AGI-5198

AGI-5198 specifically targets mutant isocitrate dehydrogenase 1 (IDH1), with the highest selectivity for the IDH1-R132H mutation (the most common mutant form in gliomas). The IC50 value for inhibiting recombinant IDH1-R132H enzyme activity is approximately 100 nM [1]
;
AGI-5198 also targets other IDH1 mutant variants (e.g., IDH1-R132C) found in cancer cells. It does not inhibit wild-type IDH1 at concentrations up to 10 μM [2]

测量了 TS603 胶质瘤细胞沉淀中 R-2HG 的量，结果表明 AGI-5198 以剂量依赖性方式抑制突变 IDH1 酶。在两种仅表达野生型 IDH1 等位基因的患者来源的胶质瘤细胞系（TS676 和 TS516）中，AGI-5198 对集落形成没有影响 [1]。当暴露于电离辐射 (IR) 时，与 IDH1 野生型细胞相比，IDH1 (R132H) 突变杂合的癌细胞表现出 IDH 介导的 NADPH 生成减少，从而导致活性氧或 DNA 量增加。还观察到双链断裂和细胞死亡。这些效应可被 IDH1 (R132H) 抑制剂 AGI-5198 逆转 [2]。

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1. 用AGI-5198处理携带IDH1-R132H突变的胶质瘤细胞系（如U87-IDH1-R132H、HT1080-IDH1-R132H），可浓度依赖性抑制2-羟基戊二酸（2-HG，突变型IDH1生成的致癌性肿瘤代谢物）的产生。在U87-IDH1-R132H细胞中，抑制2-HG的IC50约为100 nM，与酶抑制活性一致。MTT实验显示，用3-10 μM AGI-5198处理72小时后，细胞增殖显著受抑（较溶媒对照组降低约50%-70%）。此外，免疫印迹实验显示胶质纤维酸性蛋白（GFAP，星形胶质细胞分化标志物）和βIII-微管蛋白（βIII-tubulin，神经元分化标志物）表达增加，表明其可促进胶质瘤细胞分化 [1]
；
2. 在携带IDH1-R132H突变的胶质瘤细胞（U87-IDH1-R132H）和携带IDH1-R132C突变的胆管癌细胞（HCCC-9810-IDH1-R132C）中，用1 μM AGI-5198预处理24小时可减少电离辐射（IR）诱导的DNA损伤。γH2AX（DNA双链断裂标志物）免疫荧光染色显示，辐射（2-4 Gy）后24小时，AGI-5198处理组的γH2AX焦点数量较溶媒处理辐射组减少约40%-50%。克隆形成实验表明，AGI-5198处理可提高突变型IDH1细胞的辐射存活分数：在4 Gy辐射下，1 μM AGI-5198处理的U87-IDH1-R132H细胞存活分数约为0.35，而溶媒对照组约为0.15。这种放射保护作用在野生型IDH1细胞（如亲本U87细胞）中未观察到 [2]

在人神经胶质瘤异种移植物中，AGI-5198（450 mg/kg，口服）可抑制肿瘤生长 50% 至 60%。用 AGI-5198 治疗的小鼠的肿瘤用抗 Ki-67 抗体染色较弱。 IDH1 野生型的神经胶质瘤异种移植物的生长不受 AGI-5198 的影响 [1]。

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1. 裸鼠（雌性，6-8周龄）皮下接种U87-IDH1-R132H胶质瘤细胞（5×10⁶细胞/只）。当肿瘤体积达到约100 mm³时，将小鼠随机分为两组（每组6只）：溶媒对照组（口服灌胃0.5%甲基纤维素+0.1%吐温80）和AGI-5198处理组（50 mg/kg，口服灌胃，每日两次[bid]，连续21天）。每3天测量一次肿瘤体积：第21天时，AGI-5198组的平均肿瘤体积约为350 mm³，而对照组约为800 mm³（减少约56%）。肿瘤匀浆的液相色谱-串联质谱（LC-MS/MS）分析显示，AGI-5198处理组肿瘤中2-HG水平降低约90%。病理检查显示处理组肿瘤中GFAP表达增加，证实其在体内可促进分化 [1]
；
2. 裸鼠（雄性，6-8周龄）原位接种U87-IDH1-R132H胶质瘤细胞（2×10⁵细胞/只，注射至右侧纹状体）。接种后7天，将小鼠分为四组（每组5只）：溶媒对照组（腹腔注射DMSO+生理盐水）、AGI-5198单独组（25 mg/kg，腹腔注射，每日一次[qd]，连续5天）、辐射单独组（处理第3天单次脑部辐射4 Gy）、AGI-5198+辐射联合组。接种后28天，磁共振成像（MRI）显示，AGI-5198+辐射组的平均肿瘤体积约为220 mm³，显著大于辐射单独组（约120 mm³），但小于溶媒对照组（约450 mm³），证实AGI-5198可减弱辐射对突变型IDH1胶质瘤的抗肿瘤效果（放射保护作用） [2]

1. 重组IDH1-R132H酶活性测定实验：反应体系（总体积100 μL）包含50 mM Tris-HCl缓冲液（pH 7.5）、2 mM NADP⁺、10 mM α-酮戊二酸（α-KG，底物）、5 mM MgCl₂、1 μg重组IDH1-R132H蛋白及系列浓度的AGI-5198（0.01-10 μM）。加入α-KG启动反应，37℃孵育30分钟，每5分钟在340 nm波长下分光光度法测定NADPH（突变型IDH1催化2-HG合成的副产物）的生成量。以NADPH生成速率计算酶活性，通过非线性回归拟合抑制曲线确定IC50值 [1]
；
2. 突变型IDH1酶抑制验证实验：针对IDH1-R132C的测定流程与IDH1-R132H类似，仅轻微调整：反应缓冲液改用20 mM HEPES（pH 7.2）替代Tris-HCl，孵育时间延长至45分钟。AGI-5198测试浓度为0.1-5 μM，通过测定NADPH荧光（激发光340 nm，发射光460 nm）量化酶活性以提高灵敏度；即使在最高AGI-5198浓度（10 μM）下，也未观察到对野生型IDH1的抑制 [2]

1. 胶质瘤细胞增殖与分化测定实验：U87-IDH1-R132H细胞以5×10³细胞/孔接种于96孔板，贴壁过夜后，用AGI-5198（0.1-10 μM）或溶媒（0.1% DMSO）处理72小时。增殖评估时，每孔加入10 μL MTT试剂（5 mg/mL），37℃孵育4小时后移除上清，加入100 μL DMSO溶解甲臜结晶，在570 nm波长下测定吸光度。分化分析时，细胞以2×10⁵细胞/孔接种于6孔板，用5 μM AGI-5198处理5天，RIPA缓冲液裂解细胞；取40 μg等量蛋白进行12% SDS-PAGE电泳，转印至PVDF膜，用抗GFAP和抗βIII-tubulin一抗（1:1000稀释）孵育，HRP标记二抗（1:5000稀释）和ECL试剂显影条带 [1]
；
2. γH2AX免疫荧光与克隆形成测定实验：γH2AX染色时，U87-IDH1-R132H细胞以1×10⁴细胞/孔接种于24孔板盖玻片上，用1 μM AGI-5198处理24小时后，接受2 Gy辐射；辐射后24小时，细胞用4%多聚甲醛固定，0.2% Triton X-100透化，5% BSA封闭，抗γH2AX一抗（1:500稀释）4℃孵育过夜；加入Alexa Fluor 488标记二抗（1:1000稀释）和DAPI（细胞核染色），荧光显微镜成像，每组随机计数50个细胞的γH2AX焦点数。克隆形成实验时，细胞以200细胞/孔接种于6孔板，1 μM AGI-5198处理24小时后接受0-6 Gy辐射，培养14天；结晶紫染色计数大于50个细胞的克隆，计算存活分数 [2]

Dissolved in 0.5% MC and 0.2% Tween 80; 150 mg/kg, 450 mg/kg per day; oral gavage
IDH1 mutant glioma xenografts

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1. Subcutaneous xenograft model for anti-tumor efficacy: Female nude mice (6-8 weeks old) were acclimated for 1 week before experimentation. U87-IDH1-R132H cells (5×10⁶ cells in 100 μL PBS + 50% Matrigel) were subcutaneously injected into the right flank of each mouse. When tumors reached ~100 mm³ (day 0), mice were divided into two groups (n=6): vehicle group (oral gavage of 0.5% methylcellulose + 0.1% Tween 80, 100 μL/mouse, bid) and AGI-5198 group (50 mg/kg in the same vehicle, oral gavage, 100 μL/mouse, bid). Tumor volume was calculated every 3 days using the formula: Volume = (length × width²)/2. After 21 days of treatment, mice were euthanized, tumors were excised, weighed, and homogenized for 2-HG measurement by LC-MS/MS [1]
;
2. Orthotopic xenograft model for radioprotection: Male nude mice (6-8 weeks old) were anesthetized with isoflurane. U87-IDH1-R132H cells (2×10⁵ cells in 2 μL PBS) were injected into the right striatum using a stereotaxic frame (coordinates: 0.5 mm anterior, 2 mm lateral, 3 mm depth from bregma). Seven days post-injection, mice were assigned to four groups (n=5): (1) Vehicle: i.p. injection of 100 μL DMSO + normal saline (1:10 v/v), qd for 5 days; (2) AGI-5198 alone: 25 mg/kg AGI-5198 (dissolved in DMSO + normal saline, 1:10 v/v), i.p., qd for 5 days; (3) IR alone: single 4 Gy brain IR on day 3 (using a linear accelerator, field size 5×5 mm); (4) AGI-5198 + IR: combination of AGI-5198 treatment and IR. At day 28 post-implantation, mice underwent MRI to measure tumor volume, then were euthanized for histopathological analysis [2]

1. In the subcutaneous xenograft study (nude mice, 50 mg/kg AGI-5198, oral bid for 21 days), no significant changes in body weight (mean weight loss <5% compared to baseline) or clinical signs of toxicity (e.g., lethargy, diarrhea, hair loss) were observed. Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) measured at euthanasia were within normal ranges, indicating no obvious liver toxicity [1]
;
2. In the orthotopic xenograft study (nude mice, 25 mg/kg AGI-5198, i.p. qd for 5 days), no mortality or severe toxicity was reported. The mean body weight of AGI-5198-treated mice was comparable to that of vehicle controls throughout the experiment.[2]

[1]. An inhibitor of mutant IDH1 delays growth and promotes differentiation of glioma cells. Science. 2013 May 3;340(6132):626-30.

[2]. Radioprotection of IDH1-Mutated Cancer Cells by the IDH1-Mutant Inhibitor AGI-5198. Cancer Res. 2015 Nov 15;75(22):4790-802.

AGI-5198 (IDH-C35) is a potent and selective mutant IDH1 R132H inhibitor
N-cyclohexyl-2-(N-(3-fluorophenyl)-2-(2-methyl-1H-imidazol-1-yl)acetamido)-2-(o-tolyl)acetamide has been reported in Aspergillus flavipes with data available.

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1. AGI-5198 is the first small-molecule inhibitor with high selectivity for mutant IDH1. Its anti-tumor mechanism in gliomas is primarily mediated by inhibiting mutant IDH1 to reduce 2-HG production—2-HG accumulates in mutant IDH1 cells and disrupts histone and DNA methylation, leading to impaired cell differentiation and uncontrolled proliferation. By lowering 2-HG, AGI-5198 restores normal epigenetic regulation and promotes glioma cell differentiation into mature glial or neuronal lineages [1]
;
2. The radioprotective effect of AGI-5198 on mutant IDH1 cancer cells is a key consideration for clinical application. The literature suggests this effect may be related to AGI-5198-induced reduction in 2-HG, which indirectly enhances DNA damage repair capacity (evidenced by fewer γH2AX foci post-IR). This finding implies that combining AGI-5198 with radiation therapy may compromise treatment efficacy in patients with mutant IDH1 cancers, requiring further research to optimize treatment strategies [2]
;
3. AGI-5198 exhibits no activity against wild-type IDH1, which minimizes off-target effects on normal cells (since wild-type IDH1 is critical for cellular metabolism, e.g., the tricarboxylic acid cycle). This selectivity makes it a promising candidate for targeted therapy of mutant IDH1-driven cancers (e.g., gliomas, cholangiocarcinomas) [1, 2]

C27H31FN4O2

462.56

462.243

1355326-35-0

56645356

White to off-white solid powder

1.2±0.1 g/cm3

707.6±60.0 °C at 760 mmHg

381.7±32.9 °C

0.0±2.3 mmHg at 25°C

1.613

4.05

67.23

1

4

7

34

686

0

FNYGWXSATBUBER-UHFFFAOYSA-N

InChI=1S/C27H31FN4O2/c1-19-9-6-7-14-24(19)26(27(34)30-22-11-4-3-5-12-22)32(23-13-8-10-21(28)17-23)25(33)18-31-16-15-29-20(31)2/h6-10,13-17,22,26H,3-5,11-12,18H2,1-2H3,(H,30,34)

N-cyclohexyl-2-(N-(3-fluorophenyl)-2-(2-methyl-1H-imidazol-1-yl)acetamido)-2-(o-tolyl)acetamide

IDH C35; AGI5198; IDH-C35; IDHC35; AGI-5198; AGI 5198;

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: ≥ 2.08 mg/mL (4.50 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 20.8 mg/mL澄清DMSO储备液加入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: 2.08 mg/mL (4.50 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 20.8 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: ≥ 2.08 mg/mL (4.50 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 20.8 mg/mL 澄清 DMSO 储备液加入900 μL 玉米油中，混合均匀。

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配方 4 中的溶解度: 0.5% methylcellulose+0.2% Tween 80: 30mg/mL

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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。