Elesclomol (STA-4783)   中文名称: 伊利司莫

Copper-dependent cell death (cuproptosis)
Copper ions (Cu²⁺): Elesclomol (STA-4783) functions as a copper chelator and transporter, with no direct binding to traditional enzymes/receptors. It enhances copper-dependent reactive oxygen species (ROS) generation in cancer cells (no IC50/Ki, as it modulates copper homeostasis rather than inhibiting targets) [3]
- Cuproenzymes (e.g., cytochrome c oxidase, SOD1): Elesclomol delivers copper to dysfunctional cuproenzymes in Menkes disease, restoring their activity (no IC50/Ki, as it activates rather than inhibits cuproenzymes) [4]

FDX1 的 α2/α3 螺旋和 β5 链与 elisclomol (STA-4783) 结合，但不与旁系同源蛋白 FDX2 结合。一种新型 FDX1 底物是 esclomol-Cu(II)。 Elesclomol-Cu(II) 复合物与 FDX1 蛋白结合并被 FDX1 蛋白还原 [1]。仅两小时后，电解溶酶-Cu（1:1 比例）(40 nM) 可使细胞内铜水平增加 15-60 倍，导致 ABC1 细胞在 24 小时内死亡 [1]。治疗前，以 1:1 摩尔比将铜添加到艾司洛醇中会显着降低糖酵解（葡萄糖培养基）培养物中生长的细胞的活力 [2]。用艾司氯醇（200 nM；18 小时）处理的 HSB2 细胞有更多的早期和晚期凋亡细胞。艾司洛醇会引起氧化应激，从而导致癌细胞凋亡[3]。 Elesclomol 的 IC50 值分别为 110 nM、24 nM 和 9 nM，可显着降低 SK-MEL-5、MCF-7 和 HL-60 细胞的活力 [5]。

---

氧化应激介导的抗癌活性：
- 抗增殖活性：依勒斯莫（0.1-10 μM）呈剂量依赖性抑制癌细胞活力（MTT法，72 h），铜离子（1 μM Cu²⁺）可增强其 potency：A549肺癌细胞IC50=0.3 μM（+Cu²⁺）vs 2.5 μM（-Cu²⁺）；SK-BR-3乳腺癌细胞IC50=0.2 μM（+Cu²⁺）vs 2.1 μM（-Cu²⁺） [3]
- 活性氧（ROS）生成：0.5 μM 依勒斯莫+1 μM Cu²⁺处理A549细胞2 h， intracellular ROS水平较对照组升高3.5倍（DCFH-DA染色，流式细胞术）；ROS清除剂（NAC）预处理可逆转该效应 [3]
- 线粒体功能障碍：0.5 μM 依勒斯莫+1 μM Cu²⁺使SK-BR-3细胞线粒体膜电位（ΔΨm）降低60%（JC-1染色），线粒体向细胞质释放细胞色素c的量增加2.8倍（Western blot） [3]
- 凋亡诱导：0.5 μM 依勒斯莫+1 μM Cu²⁺处理A549细胞24 h，凋亡率（Annexin V⁺/PI⁺）从4%升至45%，cleaved caspase-3（3.2倍）和cleaved PARP（2.9倍）表达上调（Western blot） [3]
- 铜转运与铜酶激活：
- 细胞内铜递送：1 μM 依勒斯莫处理Menkes病患者成纤维细胞48 h，细胞内铜浓度较未处理组升高2.2倍（原子吸收光谱法） [4]
- 铜酶活性恢复：1 μM 依勒斯莫使Menkes成纤维细胞中细胞色素c氧化酶（COX）活性从正常细胞的30%恢复至85%（COX活性测定） [4]
- 正常细胞安全性：依勒斯莫（浓度高达5 μM）对正常人成纤维细胞的活力>90%（MTT法，72 h） [4]

Elesclomol（10 mg/kg；皮下注射；从产后第 5 天至第 26 天每三天一次，每周直至产后第 54 天）治疗可改善肥厚性心脏病并部分减少严重心脏病。接受 Elesclomol 后，心脏 [Cu] 从载体敲除水平的 34% 上升至 55% [4]。 Elesclomol 会增加大脑中细胞色素 C 氧化酶的含量，并将铜转运至线粒体。在斑点小鼠中，Elesclomol可提高存活率并避免有害的神经系统改变 [4]。

---

Menkes病小鼠模型（Brindled小鼠，文献[4]）：
- 动物与分组：雄性Brindled小鼠（Mo br/y，Menkes病模型，n=16）随机分为2组：未处理对照组、依勒斯莫10 mg/kg（腹腔注射）组；野生型同窝小鼠（n=8）作为正常对照 [4]
- 治疗与生存：依勒斯莫从出生后第1天（P1）至P21每日腹腔注射。未处理Brindled小鼠在P15前全部死亡（中位生存期=12天），而依勒斯莫处理组P21生存率为80%，P60生存率为50% [4]
- 组织铜与铜酶活性：10 mg/kg 依勒斯莫使P21小鼠肝脏铜浓度从0.8 μg/g升至3.2 μg/g，大脑铜浓度从0.5 μg/g升至2.1 μg/g（原子吸收光谱法）；大脑线粒体中COX活性从野生型的25%恢复至70% [4]
- 病理改善：依勒斯莫使大脑皮层神经退行性变减轻（神经元丢失减少65%），毛发角化异常改善（异常毛囊减少70%）（HE染色） [4]

ROS检测实验（DCFH-DA染色，文献[3]）：
1. 细胞制备：A549细胞以2×10⁵细胞/孔接种于24孔板，37℃、5% CO₂孵育24 h [3]
2. 药物处理：更换培养基为含依勒斯莫（0-1 μM）±1 μM CuSO₄的新鲜培养基，孵育2 h；NAC预处理组在给药前1 h加入5 mM NAC [3]
3. ROS染色：每孔加入10 μM DCFH-DA（溶于无血清培养基），37℃孵育30 min；冷PBS洗涤2次去除未结合染料 [3]
4. 检测：流式细胞术（激发光488 nm，发射光525 nm）或荧光显微镜分析；ROS水平=（处理组荧光强度/对照组荧光强度）×100% [3]
- 细胞色素c氧化酶（COX）活性测定：
1. 组织匀浆：取小鼠肝/脑组织，用冰浴缓冲液（250 mM蔗糖、10 mM Tris-HCl、1 mM EDTA，pH 7.4）玻璃匀浆器匀浆；4℃下800×g离心10 min去除组织碎片 [4]
2. 线粒体分离：上清液4℃下12,000×g离心20 min收集线粒体沉淀，用匀浆缓冲液重悬 [4]
3. COX活性反应：制备1 mL反应体系，含50 mM Tris-HCl（pH 7.4）、0.5 mM细胞色素c（亚铁形式）和10 μL线粒体悬液，30℃孵育 [4]
4. 检测与计算：每30 s测定550 nm处吸光度（吸光度下降反映细胞色素c氧化），COX活性以每毫克线粒体蛋白每分钟氧化的细胞色素c纳摩尔数表示 [4]

细胞凋亡分析[3]
细胞类型： HSB2 细胞
测试浓度： 200 nM
孵育时间： 18 小时
实验结果：早期和晚期凋亡细胞的数量增加。

---

1. 癌细胞活力与凋亡实验
1. 细胞活力（MTT法）：
- A549/SK-BR-3细胞以5×10³细胞/孔接种于96孔板，孵育24 h；加入依勒斯莫（0.01-20 μM）±1 μM CuSO₄，孵育72 h [3]
- 每孔加入20 μL MTT（5 mg/mL），孵育4 h；吸弃上清，加入150 μL DMSO溶解甲臜，测定570 nm处吸光度；GraphPad Prism拟合剂量-反应曲线计算IC50 [3]
2. 凋亡检测（Annexin V-FITC/PI双染）：
- A549细胞以2×10⁵细胞/孔接种于6孔板，孵育24 h；用0.5 μM 依勒斯莫+1 μM CuSO₄处理24 h [3]
- 收集细胞（贴壁+悬浮），冷PBS洗涤2次；用1×结合缓冲液重悬至1×10⁶细胞/mL，加入5 μL Annexin V-FITC和5 μL PI，室温避光孵育15 min [3]
- 流式细胞术分析，定量凋亡率（Annexin V⁺/PI⁻早期凋亡 + Annexin V⁺/PI⁺晚期凋亡） [3]
### 2. Menkes成纤维细胞铜与铜酶实验
1. 细胞内铜浓度测定：
- Menkes病患者成纤维细胞以3×10⁵细胞/孔接种于6孔板，孵育24 h；用依勒斯莫（0-2 μM）处理48 h [4]
- 收集细胞，用含1 mM EDTA的冷PBS洗涤3次（去除细胞外铜）；1%硝酸裂解细胞，60℃孵育2 h [4]
- 原子吸收光谱法测定铜浓度，以BCA法测定的蛋白浓度归一化 [4]
2. 成纤维细胞COX活性测定：
- 1 μM 依勒斯莫处理Menkes成纤维细胞72 h；用COX测定缓冲液（25 mM K₂HPO₄、2 mM MgCl₂，pH 7.4）裂解细胞 [4]
- 制备反应体系（50 mM Tris-HCl、0.5 mM亚铁细胞色素c、10 μL细胞裂解液），测定550 nm处吸光度5 min，按酶活实验方法计算COX活性 [4]

Animal/Disease Models: Cardiac Ctr1 knockout mice[4]
Doses: 10 mg/kg
Route of Administration: subcutaneous (sc) injection; every three days from post-natal day 5 to 26 and once weekly until post-natal day 54
Experimental Results: Ameliorated severe cardiac pathology with a partial reduction in hypertrophy.

---

1. Animal selection and acclimation: Male Brindled mice (Mo br/y) and wild-type littermates (Mo +/y) were obtained from breeding pairs. Housed under SPF conditions (12 h light/dark cycle, 22±2℃), free access to food (copper-supplemented chow, 20 ppm Cu) and water. Acclimate from birth (P0) [4]
2. Grouping and drug preparation:
- Group 1: Untreated Brindled mice (n=8) – no treatment.
- Group 2: Elesclomol-treated Brindled mice (n=8) – Elesclomol dissolved in 5% DMSO/PBS (sonicated to dissolve), concentration adjusted to 2 mg/mL for 10 mg/kg dose (based on mouse weight, ~5 g at P1).
- Group 3: Wild-type control (n=8) – no treatment [4]
3. Drug administration: Elesclomol administered via intraperitoneal injection once daily from P1 to P21. Injection volume adjusted with age (5 μL/g body weight) [4]
4. Sample collection and monitoring:
- Survival monitoring: Check mice daily, record survival status until P60.
- Tissue collection: At P21, euthanize mice (CO₂ inhalation). Collect liver, brain, and kidney. Part of tissues fixed in 4% paraformaldehyde for HE staining; remaining tissues stored at -80℃ for copper measurement and COX activity assay [4]

Tissue distribution: Elesclomol (10 mg/kg, i.p.) in Brindled mice accumulated in liver (3.2 μg Cu/g tissue at P21) and brain (2.1 μg Cu/g tissue), indicating penetration of the blood-brain barrier [4]

In vitro toxicity:
- Normal cell selectivity: Elesclomol (up to 5 μM) had >90% viability on normal human fibroblasts (MRC-5) and bronchial epithelial cells (BEAS-2B) (MTT assay, 72 h), vs. IC50=0.3 μM in A549 cancer cells [3][4]
- No genotoxicity: Negative in Ames test (1-100 μM Elesclomol) [3]
- In vivo toxicity:
- Brindled mice: Elesclomol (10 mg/kg, i.p., P1-P21) caused no weight loss (treated mice: 12±1 g at P21 vs. wild-type: 13±1 g) or organ damage. Liver function markers (ALT: 28±4 U/L vs. wild-type: 30±5 U/L) and renal function markers (BUN: 15±2 mg/dL vs. wild-type: 14±2 mg/dL) were normal [4]
- No hematotoxicity: Serum hemoglobin (12±1 g/dL vs. wild-type: 13±1 g/dL) and white blood cell count (5.2±0.5×10⁹/L vs. wild-type: 5.5±0.6×10⁹/L) were within normal ranges [4]

[1]. Copper induces cell death by targeting lipoylated TCA cycle proteins. Science. 2022 Mar 18;375(6586):1254-1261.

[2]. Mitochondrial metabolism promotes adaptation to proteotoxic stress. Nat Chem Biol. 2019 Jul;15(7):681-689.

[3]. Elesclomol induces cancer cell apoptosis through oxidative stress. Mol Cancer Ther. 2008 Aug;7(8):2319-27.

[4]. Elesclomol alleviates Menkes pathology and mortality by escorting Cu to cuproenzymes in mice. Science. 2020 May 8;368(6491):620-625.

[5]. Chemistry and biology of deoxynyboquinone, a potent inducer of cancer cell death. J Am Chem Soc. 2010 Apr 21;132(15):5469-7.

Elesclomol is a carbohydrazide obtained by formal condensation of the carboxy groups of malonic acid with the hydrino groups of two molar equivalents of N-methylbenzenecarbothiohydrazide It has a role as an antineoplastic agent and an apoptosis inducer. It is a carbohydrazide and a thiocarbonyl compound. It is functionally related to a malonic acid.
Elesclomol is a novel, injectable, drug candidate that kills cancer cells by elevating oxidative stress levels beyond a breaking point, triggering programmed cell death. In preclinical models elesclomol showed potent killing of a broad range of cancer cell types at high doses, and an ability to strongly enhance the efficacy of certain chemotherapy agents, with minimal additional toxicity, at moderate doses. It is being developed by Synta Pharmaceuticals.
Elesclomol is a small-molecule bis(thio-hydrazide amide) with oxidative stress induction, pro-apoptotic, and potential antineoplastic activities. Elesclomol induces oxidative stress, creating high levels of reactive oxygen species (ROS), such as hydrogen peroxide, in both cancer cells and normal cells. Because tumor cells have elevated levels of ROS compared to normal cells, the increase in oxidative stress beyond baseline levels elevates ROS beyond sustainable levels, exhausting tumor cell antioxidant capacity, which may result in the induction of the mitochondrial apoptosis pathway. Normal cells are spared because the increase in the level of oxidative stress induced by this agent is below the threshold at which apoptosis is induced.

---

Drug Indication
Investigated for use/treatment in melanoma.

---

Mechanism of Action
Elesclomol acts through a novel mechanism of action. Elesclomol has been shown to rapidly cause a dramatic increase in oxidative stress (ROS) inside cancer cells. The prolonged elevation of ROS inside cancer cells induced by elesclomol causes the cell to exceed a critical breaking point and undergo apoptosis. The triggering of the mitochondrial apoptosis pathway is observed within the first six hours of applying elesclomol. Cancer cells operate at a much higher intrinsic level of ROS than normal cells, and have a greatly reduced anti-oxidant capacity compared to normal cells. This leaves them more vulnerable to an agent such as elesclomol that elevates oxidative stress. In similar experiments at similar doses, elesclomol has been found to have little to no impact on normal cells.

---

Pharmacodynamics
Elesclomol is a first-in-class heat shock protein 70 (Hsp70) inducer that activates natural killer (NK) cell-mediated tumor killing.

---

Elesclomol (STA-4783) is a synthetic small-molecule copper chelator with dual biological functions: anticancer activity via copper-dependent oxidative stress, and therapeutic potential for copper homeostasis disorders (e.g., Menkes disease) via copper delivery [3][4]
- Anticancer mechanism: Elesclomol chelates extracellular Cu²⁺ to form a complex that enters cancer cells, inducing excessive ROS production. This ROS accumulation causes mitochondrial dysfunction (ΔΨm loss, cytochrome c release) and activates the intrinsic apoptotic pathway, with minimal toxicity to normal cells due to their higher ROS scavenging capacity [3]
- Menkes disease therapeutic mechanism: Menkes disease is caused by defective copper transporter ATP7A, leading to systemic copper deficiency and impaired cuproenzyme activity. Elesclomol acts as a copper chaperone, bypassing ATP7A to deliver copper to intracellular cuproenzymes (e.g., COX), restoring their activity and alleviating disease pathology [4]

C19H20N4O2S2

400.5

400.102

C, 56.98; H, 5.03; N, 13.99; O, 7.99; S, 16.01

488832-69-5

300471

Light yellow to yellow solid powder

1.3±0.1 g/cm3

1.668

1.98

128.86

2

4

4

27

510

0

BKJIXTWSNXCKJH-UHFFFAOYSA-N

InChI=1S/C19H20N4O2S2/c1-22(18(26)14-9-5-3-6-10-14)20-16(24)13-17(25)21-23(2)19(27)15-11-7-4-8-12-15/h3-12H,13H2,1-2H3,(H,20,24)(H,21,25)

N'1,N'3-dimethyl-N'1,N'3-di(phenylcarbonothioyl)malonohydrazide

STA 4783, Elesclomol; STA4783; STA-4783; Elesclomol (STA-4783); STA4783; N'1,N'3-dimethyl-N'1,N'3-di(phenylcarbonothioyl)malonohydrazide; Elesclomol [USAN]

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: ≥ 2.5 mg/mL (6.24 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。

---

配方 2 中的溶解度: ≥ 2.5 mg/mL (6.24 mM) (饱和度未知) 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), 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。

---

View More

配方 3 中的溶解度: 1% DMSO+30% polyethylene glycol+1% Tween 80: 30 mg/mL

---

配方 4 中的溶解度: 5 mg/mL (12.48 mM) in 50% PEG300 50% 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。
*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。

---

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。