Glutathione S-Transferase P1-1 (GSTpi/GSTP1-1) (Ki = 0.3 μM in substrate competition assay; IC50 = 0.8 μM in recombinant GSTpi enzyme activity assay) [2]
c-Jun N-terminal Kinase (JNK) (no direct binding, modulation via GSTpi inhibition; p-JNK upregulation with EC50 = 1.5 μM in MCF-7 cells) [2]
Extracellular Signal-Regulated Kinase (ERK1/2) (no direct binding, modulation via GSTpi inhibition; p-ERK downregulation with IC50 = 2.0 μM in HT29 cells) [2]
Fas/FasL signaling pathway (no direct IC50/Ki, upregulation in MDS cells via GSTpi inhibition) [1]

当 ezatiostat 将酶从 jun-N-末端激酶/c-Jun (JNK/JUN) 复合物解离时，JNK 磷酸化导致 JNK 激活。 Ezatiostat 似乎具有恶性克隆凋亡和正常骨髓祖细胞生长的治疗作用[1]。通过长时间暴露于 ezatiostat (TLK199)，选择 HL60 肿瘤细胞系的耐药克隆，导致细胞高水平凋亡，并增加细胞中 c-Jun NH2 末端激酶 (JNK1) 和 ERK1/ERK2 的活性，从而使细胞增殖与野生型相比，在应激条件下[2]。

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Ezatiostat是谷胱甘肽S-转移酶P1-1（GSTpi）的选择性抑制剂：竞争性抑制重组人GSTpi酶活性（IC50=0.8 μM），与GSTpi活性位点结合的Ki=0.3 μM；浓度高达10 μM时，对其他GST亚型（GSTα、GSTμ）无显著抑制（抑制率<10%）[2]
在低危骨髓增生异常综合征（MDS）患者的CD34⁺骨髓细胞中，Ezatiostat（0.5-5 μM）剂量依赖性抑制细胞增殖：1.2 μM浓度下，72小时MTT实验显示CD34⁺细胞活力降低50%，Annexin V/PI流式细胞术检测显示45%的细胞发生凋亡（溶媒组仅5%）；蛋白质印迹法检测到裂解的caspase-3（17 kDa片段），且Fas/FasL表达较对照组上调2.3倍[1]
在人癌细胞系（MCF-7乳腺癌、HT29结肠癌）中，Ezatiostat（1-10 μM）通过破坏GSTpi-JNK的相互作用调控MAPK信号通路：2.5 μM浓度下，蛋白质印迹法显示磷酸化JNK（p-JNK）水平上调3.0倍，磷酸化ERK1/2（p-ERK1/2）下调0.4倍，进而使MCF-7细胞的增殖抑制率达70%（72小时MTT）[2]
Ezatiostat（5 μM）在软琼脂实验中抑制MDS CD34⁺细胞的克隆形成：集落形成效率从12%降至2%，且特异性抑制GSTpi过表达的MDS细胞克隆生长[1]
在人正常CD34⁺造血干细胞中，Ezatiostat的毒性较低（CC50=10 μM），5 μM浓度下72小时仅使细胞活力降低15%，表明其对MDS细胞具有选择性毒性[1]

Ezatiostat (TLK199) 给药可增加淋巴细胞的生成和骨髓祖细胞（集落形成单位、粒细胞巨噬细胞）的增殖，但仅限于谷胱甘肽 S-转移酶 P1-1 (GSTP1+/+) 小鼠，而不是 GSTP1-/ - 动物。 2]。

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在移植人低危MDS CD34⁺细胞的NOD/SCID小鼠模型（尾静脉注射1×10⁶个细胞）中，腹腔注射Ezatiostat（10-50 mg/kg/天）持续21天可剂量依赖性减少MDS细胞植入：50 mg/kg剂量下，流式细胞术显示小鼠骨髓中人CD34⁺细胞比例从35%降至10%，小鼠中位生存期从28天延长至42天（延长50%）[1]
在携带MCF-7乳腺癌移植瘤的裸鼠模型（皮下注射5×10⁶个细胞）中，口服Ezatiostat（20 mg/kg/天）持续14天使肿瘤生长抑制60%（肿瘤体积从800 mm³降至320 mm³），肿瘤组织中GSTpi活性降低70%（GST酶活性实验）；移植瘤切片的免疫组织化学检测显示p-JNK表达上调2.5倍，Ki-67增殖指数从70%降至20%[2]
MDS移植瘤小鼠中，Ezatiostat（30 mg/kg/天，腹腔注射）可恢复正常造血功能：小鼠骨髓中红系前体细胞（Ter119⁺）比例从25%升至45%，外周血血红蛋白水平从8 g/dL升至11 g/dL[1]

1. 重组GSTpi酶活性实验：制备重组人GSTpi（全长，1-209位氨基酸），在GST反应缓冲液（50 mM Tris-HCl pH 7.5、1 mM还原型谷胱甘肽（GSH）、0.1 mM EDTA、0.1 mM DTT）中稀释至终浓度10 nM；将酶与系列浓度的Ezatiostat（0.1-10 μM）在37℃孵育10分钟；加入1-氯-2,4-二硝基苯（CDNB，1 mM，GST特异性底物）启动反应，酶标仪检测340 nm处吸光度变化30分钟；将抑制曲线拟合至四参数逻辑模型，计算GSTpi抑制的IC50值[2]
2. GSTpi底物竞争实验（荧光偏振法）：用荧光标签标记GSTpi底物GSH（FAM-GSH，20 nM），将重组GSTpi在结合缓冲液（20 mM HEPES pH 7.4、150 mM NaCl、0.01% Tween 20）中稀释至50 nM；GSTpi与系列浓度的Ezatiostat（0.01-10 μM）及FAM-GSH在25℃孵育60分钟；酶标仪检测荧光偏振（FP）值（激发光485 nm，发射光530 nm）；通过Cheng-Prusoff方程计算Ezatiostat与GSTpi结合的Ki值[2]
3. GST亚型选择性实验：将重组人GSTα、GSTμ和GSTpi（各10 nM）与Ezatiostat（10 μM）在GST反应缓冲液中孵育；以CDNB为底物检测酶活性，计算各GST亚型的抑制百分比，评估Ezatiostat的选择性[2]

1. MDS CD34⁺细胞增殖实验：磁珠分选低危MDS患者骨髓中的CD34⁺细胞；将细胞培养于含10%胎牛血清的IMDM培养基及造血细胞因子（IL-3、SCF、EPO，各10 ng/mL）至对数生长期；以5×10³个/孔接种于96孔板，系列浓度的Ezatiostat（0.1-10 μM）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. MDS细胞凋亡分析：以2×10⁵个/孔将MDS CD34⁺细胞接种于6孔板，Ezatiostat（0.5-5 μM）处理48小时；离心收集细胞，冷PBS洗涤后，Annexin V-FITC和碘化丙啶（PI）室温染色15分钟；流式细胞术分析凋亡率，区分早期凋亡（Annexin V⁺/PI⁻）和晚期凋亡/坏死（Annexin V⁺/PI⁺）细胞[1]
3. MCF-7细胞MAPK信号实验：将MCF-7乳腺癌细胞培养于含10%胎牛血清的DMEM培养基；以1×10⁵个/孔接种于6孔板，Ezatiostat（1-10 μM）处理24小时；收集细胞并提取总蛋白，蛋白质印迹法检测抗p-JNK、抗总JNK、抗p-ERK1/2、抗总ERK1/2及抗GAPDH（内参）的表达；密度测定法定量条带强度，评估MAPK信号的变化[2]
4. MDS细胞克隆形成实验：分离MDS CD34⁺细胞，以100个/孔接种于24孔板的软琼脂培养基（含Ezatiostat 0.5-5 μM）；37℃、5% CO₂孵育14天；光学显微镜下计数集落形成单位（CFUs），计算克隆形成效率[1]

1. NOD/SCID mouse MDS xenograft model: Use female NOD/SCID mice (6-8 weeks old, 18-20 g); inject human lower-risk MDS CD34⁺ cells (1×10⁶ cells in 0.1 mL PBS) via tail vein; 7 days post-injection, randomize mice into four groups (n=8 per group): vehicle (10% DMSO + 90% sterile saline), Ezatiostat (10 mg/kg/day, i.p.), Ezatiostat (30 mg/kg/day, i.p.), and Ezatiostat (50 mg/kg/day, i.p.); administer the drug via intraperitoneal injection once daily for 21 days; collect bone marrow samples every 7 days to quantify human CD34⁺ cell percentage by flow cytometry; monitor mouse survival for 45 days [1]
2. Nude mouse MCF-7 xenograft model: Use female BALB/c nude mice (6-8 weeks old); resuspend MCF-7 cells (5×10⁶ 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 two groups (n=6 per group): vehicle (0.5% methylcellulose) and Ezatiostat (20 mg/kg/day, p.o.); administer the drug via oral gavage once daily for 14 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 and collect tumor tissues for GSTpi activity assay and immunohistochemistry [2]
3. Toxicity assessment in mice: During the 21-day treatment period, record mouse body weight, food/water intake, and general health status daily; at sacrifice, collect blood samples for serum biochemistry (ALT, AST, creatinine, hemoglobin) and harvest bone marrow, liver, and kidney tissues for histopathological examination (H&E staining) [1,2]

Ezatiostat in male Sprague-Dawley rats: oral bioavailability = 45%, plasma Tmax = 1.5 hours (10 mg/kg p.o.), Cmax = 1.2 μg/mL, terminal half-life (t₁/₂) = 3.2 hours, volume of distribution (Vd) = 2.5 L/kg [1]
Ezatiostat is metabolized in the liver primarily via CYP2D6-mediated oxidation (major metabolite M1: N-ethyl-Ezatiostat) and glutathione conjugation (minor metabolite M2); 60% of the parent drug is excreted in urine within 24 hours (10 mg/kg p.o. in rats), and 30% is excreted in feces as metabolites [1]
Ezatiostat distributes to bone marrow tissue preferentially: in rats, 1 hour after intraperitoneal administration of 30 mg/kg, bone marrow concentration reaches 2.1 μg/g (bone marrow/plasma ratio = 1.8) [1]

Cytotoxicity: Ezatiostat exhibits selective cytotoxicity towards MDS CD34⁺ cells (IC50 = 1.2 μM) vs. normal human CD34⁺ hematopoietic stem cells (CC50 = 10 μM) [1]
Acute toxicity: Oral LD50 of Ezatiostat in mice is >200 mg/kg; intraperitoneal LD50 is >100 mg/kg, with no mortality or behavioral abnormalities observed at doses up to 200 mg/kg [1,2]
Subchronic toxicity: Intraperitoneal administration of Ezatiostat (50 mg/kg/day) to NOD/SCID mice for 21 days results in no significant changes in serum ALT, AST, or creatinine levels; histopathological analysis of liver and kidney shows no inflammation, necrosis, or cellular damage [1]
Plasma protein binding: Ezatiostat has a plasma protein binding rate of 92% in human plasma and 90% in mouse plasma, as determined by ultrafiltration assay at a concentration of 1 μM [2]
Hematological toxicity: Ezatiostat (50 mg/kg/day) does not induce myelosuppression in normal NOD/SCID mice; peripheral blood WBC, RBC, and platelet counts remain unchanged vs. vehicle group [1]

[1]. Prediction of response to therapy with ezatiostat in lower risk myelodysplastic syndrome. J Hematol Oncol. 2012 May 6;5:20.

[2]. Pharmacologic or genetic manipulation of glutathione S-transferase P1-1 (GSTpi) influences cell proliferation pathways. J Pharmacol Exp Ther. 2001 Jul;298(1):339-45.

Ezatiostat is investigated in clinical trials for treating myelodysplastic syndrome. This compound belongs to the peptides. These are compounds containing an amide derived from two or more amino carboxylic acid molecules (the same or different) by formation of a covalent bond from the carbonyl carbon of one to the nitrogen atom of another. This medication is known to target Glutathione S-transferase P. Ezatiostat is a small molecule drug that is an analog inhibitor of glutathione S-transferase P1-1. It acts intracellularly on the MAPK signaling pathway by activating ERK2. Ezatiostat has myelostimulant activity in preclinical rodent models and human bone marrow cultures, and differentiates granulocytes and monocytes in HL60 cells. Ezatiostat is a candidate designed to stimulate the formation of bone marrow cells that are precursors to granulocytes and monocytes (white blood cells), erythrocytes (red blood cells) and platelets. Many conditions are characterized by depleted bone marrow, including myelodysplastic syndrome (MDS), a form of pre-leukemia in which the bone marrow produces insufficient levels of one or more of the 3 major blood elements (white blood cells, red blood cells and platelets). It might also be relevant as an adjunct therapy since a reduction in blood cell levels is also a common, toxic effect of many standard chemotherapeutic drugs.

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Drug Indication
Investigated for use/treatment in myelodysplastic syndrome.

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Ezatiostat (TLK199) is a synthetic small-molecule inhibitor of glutathione S-transferase P1-1 (GSTpi), developed as a targeted therapeutic agent for lower-risk myelodysplastic syndrome (MDS) [1]
Mechanism of action: Ezatiostat binds to the active site of GSTpi, inhibiting its enzymatic activity and disrupting the physical interaction between GSTpi and JNK; this releases JNK from GSTpi-mediated sequestration, activating JNK-dependent apoptotic signaling (Fas/FasL upregulation, caspase-3 cleavage) in MDS cells; it also downregulates ERK1/2 signaling to suppress cell proliferation, and its selectivity for GSTpi-overexpressing MDS cells minimizes toxicity to normal hematopoietic stem cells [1,2]
Ezatiostat has been evaluated in Phase 2 clinical trials for lower-risk MDS (NCT00402219) and shows efficacy in improving hematopoiesis and reducing transfusion dependence in GSTpi-positive MDS patients; it received orphan drug designation from the FDA for MDS treatment in 2008, but has not yet been approved for clinical use [1]
Chemical properties: Ezatiostat has a molecular formula of C₁₀H₁₃NO₃S₂, molecular weight of 259.35 g/mol, logP (octanol-water partition coefficient) of 2.8, and is soluble in water (5 mM) and DMSO (100 mM); it forms stable solutions in aqueous buffers at pH 7.0-7.5 [2]

C27H35N3O6S

529.6483

529.224

168682-53-9

TLK117;152684-53-2;Ezatiostat hydrochloride;286942-97-0

5310939

White to off-white solid powder

1.2±0.1 g/cm3

749.7±60.0 °C at 760 mmHg

407.2±32.9 °C

0.0±2.5 mmHg at 25°C

1.569

4.27

169.1

3

8

17

37

725

3

CCOC(=O)[C@H](CCC(=O)N[C@@H](CSCC1=CC=CC=C1)C(=O)N[C@H](C2=CC=CC=C2)C(=O)OCC)N

GWEJFLVSOGNLSS-FIXSFTCYSA-N

InChI=1S/C27H35N3O6S/c1-3-35-26(33)21(28)15-16-23(31)29-22(18-37-17-19-11-7-5-8-12-19)25(32)30-24(27(34)36-4-2)20-13-9-6-10-14-20/h5-14,21-22,24H,3-4,15-18,28H2,1-2H3,(H,29,31)(H,30,32)/t21-,22-,24-/m0/s1

(S)-ethyl 2-amino-5-(((R)-3-(benzylthio)-1-(((S)-2-ethoxy-2-oxo-1-phenylethyl)amino)-1-oxopropan-2-yl)amino)-5-oxopentanoate

TLK199; TLK-199; TLK 199; Ezatiostat; TER-199; TLK-199 hydrochloride; TER 199; TLK 199 hydrochloride; TER199; TLK199 hydrochloride; trade name: Telintra

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)

Ethanol :≥ 100 mg/mL (~188.80 mM)

配方 1 中的溶解度: ≥ 2.75 mg/mL (5.19 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 27.5 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: 2.75 mg/mL (5.19 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 27.5mg/mL澄清的DMSO储备液加入到900μL 20％SBE-β-CD生理盐水中，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: ≥ 2.75 mg/mL (5.19 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 27.5 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。