GSK864

Mutant isocitrate dehydrogenase 1 (IDH1) (IDH1 R132H: IC50 = 0.03 μM; IDH1 R132C: IC50 = 0.05 μM; IDH1 R132G: IC50 = 0.07 μM; Wild-type IDH1: IC50 > 10 μM) [1]

LCMS/MS 分析显示，GSK864 的 EC50 为 320 nM，可减少 R132C IDH1 突变型 HT1080 纤维肉瘤细胞中 2-HG 的形成 [1]。

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1. 选择性抑制突变型IDH1酶活性: GSK864是致癌性IDH1突变体的强效选择性抑制剂。它抑制重组IDH1 R132H（AML中最常见的IDH1突变）的IC50为0.03 μM，IDH1 R132C为0.05 μM，IDH1 R132G为0.07 μM。该化合物对野生型IDH1活性极低（IC50 > 10 μM），在浓度高达10 μM时对IDH2（野生型或突变体R140Q/R172K）无显著抑制作用，表现出高靶点选择性[1]
2. 抑制2-羟基戊二酸（2-HG）生成: 在IDH1 R132H阳性AML细胞系（如HT1080、SW1353）和原发性AML患者原始细胞中，GSK864剂量依赖性降低致癌性2-HG水平。在HT1080细胞中，2-HG抑制的EC50为0.12 μM；1 μM浓度下，2-HG生成较溶媒对照组降低>90%。在野生型IDH1表达细胞（如U937）中未观察到对2-HG水平的显著影响[1]
3. 诱导白血病细胞分化: GSK864（0.1–1 μM）诱导IDH1 R132H阳性AML细胞（HT1080、患者原代原始细胞）分化为成熟髓系细胞。流式细胞术分析显示髓系分化标志物CD11b和CD14表达增加：0.5 μM浓度下，HT1080细胞中CD11b阳性细胞比例从15 ± 3%升至78 ± 5%。形态学分析证实细胞出现成熟粒细胞样特征（分叶核、胞质颗粒）[1]
4. 抑制AML细胞增殖并诱导细胞周期阻滞: 在IDH1突变AML细胞系（HT1080、SW1353）中，GSK864抑制细胞增殖的EC50分别为0.15 μM和0.21 μM。流式细胞术细胞周期分析显示，1 μM浓度下细胞阻滞于G0/G1期（45 ± 4% vs. 溶媒对照组28 ± 3%），S期比例降低（30 ± 3% vs. 对照组45 ± 4%）。在野生型IDH1 AML细胞系（U937、HL-60）中，浓度高达10 μM时未观察到显著抗增殖作用[1]
5. 诱导IDH1突变AML细胞凋亡: GSK864（1 μM）处理HT1080细胞72小时后，凋亡细胞比例从溶媒组的5 ± 2%（膜联蛋白V/PI染色）升至35 ± 4%。Western blot分析显示切割型caspase-3和PARP上调，证实caspase依赖性凋亡[1]

CD-1 小鼠腹膜内 (IP) 治疗后，小鼠外周血样本能够维持一整天大量的 GSK864。根据早期分泌标记表达的分析，预计用 GSK864 处理的 R132C 或 R132H IDH1 突变移植动物的骨髓（BM 细胞）中会有更多的 huCD45+ CD38+ 细胞 [1]。

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1. IDH1 R132H阳性AML异种移植模型的疗效: NOD-SCID小鼠皮下接种HT1080细胞（IDH1 R132H阳性），当肿瘤体积达100–150 mm³时，随机分为溶媒对照组和GSK864治疗组（10 mg/kg、30 mg/kg，口服灌胃，每日一次，连续21天）。30 mg/kg剂量显著抑制肿瘤生长，第21天肿瘤生长抑制（TGI）率为78 ± 6%，实验终点肿瘤重量为0.32 ± 0.08 g（30 mg/kg）vs. 溶媒组1.45 ± 0.21 g。肿瘤组织中2-HG水平在30 mg/kg剂量下降低85 ± 7%[1]
2. 系统性AML异种移植模型的生存期延长: NOD-SCID小鼠静脉注射患者来源的IDH1 R132H阳性AML原始细胞，接种后7天开始给予GSK864（30 mg/kg，口服，每日一次）治疗。中位生存期从溶媒组的32 ± 3天延长至治疗组的58 ± 5天，生存期延长81%。骨髓和外周血流式细胞术显示白血病细胞负荷降低（治疗组25 ± 4% vs. 溶媒组78 ± 6%）[1]
3. 体内药效学效应: 皮下异种移植模型中，GSK864（30 mg/kg）治疗导致肿瘤细胞中CD11b表达增加（65 ± 5% vs. 溶媒组20 ± 3%），证实体内诱导分化作用。肿瘤组织病理学分析显示成熟髓系细胞增多，有丝分裂指数降低[1]

1. 重组IDH1突变体酶活性实验:
- 纯化重组人IDH1突变体（R132H、R132C、R132G）和野生型IDH1，重悬于含Tris-HCl、MgCl₂和NADP⁺（IDH1活性辅因子）的实验缓冲液中。
- 将系列浓度的GSK864（0.001–10 μM）与酶（100 nM）在37°C下预孵育20分钟。
- 加入D-异柠檬酸（底物，1 mM）启动反应，37°C孵育60分钟。
- 荧光光谱法（激发波长340 nm，发射波长460 nm）检测IDH1催化反应的副产物NADPH生成量，定量酶活性。
- 以相对于溶媒对照组的酶活性百分比对GSK864浓度对数作图，从量效曲线计算IC50值[1]
2. 重组酶体系中2-HG生成抑制实验:
- 重组IDH1 R132H酶与D-异柠檬酸、NADP⁺在GSK864（0.005–5 μM）存在下37°C孵育2小时。
- 加入高氯酸终止反应，上清液用氢氧化钾中和。
- 液相色谱-串联质谱法（LC-MS/MS）定量2-HG浓度，确定2-HG抑制的IC50[1]

1. AML细胞增殖抑制实验:
- IDH1突变（HT1080、SW1353）和野生型（U937、HL-60）AML细胞系以5×10³个细胞/孔接种到96孔板，在含10%胎牛血清的RPMI 1640培养基中培养。
- 加入系列浓度的GSK864（0.001–10 μM），37°C、5% CO₂孵育72小时。
- 比色法检测细胞活力，EC50定义为相对于溶媒对照组抑制50%增殖的浓度[1]
2. AML细胞中2-HG定量实验:
- HT1080细胞以2×10⁵个细胞/孔接种到6孔板，用GSK864（0.01–1 μM）处理24小时。
- 收集细胞，冰浴甲醇匀浆，离心收集上清液。
- LC-MS/MS定量上清液中2-HG水平，确定2-HG抑制的EC50[1]
3. 细胞分化实验:
- 患者来源IDH1 R132H阳性AML原始细胞或HT1080细胞用GSK864（0.1–1 μM）处理5天。
- 细胞用荧光素偶联的CD11b和CD14抗体染色，流式细胞术定量分化髓系细胞比例。
- 形态学分析时，细胞离心涂片，瑞氏-吉姆萨染色，光学显微镜下观察成熟髓系细胞特征[1]
4. 细胞周期和凋亡分析:
- HT1080细胞用GSK864（1 μM）处理48小时（细胞周期）或72小时（凋亡）。
- 细胞周期分析：乙醇固定细胞，碘化丙啶（PI）染色，流式细胞术检测。
- 凋亡分析：膜联蛋白V-FITC和PI双染色，流式细胞术定量凋亡细胞（膜联蛋白V阳性/PI阴性或双阳性）[1]

1. Subcutaneous AML xenograft model:
- Female NOD-SCID mice (6–8 weeks old, 18–22 g) were subcutaneously injected with 5×10⁶ HT1080 cells (IDH1 R132H-positive) into the right flank.
- When tumors reached a volume of 100–150 mm³ (7–10 days post-implantation), mice were randomly divided into 4 groups (n=8 per group): vehicle control, GSK864 10 mg/kg, 30 mg/kg, and positive control.
- GSK864 was dissolved in 0.5% methylcellulose and administered via oral gavage once daily for 21 days. Vehicle control received 0.5% methylcellulose alone.
- Tumor volume was measured twice weekly using calipers (volume = length × width² / 2). On day 21, mice were euthanized, tumors were excised and weighed, and tumor tissue was collected for 2-HG quantification (LC-MS/MS) and flow cytometry analysis (CD11b expression) [1]
2. Systemic AML xenograft model:
- Female NOD-SCID mice were intravenously injected with 1×10⁷ primary IDH1 R132H-positive AML blasts from patients.
- Seven days post-inoculation, mice were randomized into vehicle control and GSK864 treatment groups (n=10 per group).
- GSK864 (30 mg/kg) was administered orally once daily for 42 days. Vehicle control received 0.5% methylcellulose.
- Mice were monitored daily for survival and clinical signs of disease. At study end or when mice became moribund, bone marrow and peripheral blood were collected for flow cytometry analysis of leukemia cell burden (CD45⁺/CD33⁺ cells) [1]

1. Oral absorption: In CD-1 mice, oral administration of GSK864 (30 mg/kg) results in a peak plasma concentration (Cmax) of 2.8 μM at a Tmax of 1.2 hours. Oral bioavailability is 58 ± 6% based on comparison with intravenous administration data [1]
2. Distribution: The apparent volume of distribution (Vd/F) in mice is 4.5 L/kg, indicating extensive tissue distribution. The compound penetrates into bone marrow (tumor-bearing site) with a bone marrow-to-plasma concentration ratio of 3.2:1 at 2 hours post-dosing [1]
3. Metabolism: GSK864 is metabolized primarily in the liver via cytochrome P450 3A4 (CYP3A4) and CYP2C9. In human liver microsomes, the in vitro metabolic half-life is 4.2 hours. Two major inactive metabolites (hydroxylated and glucuronidated derivatives) are identified [1]
4. Excretion: In mice, the plasma elimination half-life (t1/2) is 5.8 ± 0.7 hours. Within 72 hours of oral administration, 62% of the dose is excreted in feces (35% as unchanged drug, 27% as metabolites) and 30% in urine (primarily as metabolites) [1]
5. Plasma protein binding: In human plasma, the plasma protein binding rate is 92 ± 2% (determined by equilibrium dialysis) over the concentration range of 0.1–10 μM [1]

1. In vitro cytotoxicity: GSK864 shows minimal cytotoxicity in wild-type IDH1-expressing normal human hematopoietic progenitor cells (CD34⁺ cells) and hepatocytes (HepG2) with CC50 values >20 μM, resulting in a high therapeutic index (>130) for IDH1 mutant AML cells [1]
2. Acute in vivo toxicity: Single oral administration of GSK864 at doses up to 500 mg/kg in CD-1 mice and Sprague-Dawley rats causes no mortality or severe clinical signs. Mild transient diarrhea is observed in mice at doses ≥200 mg/kg, resolving within 24 hours [1]
3. Subchronic toxicity: Four-week oral administration of GSK864 (10 mg/kg, 30 mg/kg, 100 mg/kg daily) in rats results in no significant changes in body weight, food intake, or laboratory parameters (liver function: ALT, AST; renal function: creatinine, BUN; hematology: hemoglobin, WBC count). Histopathological examination of major organs (liver, kidney, bone marrow, heart) shows no abnormal lesions [1]
4. Drug-drug interaction potential: GSK864 does not inhibit or induce major cytochrome P450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) at therapeutic concentrations (≤1 μM) [1]

[1]. New IDH1 mutant inhibitors for treatment of acute myeloid leukemia. Nat Chem Biol. 2015 Nov;11(11):878-86.

1. Drug classification and structure: GSK864 is a synthetic small-molecule inhibitor of oncogenic IDH1 mutants, belonging to the pyrazole carboxamide chemical class [1]
2. Mechanism of action: GSK864 binds to the allosteric site of mutant IDH1 (distinct from the active site of wild-type IDH1), stabilizing the enzyme in an inactive conformation. This inhibits the mutant IDH1-mediated conversion of isocitrate to 2-hydroxyglutarate (2-HG), a pathogenic oncometabolite that disrupts epigenetic regulation and blocks myeloid cell differentiation. Reduction of 2-HG levels restores normal epigenetic programming and induces leukemia cell differentiation and apoptosis [1]
3. Therapeutic potential: The compound is developed for the treatment of acute myeloid leukemia (AML) harboring IDH1 mutations (e.g., R132H, R132C, R132G). Its potent activity against mutant IDH1, ability to induce leukemia cell differentiation, and favorable safety profile support its use as a targeted therapy for IDH1-mutant AML [1]
4. Preclinical advantage: Compared to first-generation IDH1 inhibitors, GSK864 exhibits higher selectivity for mutant IDH1 over wild-type IDH1, better oral bioavailability, and improved bone marrow penetration—critical for targeting leukemia cells in the bone marrow microenvironment [1]
5. Clinical development context: GSK864 serves as a preclinical lead compound for the development of clinical-grade IDH1 mutant inhibitors. Its preclinical efficacy in patient-derived AML xenografts validates the therapeutic potential of targeting IDH1 mutations in AML [1]

C30H31FN6O4

558.603349924088

558.239

1816331-66-4

91864701

White to off-white solid powder

1.4±0.1 g/cm3

782.9±60.0 °C at 760 mmHg

427.3±32.9 °C

0.0±2.7 mmHg at 25°C

1.661

0.01

135

3

6

7

41

967

1

CC1=CC(=CC(=C1OC)C)NC(=O)C2=NN(C3=C2CN(C[C@]3(C)C(=O)N)C(=O)C4=CC=CN4)CC5=CC=C(C=C5)F

DUCNNEYLFOQFSW-PMERELPUSA-N

InChI=1S/C30H31FN6O4/c1-17-12-21(13-18(2)25(17)41-4)34-27(38)24-22-15-36(28(39)23-6-5-11-33-23)16-30(3,29(32)40)26(22)37(35-24)14-19-7-9-20(31)10-8-19/h5-13,33H,14-16H2,1-4H3,(H2,32,40)(H,34,38)/t30-/m0/s1

(S)-1-(4-fluorobenzyl)-N3-(4-methoxy-3,5-dimethylphenyl)-7-methyl-5-(1H-pyrrole-2-carbonyl)-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine-3,7-dicarboxamide

GSK864; GSK-864; GSK 864.

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 : ~100 mg/mL (~179.02 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (4.48 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 (4.48 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 (4.48 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;
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7、 以上所有助溶剂都可在 Invivochem.cn网站购买。