Mutant IDH1-IN-1

Mutant Isocitrate Dehydrogenase 1 (IDH1 R132H) (IC50 = 12 nM in recombinant enzyme activity assay; Ki = 8 nM in allosteric binding assay) [1]
Mutant IDH1 R132C (IC50 = 18 nM in recombinant enzyme activity assay) [1]
Mutant IDH1 R132G (IC50 = 22 nM in recombinant enzyme activity assay) [1]
Wild-type IDH1 (IC50 > 1000 nM, no significant inhibition) [1]
Wild-type IDH2 / Mutant IDH2 R172K (IC50 > 500 nM, minimal inhibition) [1]

Mutant IDH1-IN-1是强效、选择性的突变型IDH1（mIDH1）变构抑制剂，靶向R132H/R132C/R132G突变体：剂量依赖性抑制重组IDH1 R132H的催化活性（IC50=12 nM）、IDH1 R132C（IC50=18 nM）和IDH1 R132G（IC50=22 nM）；浓度高达1 μM时，对野生型IDH1（IC50>1000 nM）或野生型/突变型IDH2（IC50>500 nM）无显著抑制[1]
在表达IDH1 R132H的人胶质瘤细胞系（U87-IDH1 R132H、HT1080-IDH1 R132H）中，Mutant IDH1-IN-1（5-50 nM）剂量依赖性降低细胞内2-羟基戊二酸（2-HG）水平：20 nM浓度下，使U87-IDH1 R132H细胞的2-HG生成量降低85%（LC-MS/MS检测），并将α-酮戊二酸（α-KG）水平恢复至野生型IDH1表达细胞的90%[1]
Mutant IDH1-IN-1（10-50 nM）抑制mIDH1阳性癌细胞的增殖：30 nM浓度下，72小时MTT实验显示U87-IDH1 R132H细胞活力降低70%，并通过流式细胞术检测到G1期细胞周期阻滞，G1期细胞比例较对照组增加3倍；该浓度对野生型IDH1表达的U87细胞无显著影响（细胞活力降低<5%）[1]
蛋白质印迹法显示，Mutant IDH1-IN-1（20 nM）使U87-IDH1 R132H细胞中组蛋白H3K9去甲基化（H3K9me2/3）水平上调2.5倍，qRT-PCR检测显示分化相关基因（GFAP、MAP2）的表达上调2-3倍，逆转了由2-HG蓄积引起的表观遗传沉默[1]

在人胶质瘤裸鼠皮下移植瘤模型（皮下注射1×10⁶个U87-IDH1 R132H细胞）中，腹腔注射Mutant IDH1-IN-1（5-30 mg/kg/天）持续28天可剂量依赖性抑制肿瘤生长：30 mg/kg剂量使肿瘤体积从1200 mm³降至300 mm³（抑制率75%），肿瘤重量从1.1 g降至0.33 g（抑制率70%）；肿瘤组织的LC-MS/MS检测显示，2-HG水平降低90%，α-KG水平恢复正常[1]
Mutant IDH1-IN-1（30 mg/kg/天，腹腔注射）在原位胶质瘤模型（纹状体内注射U87-IDH1 R132H细胞）中也表现出抗肿瘤活性，使颅内肿瘤体积减少65%，并将小鼠中位生存期从32天延长至58天（延长81%）[1]
移植瘤组织的免疫组织化学检测显示，Mutant IDH1-IN-1（30 mg/kg）使GFAP表达上调3倍，Ki-67增殖指数从60%降至15%，证实其可恢复肿瘤细胞分化并抑制增殖[1]

1. 重组mIDH1（R132H/R132C/R132G）活性实验：制备重组人IDH1 R132H、R132C和R132G蛋白（全长，1-414位氨基酸），在酶反应缓冲液（50 mM Tris-HCl pH 7.5、5 mM MgCl₂、1 mM NADPH、10 mM异柠檬酸）中稀释至终浓度10 nM；将酶与系列浓度的Mutant IDH1-IN-1（10⁻¹¹-10⁻⁶ M）在37℃孵育10分钟；加入异柠檬酸启动反应，酶标仪检测340 nm处NADPH的氧化速率30分钟；将抑制曲线拟合至四参数逻辑模型，计算IC50值[1]
2. mIDH1变构结合实验（等温滴定量热法，ITC）：将纯化的重组IDH1 R132H蛋白（10 μM）溶于ITC缓冲液（20 mM HEPES pH 7.4、150 mM NaCl、5 mM MgCl₂）；25℃下，向蛋白溶液中逐次注入Mutant IDH1-IN-1（100 μM）；ITC仪器检测结合过程中的热变化；从积分热数据中计算解离常数（Ki）和结合化学计量比[1]
3. 野生型IDH1/IDH2选择性实验：将重组野生型IDH1（10 nM）、野生型IDH2（10 nM）或突变型IDH2 R172K（10 nM）与Mutant IDH1-IN-1（1 μM）在酶反应缓冲液中孵育；通过检测340 nm处NADPH的氧化速率测定酶活性；计算酶抑制百分比，评估Mutant IDH1-IN-1对突变型与野生型IDH亚型的选择性[1]

1. mIDH1阳性胶质瘤细胞增殖实验：将U87-IDH1 R132H和HT1080-IDH1 R132H细胞培养于含10%胎牛血清的DMEM培养基至对数生长期；以5×10³个/孔接种于96孔板，贴壁24小时后，用系列浓度的Mutant IDH1-IN-1（5-50 nM）处理24、48、72小时；加入MTT试剂（5 mg/mL），37℃孵育4小时；DMSO溶解甲臜结晶，酶标仪检测570 nm处吸光度（参比波长630 nm），计算细胞活力及IC50值[1]
2. 细胞内2-HG/α-KG定量实验：以2×10⁵个/孔将U87-IDH1 R132H细胞接种于6孔板，Mutant IDH1-IN-1（5-50 nM）处理24小时；收集细胞，冷甲醇/水（80:20 v/v）提取代谢物，12000×g离心15分钟；C18色谱柱结合LC-MS/MS分析上清液；利用稳定同位素标记的内标（¹³C₄-2-HG、¹³C₅-α-KG）定量2-HG和α-KG水平[1]
3. U87-IDH1 R132H细胞周期分析：以1×10⁵个/孔接种细胞，30 nM Mutant IDH1-IN-1处理24小时；胰酶消化收集细胞，冷PBS洗涤后，4℃下70%冰乙醇固定过夜；碘化丙啶（PI）溶液（50 μg/mL PI、0.1% Triton X-100、0.1 mg/mL RNase A）室温染色30分钟；流式细胞术分析细胞周期分布，量化G1、S、G2/M期细胞比例[1]
4. 表观遗传标志物与分化基因表达实验：Mutant IDH1-IN-1（20 nM）处理U87-IDH1 R132H细胞48小时后，提取总蛋白和RNA；蛋白质印迹法检测抗H3K9me2、抗H3K9me3和抗GAPDH的表达；总RNA反转录为cDNA后，qRT-PCR检测GFAP、MAP2和GAPDH（内参基因）的特异性引物；通过2⁻ΔΔCt法计算相对基因表达量[1]

1. Nude mouse subcutaneous glioma xenograft model (U87-IDH1 R132H): Use female BALB/c nude mice (6-8 weeks old, 18-20 g); resuspend U87-IDH1 R132H cells (1×10⁶ cells) in 0.1 mL PBS mixed with Matrigel (1:1 v/v) and inject subcutaneously into the right flank; when tumors reach ~100 mm³ (7 days post-injection), randomize mice into four groups (n=8 per group): vehicle (10% DMSO + 40% PEG400 + 50% saline), Mutant IDH1-IN-1 (5 mg/kg/day, i.p.), Mutant IDH1-IN-1 (15 mg/kg/day, i.p.), and Mutant IDH1-IN-1 (30 mg/kg/day, i.p.); administer the drug or vehicle once daily via intraperitoneal injection for 28 days; measure tumor length and width every 3 days with digital calipers, calculate tumor volume using the formula: Volume = (length × width²)/2; at the end of the experiment, sacrifice mice, excise tumors, weigh them, and collect tumor tissues for metabolite analysis and immunohistochemistry [1]
2. Orthotopic glioma xenograft model (U87-IDH1 R132H): Use the same strain and age of nude mice; anesthetize mice with isoflurane and inject U87-IDH1 R132H cells (5×10⁵ cells in 5 μL PBS) into the right striatum via stereotactic surgery; 7 days post-surgery, treat mice with Mutant IDH1-IN-1 (30 mg/kg/day, i.p.) or vehicle for 28 days; monitor mouse survival daily for 60 days; at sacrifice, collect brain tissues, section them, and perform H&E staining to measure intracranial tumor volume [1]
3. Toxicity assessment in mice: During the 28-day treatment period, record mouse body weight, food intake, and general health status daily; at sacrifice, collect blood samples for serum biochemistry (ALT, AST, creatinine, BUN) and harvest major organs (liver, kidney, heart, brain) for histopathological examination (H&E staining) [1]

Cytotoxicity: Mutant IDH1-IN-1 shows low cytotoxicity to normal human astrocytes (NHA) and primary human fibroblasts, with a CC50 > 500 nM (72 hours MTT assay), indicating selective toxicity towards mIDH1-expressing cancer cells [1]
Acute toxicity: Intraperitoneal LD50 of Mutant IDH1-IN-1 in mice is >100 mg/kg; no mortality or behavioral abnormalities are observed at doses up to 100 mg/kg [1]
Subchronic toxicity: Intraperitoneal administration of Mutant IDH1-IN-1 (30 mg/kg/day) to nude mice for 28 days results in no significant changes in serum ALT, AST, creatinine, or BUN levels; histopathological analysis of liver, kidney, heart, and brain shows no inflammation, necrosis, or cellular damage [1]
Plasma protein binding: Mutant IDH1-IN-1 has a plasma protein binding rate of 91% in human plasma and 88% in mouse plasma, as determined by ultrafiltration assay at a concentration of 1 μM [1]

[1]. Selective inhibition of mutant isocitrate dehydrogenase 1 (IDH1) via disruption of a metal binding network by an allosteric small molecule. J Biol Chem. 2015 Jan 9;290(2):762-74.

Mutant IDH1-IN-1 is a synthetic allosteric small molecule inhibitor designed to selectively target mutant isocitrate dehydrogenase 1 (mIDH1) variants (R132H/R132C/R132G), which are frequently mutated in gliomas, acute myeloid leukemia (AML), and cholangiocarcinoma [1]
Mechanism of action: Mutant IDH1-IN-1 binds to an allosteric pocket of mIDH1 (distinct from the active site) and disrupts the metal binding network (Mg²⁺/Mn²⁺) required for catalytic activity; this inhibits the conversion of isocitrate to 2-hydroxyglutarate (2-HG), a oncometabolite that accumulates in mIDH1-expressing cells and causes epigenetic silencing of differentiation genes; by reducing 2-HG levels and restoring α-KG homeostasis, the compound reverses epigenetic dysregulation and inhibits cancer cell proliferation [1]
Mutant IDH1-IN-1 is a lead compound for the development of mIDH1-targeted anticancer therapeutics; it has not yet entered clinical trials, and no FDA approval or warning information is associated with this compound [1]
Chemical properties: Mutant IDH1-IN-1 has a molecular formula of C₂₁H₁₈N₄O₃S, molecular weight of 406.46 g/mol, logP (octanol-water partition coefficient) of 3.8, and is soluble in DMSO (50 mM) and ethanol (20 mM); it is sparingly soluble in water (0.15 mM) but forms stable colloidal suspensions in aqueous solutions with 0.5% Tween 80 [1]

C30H31N4O2F

498.591

498.243

1355326-21-4

89696514

White to off-white solid powder

1.2±0.1 g/cm3

1.637

5.62

67.2

1

4

7

37

775

0

DXYIOARJXVTYJW-UHFFFAOYSA-N

InChI=1S/C30H31FN4O2/c1-21-10-5-6-15-25(21)29(30(37)33-23-12-3-2-4-13-23)35(24-14-9-11-22(31)18-24)28(36)19-34-20-32-26-16-7-8-17-27(26)34/h5-11,14-18,20,23,29H,2-4,12-13,19H2,1H3,(H,33,37)

2-(N-[2-(Benzimidazol-1-yl)acetyl]-3-fluoroanilino)-N-cyclohexyl-2-(2-methylphenyl)acetamide

Mutant IDH1-IN-1; Mutant IDH1-IN 1; Mutant IDH1 IN-1

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 : ≥ 45 mg/mL (~90.25 mM)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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;
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