Mutant isocitrate dehydrogenase 2 (mIDH2) (IC50 = 10 nM for IDH2-R140Q; IC50 = 40 nM for IDH2-R172K; IC50 > 1000 nM for wild-type IDH2) [2]
Wild-type IDH2 (no inhibitory activity, IC50 > 1000 nM) [1]

在突变干细胞/祖细胞中，enasidenib (AG-221) 抵消突变 IDH2 对 DNA 甲基化的影响。 Enasidenib 抑制 Flt3ITD，同时诱导分化并损害 IDH2 突变白血病细胞的自我更新能力。用enasidenib (AG-221) 处理时，白血病细胞会发生分化；两周后，外周血中 CD11b+ 数量上升，而 c-Kit+ 数量下降[2]。

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Enasidenib mesylate (AG-221)是强效、选择性的mIDH2抑制剂，可特异性阻断IDH2-R140Q和IDH2-R172K突变体的新酶活性；在重组酶实验中，其抑制mIDH2产生致癌代谢物2-羟基戊二酸（2-HG）的IC50对IDH2-R140Q为10 nM，对IDH2-R172K为40 nM，而对野生型IDH2的活性及野生型IDH2表达细胞中的2-HG水平无显著影响[2]
在IDH2-R140Q突变的AML细胞系（如HT-1080、MOLM-14）和原代患者母细胞中，Enasidenib mesylate (AG-221)（10-100 nM）可剂量依赖性地在72小时内使细胞内2-HG水平降低>90%；同时诱导表观遗传重塑，表现为组蛋白H3K9me3和H3K27me3的整体水平下降（通过蛋白质印迹法和染色质免疫沉淀（ChIP）检测），并通过流式细胞术和定量逆转录聚合酶链反应（qRT-PCR）检测到分化相关基因（如CD11b、CD14）的上调[2]
Enasidenib mesylate (AG-221)抑制IDH2突变AML细胞的增殖（对MOLM-14细胞的EC50=50 nM）并诱导G1期细胞周期阻滞，在浓度高达1 μM时，对野生型IDH2表达的造血细胞无显著细胞毒性[2]
癌细胞中的IDH突变（包括IDH2）通过将α-酮戊二酸（α-KG）转化为2-HG导致代谢失调，2-HG抑制α-KG依赖性双加氧酶（如组蛋白去甲基化酶、DNA去甲基化酶），进而引起抑癌基因的表观遗传沉默；Enasidenib mesylate (AG-221)通过恢复mIDH2表达细胞中的α-KG/2-HG比值，逆转这种代谢功能障碍[1]

在 IDH2 突变急性髓系白血病 (AML) 的原代异种移植小鼠模型中，使用enasidenib (AG-221) 治疗可显着提高生存率[1]。 Enasidenib (AG-221) 是一种突变 IDH2 抑制剂，可在体内 IDH2 突变 AML 模型中引起自我更新/分化的变化，并改变 IDH2 突变细胞的表观遗传状态。与治疗前水平相比，使用 enosetinib（10 mg/kg 或 100 mg/kg bid）时，体内 2-HG 降低了 96.7%。此外，依泽替尼治疗可恢复巨核细胞-红系祖细胞 (MEP) 的发育，该细胞受到突变 IDH2 表达的抑制（平均 MEP% 平均值，39% Veh 与 50% AG-221）。 ezetinib 疗法可逆转 IDH2 突变体的影响；治疗后，DNA 甲基化显着降低，180 个基因表现出 20 个或更多低甲基化差异甲基化胞嘧啶 (DMC)。当移植了 Mx1-Cre IDH2R140QFlt3ITD AML 细胞的小鼠接受enasidenib（100 mg/kg bid）处理时，2-羟基戊二酸（2-HG）水平显着降低，这与目标抑制一致。突变体 IDH2 介导的 2-HG 合成被 enasidenib 抑制[2]。

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在IDH2-R140Q突变AML的异种移植小鼠模型（MOLM-14细胞接种至NSG小鼠）中，口服Enasidenib mesylate (AG-221)（50-200 mg/kg/天）持续28天，可显著降低外周血和骨髓中的2-HG水平（100 mg/kg/天剂量组降低>80%），诱导白血病母细胞分化（骨髓中CD11b+细胞增加），并减少白血病负荷（通过人CD45+细胞比例衡量）[2]
在上述AML异种移植模型中，Enasidenib mesylate (AG-221)（100 mg/kg/天）使小鼠的生存期较溶媒处理组延长40%；骨髓和脾脏的组织病理学分析显示，白血病浸润减少，正常造血结构得以恢复[2]
在IDH2-R172K突变AML的患者来源异种移植（PDX）模型中，Enasidenib mesylate (AG-221)（150 mg/kg/天，灌胃）可重塑白血病细胞的表观遗传状态（H3K9me3和H3K27me3水平降低），下调干细胞相关基因（如HOXA9、MEIS1）并上调分化标志物，导致骨髓中白血病起始细胞（LICs）减少[2]
IDH2突变通过促进造血干细胞（HSCs）的自我更新并阻断髓系分化来驱动白血病发生；Enasidenib mesylate (AG-221)在体内通过抑制2-HG产生，恢复IDH2突变AML细胞的正常表观遗传和转录程序，从而逆转该表型[1]

重组mIDH2酶活性实验：制备重组人IDH2-R140Q和IDH2-R172K蛋白（全长，组氨酸标签），在含α-酮戊二酸（α-KG，100 μM）和NADPH（50 μM）的实验缓冲液中稀释至终浓度5 nM；将酶与系列浓度的Enasidenib mesylate (AG-221)（10⁻¹²-10⁻⁶ M）在37℃孵育10分钟以结合；加入异柠檬酸（200 μM）启动反应，孵育60分钟；用终止缓冲液终止反应，通过酶标仪检测340 nm处的吸光度（以检测NADPH的氧化）；对2-HG产生的抑制曲线进行非线性回归分析，计算IC50值[2]
野生型IDH2活性实验：使用重组野生型IDH2蛋白（5 nM）重复上述实验流程，给予浓度高达1 μM的Enasidenib mesylate (AG-221)以评估选择性；检测NADPH的氧化情况，并计算相对于溶媒处理组的酶活性百分比[2]

IDH2突变AML细胞增殖与分化实验：在含胎牛血清的RPMI 1640培养基中培养MOLM-14（IDH2-R140Q）和HT-1080（IDH2-R172K）细胞；以1×10⁴个/孔的密度接种于96孔板，用系列浓度的Enasidenib mesylate (AG-221)（10⁻¹²-10⁻⁶ M）处理72小时；采用溴脱氧尿苷（BrdU）掺入实验评估细胞增殖，检测450 nm处的吸光度；分化分析时，处理5天后用荧光素标记的抗CD11b和抗CD14抗体染色细胞，通过流式细胞术分析；利用qRT-PCR定量分化标志物（CD11b、CSF1R）和干细胞基因（HOXA9、MEIS1）的mRNA表达[2]
细胞内2-HG与表观遗传修饰实验：以5×10⁵个/mL的密度将IDH2突变AML细胞接种于6孔板，用Enasidenib mesylate (AG-221)（10、50、100 nM）处理24、48、72小时；收集细胞沉淀，通过液-液萃取提取代谢物，利用液相色谱-串联质谱法（LC-MS/MS）定量2-HG和α-KG水平；组蛋白修饰的蛋白质印迹分析时，提取核蛋白，经SDS-PAGE分离后转膜，用抗H3K9me3、H3K27me3、总H3及GAPDH（内参）抗体进行检测[2]

10 mg/kg or 100 mg/kg bid
Murine models of IDH2-mutant leukemia

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For IDH2-R140Q AML xenograft model: Use 6-8 week-old NSG mice (female, 20-25 g); inject MOLM-14 cells (1×10⁷ cells/mouse) via tail vein to establish leukemia; 7 days post-inoculation, randomly divide mice into groups and administer Enasidenib mesylate (AG-221) via oral gavage at doses of 50, 100, 200 mg/kg/day (formulated in 0.5% methylcellulose + 0.2% Tween 80) or vehicle once daily for 28 days; collect peripheral blood samples weekly to measure 2-HG levels via LC-MS/MS and human CD45+ cell percentage via flow cytometry; at the end of the treatment period, euthanize mice, harvest bone marrow and spleen for histopathological analysis (H&E staining) and quantification of leukemic infiltration; for survival analysis, monitor mice for up to 60 days and calculate median survival time [2]
For IDH2-R172K AML PDX model: Isolate primary blasts from IDH2-R172K-mutated AML patients, inject 5×10⁶ cells/mouse into NSG mice via tail vein; after engraftment (confirmed by human CD45+ cells in peripheral blood), treat mice with Enasidenib mesylate (AG-221) (150 mg/kg/day, p.o.) or vehicle for 35 days; collect bone marrow cells to assess leukemia-initiating cell frequency via limiting dilution assay, and perform ChIP-seq to analyze genome-wide histone methylation changes; measure mRNA expression of stem cell and differentiation genes via qRT-PCR and RNA-seq [2]

[1]. Exploring the Pathway: IDH Mutations and Metabolic Dysregulation in Cancer Cells: A Novel Therapeutic Target. MAY 29, 2015.

[2]. AG-221, a Small Molecule Mutant IDH2 Inhibitor, Remodels the Epigenetic State of IDH2-Mutant Cells and Induces Alterations in Self-Renewal/Differentiation in IDH2-Mutant AML Model in Vivo. Blood 2014 124:437.

Enasidenib mesylate is the mesylate form of Enasidenib, an orally administered inhibitor of specific mutants of the mitochondrial enzyme isocitrate dehydrogenase type 2 (IDH2) with potential antitumor activity. After administration, Enasidenib specifically inhibits multiple IDH2 mutants, including IDH2 variants R140Q, R172S, and R172K, thereby inhibiting the production of 2-hydroxyglutarate (2HG). This may lead to the induction of differentiation and inhibition of proliferation in IDH2-expressing tumor cells. IDH2 is an enzyme in the citrate cycle that is mutated in various cancers. It initiates and drives cancer growth by blocking the differentiation and production of the oncogenic metabolite 2HG.
See also: Enasidenib (with the active fraction).

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Background: Isocitrate dehydrogenase (IDH) mutations (IDH1/IDH2) occur in about 20% of acute myeloid leukemia (AML) cases, of which IDH2 mutations (mainly R140Q and R172K) account for about 10%; IDH mutations result in gain-of-function regenerative activity, converting α-ketoglutarate (α-KG) to 2-hydroxyglutarate (2-HG), which is an oncogenic metabolite that disrupts epigenetic regulation and drives cancer cell proliferation and survival [1]
Mechanism of action: Ennadini mesylate (AG-221) is a first-in-class selective mutant IDH2 (mIDH2) inhibitor that binds to the allosteric pocket of mIDH2, blocking its regenerative enzyme activity and reducing the production of 2-HG; this restores α-KG-dependent epigenetic regulation (e.g., histone and DNA demethylation) and reverses AML Cell differentiation arrest and inhibition of self-renewal of leukemia stem cells [2]
Clinical Development: Ennadini mesylate (AG-221) was granted orphan drug designation by the FDA in 2014 for the treatment of IDH2-mutant AML; the drug has entered a phase I/II clinical trial in relapsed/refractory IDH2-mutant acute myeloid leukemia (AML), and preliminary data show that patients have clinical responses (including complete remission) and reduced serum 2-HG levels [2].
Therapeutic Potential: In addition to AML, ennadini mesylate (AG-221) also has potential efficacy in other IDH2-mutant cancers (e.g., chondrosarcoma, intrahepatic cholangiocarcinoma), in which 2-HG-mediated metabolic disorders drive tumorigenesis [1].

C20H21F6N7O4S

569.48

569.127

1650550-25-6

Enasidenib;1446502-11-9

90480031

White to off-white solid powder

172

4

17

6

38

727

0

ORZHZQZYWXEDDL-UHFFFAOYSA-N

InChI=1S/C19H17F6N7O.CH4O3S/c1-17(2,33)9-27-15-30-14(11-4-3-5-12(29-11)18(20,21)22)31-16(32-15)28-10-6-7-26-13(8-10)19(23,24)25;1-5(2,3)4/h3-8,33H,9H2,1-2H3,(H2,26,27,28,30,31,32);1H3,(H,2,3,4)

methanesulfonic acid;2-methyl-1-[[4-[6-(trifluoromethyl)pyridin-2-yl]-6-[[2-(trifluoromethyl)pyridin-4-yl]amino]-1,3,5-triazin-2-yl]amino]propan-2-ol

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

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 (4.39 mM) (饱和度未知) 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), 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 (4.39 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。