in the absence of the inhibitor and Y is the signal obtained in the presence of the inhibitor. The ligand concentration at which 50 % inhibition of enyme activity is observed (EC50) was determined by analysis of a concentration range of the test compounds in triplicates. Nonlinear regression fittings with sigmoidal dose–response (variable slope) were performed with Prism 4.03.

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DNMT1 and G9A assay: [2]
His-DNMT1 (182 kDa, human) was cloned, expressed and purified as described by Lee et al.[21] The assays were performed as described in the literature. The reaction was performed in a total reaction volume of 10 μL in low-volume nonbinding surface (NBSTM) 384-well microplates, containing test compound (up to 1 % DMSO), 1 μm of a S-adenosyl-l-methionine (SAM)/[methyl-3H]SAM (3 TBq mmol−1) mix in a ratio of 3:1 (isotopic dilution 1*:3), 0.3 μm of biotinylated hemimethylated DNA duplex (5′-GATmCGCmCGATGmCGmCGAATmCGmCGATmCGATGmCGAT-3′ and BIOT-5′-ATCGCATCGATCGCGATTCGCGCATCGGCGATC-3′), and 90 nm of DNMT1 in methylation buffer (20 mm HEPES (pH 7.2), 1 mm EDTA, 50 mm KCl, 25 μg mL−1 bovine serum albumin). The reaction was incubated at 37 °C for 2 h, then an aliquot (8 μL) was transferred into a streptavidin 96-well scintillant-coated FlashPlate (PerkinElmer) containing 20 μm S-adenosyl-l-homocysteine (SAH; 190 μL) in 50 mM Tris-HCl (pH 7.4). The FlashPlate was agitated at RT for 1 h, washed three times with 200 μL of 0.05 % TweenR-20 in 50 mm Tris-HCl (pH 7.4), and read in 200 μL of 50 mm Tris-HCl (pH 7.4) on TopCount NXT.

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DNMT (CpG methyltransferase) assay[1]
DNA methylase activity was assayed by measuring the incorporation of 3H1-methyl group from S-adenosylmethionine (Ado-Met) into DNA using DE-81 ion exchange filter binding assay with some modifications. The details are provided in Supplementary Data.

Cell culture and treatment with SGI-1027[1]
Human colon carcinoma cell lines (HCT116 and RKO) and human hepatocellular carcinoma cell lines (Hep3B) were obtained from the American Type Culture Collection and cultured in MEM-α according to the supplier’s protocol. Human cervical cancer cell line HeLa, breast cancer cell line MCF7, and prostate cancer cell line LNCaP were obtained from the American Type Culture Collection and cultured in DMEM and RPMI, respectively. Exponentially growing cells were treated with SGI-1027 or DMSO (vehicle) at indicated concentrations for different periods. Control cells received DMSO only.

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Toxicity screening using the rat hepatoma (H4IIE) cell line[1]
Rat hepatoma H4IIE cells were used as the test system. These cells were grown in DMEM supplemented with fetal bovine serum (10%) and calf serum (10%). Cells were seeded into 96-well plates and after 48 h exposed to SGI-1027 at concentrations ranging from 0 to 300 µmol/L. The solubility was determined by Nephalometry techniques immediately after dosing and before harvesting the cells at 24 h. Following the exposure period, the cells or their supernatant (culture medium) were analyzed for changes in cell proliferation (propidium iodide), membrane leakage (α-GST), mitochondrial function [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and cellular ATP], oxidative stress (intracellular GSH and 8-isoprostane), and apoptosis (caspase-3; ref. 25). The half-maximal toxic concentration (TC50) was determined from the dose-response curves.

SGI-1027 intravitreal injection.
DNA methyltransferase inhibitor SGI-1027 (Sigma) was dissolved in 0.05% DMSO, and injected intravitreally following the same procedure as NMDA injection (10 μM in 2 μl), 24 h before retinal injury. Front Neurosci . 2016 Nov 15:10:523. https://pubmed.ncbi.nlm.nih.gov/27895551/

[1]. A new class of quinoline-based DNA hypomethylating agents reactivates tumor suppressor genes by blocking DNA methyltransferase 1 activity and inducing its degradation. Cancer Res. 2009 May 15;69(10):4277-85.

[2]. Design, synthesis and biological evaluation of 4-amino-N- (4-aminophenyl)benzamide analogues of quinoline-based SGI-1027 as inhibitors of DNA methylation. ChemMedChem. 2014 Mar;9(3):590-601.

Reactivation of silenced tumor suppressor genes by 5-azacytidine (Vidaza) and its congener 5-aza-2′-deoxycytidine (decitabine) has provided an alternate approach to cancer therapy. We have shown previously that these drugs selectively and rapidly induce degradation of the maintenance DNA methyltransferase (DNMT) 1 by a proteasomal pathway. Because the toxicity of these compounds is largely due to their incorporation into DNA, it is critical to explore novel, nonnucleoside compounds that can effectively reactivate the silenced genes. Here, we report that a quinoline-based compound, designated SGI-1027, inhibits the activity of DNMT1, DNMT3A, and DNMT3B as well M. SssI with comparable IC50 (6–13 µ mol/L) by competing with S-adenosylmethionine in the methylation reaction. Treatment of different cancer cell lines with SGI-1027 resulted in selective degradation of DNMT1 with minimal or no effects on DNMT3A and DNMT3B. At a concentration of 2.5 to 5 µmol/L (similar to that of decitabine), complete degradation of DNMT1 protein was achieved within 24 h without significantly affecting its mRNA level. MG132 blocked SGI-1027–induced depletion of DNMT1, indicating the involvement of proteasomal pathway. Prolonged treatment of RKO cells with SGI-1027 led to demethylation and reexpression of the silenced tumor suppressor genes P16, MLH1, and TIMP3. Further, this compound did not exhibit significant toxicity in a rat hepatoma (H4IIE) cell line. This study provides a novel class of DNA hypomethylating agents that have the potential for use in epigenetic cancer therapy.[1]

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Quinoline derivative SGI-1027 (N-(4-(2-amino-6-methylpyrimidin-4-ylamino)phenyl)-4-(quinolin-4-ylamino)benzamide) was first described in 2009 as a potent inhibitor of DNA methyltransferase (DNMT) 1, 3A and 3B. Based on molecular modeling studies, performed using the crystal structure of Haemophilus haemolyticus cytosine-5 DNA methyltransferase (MHhaI C5 DNMT), which suggested that the quinoline and the aminopyridimine moieties of SGI-1027 are important for interaction with the substrates and protein, we designed and synthesized 25 derivatives. Among them, four compounds—namely the derivatives 12, 16, 31 and 32—exhibited activities comparable to that of the parent compound. Further evaluation revealed that these compounds were more potent against human DNMT3A than against human DNMT1 and induced the re-expression of a reporter gene, controlled by a methylated cytomegalovirus (CMV) promoter, in leukemia KG-1 cells. These compounds possessed cytotoxicity against leukemia KG-1 cells in the micromolar range, comparable with the cytotoxicity of the reference compound, SGI-1027. Structure–activity relationships were elucidated from the results. First, the presence of a methylene or carbonyl group to conjugate the quinoline moiety decreased the activity. Second, the size and nature of the aromatic or heterocycle subsitutents effects inhibition activity: tricyclic moieties, such as acridine, were found to decrease activity, while bicyclic substituents, such as quinoline, were well tolerated. The best combination was found to be a bicyclic substituent on one side of the compound, and a one-ring moiety on the other side. Finally, the orientation of the central amide bond was found to have little effect on the biological activity. This study provides new insights in to the structure-activity relationships of SGI-1027 and its derivative.[2]

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Müller glia (MG) is the most abundant glial type in the vertebrate retina. Among its many functions, it is capable of responding to injury by dedifferentiating, proliferating, and differentiating into every cell types lost to damage. This regenerative ability is notoriously absent in mammals. We have previously reported that cultured mammalian MG undergoes a partial dedifferentiation, but fails to fully acquire a progenitor phenotype and differentiate into neurons. This might be explained by a mnemonic mechanism comprised by epigenetic traits, such as DNA methylation. To achieve a better understanding of this epigenetic memory, we studied the expression of pluripotency-associated genes, such as Oct4, Nanog, and Lin28, which have been reported as necessary for regeneration in fish, at early times after NMDA-induced retinal injury in a mouse experimental model. We found that although Oct4 is expressed rapidly after damage (4 hpi), it is silenced at 24 hpi. This correlates with a significant decrease in the DNA methyltransferase Dnmt3b expression, which returns to basal levels at 24 hpi. By MS-PCR, we observed a decrease in Oct4 methylation levels at 4 and 12 hpi, before returning to a fully methylated state at 24 hpi. To demonstrate that these changes are restricted to MG, we separated these cells using a GLAST antibody coupled with magnetic beads. Finally, intravitreous administration of the DNA-methyltransferase inhibitor SGI-1027 induced Oct4 expression at 24 hpi in MG. Our results suggest that mammalian MG injury-induced dedifferentiation could be restricted by DNA methylation, which rapidly silences Oct4 expression, preventing multipotency acquisition.Front Neurosci . 2016 Nov 15:10:523. https://pubmed.ncbi.nlm.nih.gov/27895551/

C27H23N7O

461.52

461.196

C, 70.27; H, 5.02; N, 21.24; O, 3.47

1020149-73-8

24858111

Light yellow to yellow solid powder

1.4±0.1 g/cm3

>280℃

1.789

4.52

4

7

6

35

676

0

QSYLKMKIVWJAAK-UHFFFAOYSA-N

InChI=1S/C27H23N7O/c1-17-16-25(34-27(28)30-17)32-20-10-12-21(13-11-20)33-26(35)18-6-8-19(9-7-18)31-24-14-15-29-23-5-3-2-4-22(23)24/h2-16H,1H3,(H,29,31)(H,33,35)(H3,28,30,32,34)

N-(4-((2-amino-6-methylpyrimidin-4-yl)amino)phenyl)-4-(quinolin-4-ylamino)benzamide

DNA Methyltransferase Inhibitor II; SGI-1027; N-(4-((2-amino-6-methylpyrimidin-4-yl)amino)phenyl)-4-(quinolin-4-ylamino)benzamide; DNA Methyltransferase Inhibitor II; N-[4-[(2-amino-6-methylpyrimidin-4-yl)amino]phenyl]-4-(quinolin-4-ylamino)benzamide; CHEMBL2336409; SGI 1027; SGI1027;

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 (5.42 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 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 (5.42 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 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 (5.42 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.)