SGI-1027   中文名称: SGI 1027

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

SGI-1027 是一种 DNMT 抑制剂，当使用聚（dI-dC）作为底物时，对 DNMT3B、DNMT3A 和 DNMT1 的 IC50 分别为 7.5 μM、8 μM 和 12.5 μM。 SGI-1027 针对 DNMT1（半甲基化 DNA）的 IC50 为 6 μM。 SGI-1027（1、2.5 或 5 μM）可促进多种人类癌细胞系中 DNMT1 的优先降解，但对大鼠肝癌细胞几乎没有或没有细胞毒性作用，并且不会引发大鼠肝癌细胞凋亡 [1]。 SGI-1027 对 hDNMT3A 的 EC50 为 0.9 μM，对 KG-1 细胞具有细胞毒性，EC50 为 4.4 μM [2]。

玻璃体内注射DNA甲基转移酶抑制剂SGI-1027诱导MG在24小时表达Oct4。DNA甲基化阻断使Oct4在24小时保持表达。
为了证明DNA甲基化和Oct4沉默之间的因果关系，我们向一组小鼠（n=10，每种情况5只）玻璃体内注射了SGI-1027，这是一种DNA甲基转移酶抑制剂，已被证明可以阻断和降解DNMT1、DNMT3a和DNMT3b（Yoo等人，2013；Gros等人，2015）。我们评估了在SGI-1027存在和不存在的情况下，GLAST阳性和阴性视网膜部分Oct4的表达。在注射NMDA后24小时提取视网膜，因为在之前的实验中，上述多能性相关标志物在这个时候被沉默了。我们的结果表明，SGI-1027允许Oct4在视网膜损伤后持续表达，仅在视网膜GLAST阳性部分（图（图6A）.6A）。qPCR分析显示，这一增加具有统计学意义（学生t检验）（与对照组相比，p<0.001；与未经SGI-1027治疗的24 hpi受损视网膜相比，p<0.001）。图6B）。这些结果表明，DNA甲基化可能参与体内24小时Oct4沉默，并限制了这种对MG的反应。2016年11月15日15:10:523。https://pubmed.ncbi.nlm.nih.gov/27895551/

DNMT3A测定：[2]
DNMT3A酶抑制改编自Ceccaldi等人描述的基于限制性的荧光测定方案。简而言之，将5′标记的生物素寡核苷酸与其3′端标记有6-羧基荧光素的互补链杂交，并转移到预涂有抗生物素蛋白的384孔微孔板（黑色Optiplates；PerkinElmer）中。该双链包含一个独特的CpG位点，与甲基化敏感限制性内切酶的限制性位点重叠。将参考文献中所述制备的人C末端DNMT3A（a.a.623-908）加入每个孔（200 ng/孔）中，并与所需浓度的化合物和新鲜制备的AdoMet（最终浓度为20μm）混合，以启动总体积为50μL的反应。在37°C下孵育1小时后，用含有0.05%吐温-20和NaCl（500 mm）的磷酸盐缓冲盐水（PBS）洗涤每个孔三次，再用磷酸盐缓冲盐水吐温-20（PBST）洗涤三次。如所述，用甲基化敏感限制性内切酶HpyCH4IV（New England Biolabs，Ipswich，MA，USA）检测特定的荧光信号，并在PerkinElmer Envision检测器上测量。根据方程式（1）计算抑制百分比，其中X是在没有抑制剂的情况下确定的信号，Y是在抑制剂存在的情况下获得的信号。 通过分析三份测试化合物的浓度范围来确定观察到50%抑制enyme活性的配体浓度（EC50）。使用Prism 4.03进行了S形剂量反应（可变斜率）的非线性回归拟合。

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DNMT1和G9A测定：[2]
His-DNMT1（182 kDa，人）按照Lee等人[21]的描述进行克隆、表达和纯化。反应在总反应体积为10μL的低体积非结合表面（NBSTM）384孔微孔板中进行，该微孔板含有测试化合物（高达1%DMSO）、1μm的S-腺苷-1-甲硫氨酸（SAM）/[甲基-3H]SAM（3 TBq mmol−1）混合物，比例为3:1（同位素稀释1*:3）、0.3μm的生物素化半甲基化DNA双链体（5′-GATmCGCmCGATGmCGmCGAATmCCGATmCGATGmGCAT-3′和BIOT-5′-ATCGCATCGATCGCGCGATTCGCATCGGCGATC-3′）和90 nm的DNMT 1在甲基化缓冲液中（20 mm HEPES（pH 7.2），1 mm EDTA，50 mm KCl，25μg mL-1牛血清白蛋白）。将反应物在37°C下孵育2小时，然后将等分试样（8μL）转移到链霉抗生物素蛋白96孔闪烁剂涂层的FlashPlate（PerkinElmer）中，该FlashPlate在50 mM Tris-HCl（pH 7.4）中含有20μm S-腺苷-L-同型半胱氨酸（SAH；190μL）。将FlashPlate在室温下搅拌1小时，用200μL 0.05%吐温R-20在50 mm Tris-HCl（pH 7.4）中洗涤三次，并在TopCount NXT上读取200μL 50 mm Tris-HaCl（pH 7.3）的读数。

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DNMT（CpG甲基转移酶）测定[1]
通过使用DE-81离子交换滤膜结合分析法测量S-腺苷甲硫氨酸（Ado-Met）的3H1甲基掺入DNA，并进行一些修改，来测定DNA甲基化酶活性。详细信息见补充数据。

细胞培养和SGI-1027处理[1]
人结肠癌细胞系（HCT116和RKO）和人肝细胞癌细胞系（Hep3B）从美国典型培养物保藏中心获得，并根据供应商的方案在MEM-α中培养。人宫颈癌症细胞系HeLa、癌症细胞系MCF7和癌症前列腺细胞系LNCaP从美国典型培养物保藏中心获得，并分别在DMEM和RPMI中培养。指数生长的细胞用指定浓度的SGI-1027或DMSO（载体）处理不同时间。对照细胞仅接受DMSO。

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使用大鼠肝癌（H4IIE）细胞系进行毒性筛选[1]
使用大鼠肝癌H4IIE细胞作为测试系统。这些细胞在添加了胎牛血清（10%）和小牛血清（10%的DMEM）的DMEM中生长。将细胞接种到96孔板中，48小时后暴露于浓度在0至300µmol/L范围内的SGI-1027。给药后和24小时收获细胞前，立即通过肾功能测定技术测定溶解度。暴露期结束后，分析细胞或其上清液（培养基）的细胞增殖（碘化丙啶）、膜渗漏（α-GST）、线粒体功能[3-（4,5-二甲基噻唑-2-基）-2,5-二苯基溴化四唑和细胞ATP]、氧化应激（细胞内GSH和8-异前列烷）和凋亡（半胱氨酸天冬氨酸蛋白酶-3；参考文献25）的变化。根据剂量反应曲线确定半最大毒性浓度（TC50）。

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

125.3

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)

配方 1 中的溶解度: 2.5 mg/mL (5.42 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浮液；超声助溶。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: 2.5 mg/mL (5.42 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (5.42 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。