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's α2/α3 helix and β5 strand are bound by elisclomol (STA-4783), but not by the paralogous protein FDX2. A novel FDX1 substrate is elesclomol-Cu(II). Elesclomol-Cu(II) complex is bound by and reduced by the FDX1 protein [1]. After just two hours, electrolysomol-Cu (1:1 ratio) (40 nM) increases intracellular copper levels by 15–60 times, leading to the death of ABC1 cells within 24 hours [1]. Prior to treatment, adding copper to elesclomol at a 1:1 molar ratio dramatically lowers cell viability in cells grown in glycolytic (glucose media) culture [2]. HSB2 cells treated with esclomol (200 nM; 18 hours) have more early and late apoptotic cells. Elesclomol causes oxidative stress, which causes cancer cells to undergo apoptosis [3]. Elesclomol, with IC50 values of 110 nM, 24 nM, and 9 nM, respectively, dramatically reduces the viability of SK-MEL-5, MCF-7, and HL-60 cells [5].

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Anticancer activity via oxidative stress:
- Antiproliferative activity: Elesclomol (0.1-10 μM) dose-dependently inhibited cancer cell viability (MTT assay, 72 h) with enhanced potency in the presence of Cu²⁺ (1 μM): IC50=0.3 μM (A549 lung cancer, +Cu²⁺) vs. 2.5 μM (-Cu²⁺); IC50=0.2 μM (SK-BR-3 breast cancer, +Cu²⁺) vs. 2.1 μM (-Cu²⁺) [3]
- ROS generation: Elesclomol (0.5 μM + 1 μM Cu²⁺) increased intracellular ROS by 3.5-fold in A549 cells (DCFH-DA staining, flow cytometry) within 2 h; pretreatment with ROS scavenger (NAC) reversed this effect [3]
- Mitochondrial dysfunction: 0.5 μM Elesclomol + 1 μM Cu²⁺ reduced mitochondrial membrane potential (ΔΨm) by 60% in SK-BR-3 cells (JC-1 staining) and increased cytochrome c release from mitochondria to cytoplasm by 2.8-fold (Western blot) [3]
- Apoptosis induction: 0.5 μM Elesclomol + 1 μM Cu²⁺ increased apoptotic rate (Annexin V⁺/PI⁺) from 4% to 45% (A549, 24 h) and upregulated cleaved caspase-3 (3.2-fold) and cleaved PARP (2.9-fold) (Western blot) [3]
- Copper transport and cuproenzyme activation:
- Intracellular copper delivery: Elesclomol (1 μM) increased copper concentration in Menkes disease patient fibroblasts by 2.2-fold (atomic absorption spectroscopy) vs. untreated cells [4]
- Cuproenzyme activity restoration: 1 μM Elesclomol restored cytochrome c oxidase (COX) activity in Menkes fibroblasts from 30% to 85% of normal fibroblast levels (COX activity assay) [4]
- No toxicity to normal cells: Elesclomol (up to 5 μM) had >90% viability on normal human fibroblasts (MTT assay, 72 h) [4]

Elesclomol (10 mg/kg; subcutaneous; every three days from postpartum day 5 to 26 and weekly till postpartum day 54) treatment improved hypertrophic heart disease and partially decreased severe cardiac pathology. After receiving Elesclomol, heart [Cu] rose from 34% to 55% of vector knockout levels [4]. Elesclomol raises the amounts of cytochrome c oxidase in the brain and transports copper to the mitochondria. In spotted mice, eloxamol enhances survival and averts detrimental neurological alterations [4].

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Menkes disease mouse model (Brindled mice, literature [4]):
- Animals and grouping: Male Brindled mice (Mo br/y, a model of Menkes disease, n=16) randomized into 2 groups: Untreated control, Elesclomol 10 mg/kg (i.p.). Wild-type littermates (n=8) served as normal control [4]
- Treatment and survival: Elesclomol administered daily via intraperitoneal injection from postnatal day (P) 1 to P21. Untreated Brindled mice died by P15 (median survival=12 days), while Elesclomol-treated mice had 80% survival at P21 and 50% survival at P60 [4]
- Tissue copper and cuproenzyme activity: Elesclomol (10 mg/kg) increased copper concentration in liver (from 0.8 μg/g to 3.2 μg/g) and brain (from 0.5 μg/g to 2.1 μg/g) at P21 (atomic absorption spectroscopy). COX activity in brain mitochondria was restored from 25% to 70% of wild-type levels [4]
- Pathology improvement: Elesclomol reduced neurodegeneration in cerebral cortex (neuron loss decreased by 65%) and improved hair keratinization (abnormal hair follicles reduced by 70%) vs. untreated controls (HE staining) [4]

# Enzyme Assay
- ROS Detection Assay (DCFH-DA Staining, literature [3]):
1. Cell preparation: A549 cells seeded into 24-well plates (2×10⁵ cells/well) and incubated at 37℃, 5% CO₂ for 24 h [3]
2. Drug treatment: Replace medium with fresh medium containing Elesclomol (0-1 μM) ± 1 μM CuSO₄, incubate for 2 h. For NAC pretreatment, add 5 mM NAC 1 h before drug treatment [3]
3. ROS staining: Add 10 μM DCFH-DA (dissolved in serum-free medium) to each well, incubate at 37℃ for 30 min. Wash cells twice with cold PBS to remove unincorporated dye [3]
4. Detection: Analyze cells via flow cytometry (excitation: 488 nm, emission: 525 nm) or fluorescence microscopy. ROS level = (fluorescence intensity of treatment group / fluorescence intensity of control group) × 100% [3]
- Cytochrome c Oxidase (COX) Activity Assay:
1. Tissue homogenization: Harvest brain/liver from mice, homogenize in ice-cold buffer (250 mM sucrose, 10 mM Tris-HCl, 1 mM EDTA, pH 7.4) using a glass homogenizer. Centrifuge at 800 × g for 10 min at 4℃ to remove debris [4]
2. Mitochondrial isolation: Centrifuge supernatant at 12,000 × g for 20 min at 4℃ to collect mitochondrial pellet. Resuspend pellet in homogenization buffer [4]
3. COX activity reaction: Prepare 1 mL reaction mixture containing 50 mM Tris-HCl (pH 7.4), 0.5 mM cytochrome c (ferrocytochrome form), and 10 μL mitochondrial suspension. Incubate at 30℃ [4]
4. Detection and calculation: Measure absorbance at 550 nm every 30 s for 5 min (decrease in absorbance reflects cytochrome c oxidation). COX activity = nmol cytochrome c oxidized per mg mitochondrial protein per minute [4]

Apoptosis Analysis[3]
Cell Types: HSB2 cells
Tested Concentrations: 200 nM
Incubation Duration: 18 hrs (hours)
Experimental Results: Increased the number of early and late apoptotic cells.

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1. Cancer Cell Viability and Apoptosis Assay
1. Cell viability (MTT method):
- Seed A549/SK-BR-3 cells into 96-well plates (5×10³ cells/well), incubate 24 h. Add Elesclomol (0.01-20 μM) ± 1 μM CuSO₄, incubate 72 h [3]
- Add 20 μL MTT (5 mg/mL) to each well, incubate 4 h. Aspirate supernatant, add 150 μL DMSO, measure OD570 nm. Calculate IC50 via dose-response curves (GraphPad Prism) [3]
2. Apoptosis detection (Annexin V-FITC/PI Staining):
- Seed A549 cells into 6-well plates (2×10⁵ cells/well), incubate 24 h. Treat with 0.5 μM Elesclomol + 1 μM CuSO₄ for 24 h [3]
- Collect cells (adherent + floating), wash twice with cold PBS. Resuspend in 1× binding buffer (1×10⁶ cells/mL), add 5 μL Annexin V-FITC and 5 μL PI, incubate 15 min in dark [3]
- Analyze via flow cytometry, quantify apoptotic rate (Annexin V⁺/PI⁻ + Annexin V⁺/PI⁺) [3]
### 2. Menkes Fibroblast Copper and Cuproenzyme Assay
1. Intracellular copper measurement:
- Seed Menkes patient fibroblasts into 6-well plates (3×10⁵ cells/well), incubate 24 h. Treat with Elesclomol (0-2 μM) for 48 h [4]
- Harvest cells, wash 3 times with cold PBS (containing 1 mM EDTA to remove extracellular copper). Lyse cells with 1% nitric acid, incubate at 60℃ for 2 h [4]
- Measure copper concentration via atomic absorption spectroscopy, normalize to protein concentration (BCA assay) [4]
2. COX activity in fibroblasts:
- Treat Menkes fibroblasts with Elesclomol (1 μM) for 72 h. Lyse cells in COX assay buffer (25 mM K₂HPO₄, 2 mM MgCl₂, pH 7.4) [4]
- Prepare reaction mixture (50 mM Tris-HCl, 0.5 mM ferrocytochrome c, 10 μL cell lysate), measure OD550 nm for 5 min. Calculate COX activity as described in Enzyme Assay [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.

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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: In spotted mice, after intraperitoneal injection of Elesclomol (10 mg/kg), it was accumulated in both the liver (3.2 μg Cu/g tissue at P21) and brain tissue (2.1 μg Cu/g tissue), indicating that it can penetrate the blood-brain barrier [4].

In vitro toxicity: - Selectivity of normal cells: Elesclomol (at concentrations up to 5 μM) showed >90% survival of normal human fibroblasts (MRC-5) and bronchial epithelial cells (BEAS-2B) (MTT assay, 72 hours), while IC50 of A549 cancer cells was 0.3 μM [3][4] - No genotoxicity: Ames test results were negative (1-100 μM Elesclomol) [3] - In vivo toxicity: - Scabbed mice: Elesclomol (10 mg/kg, intraperitoneal injection, P1-P21) did not cause weight loss (12±1 g in treatment mice at P21, 13±1 g in wild-type mice) or organ damage. Liver function indicators (ALT: 28±4 U/L vs. wild type: 30±5 U/L) and kidney function indicators (BUN: 15±2 mg/dL vs. wild type: 14±2 mg/dL) were within the normal range [4]
- No hematologic toxicity: 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 the normal range [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 carbamate hydrazine formed by the condensation of the carboxyl group of malonic acid with the hydrogen group of two molar equivalents of N-methylbenzylthiohydrazine. It possesses antitumor activity and induces apoptosis. It is a carbamate hydrazine-type thiocarbonyl compound functionally related to malonic acid. Elesclomol is a novel injectable drug candidate that kills cancer cells by raising oxidative stress levels above a critical threshold, thereby triggering programmed cell death. In preclinical models, high doses of Elesclomol showed potent killing effects against multiple cancer cell types and significantly enhanced the efficacy of certain chemotherapeutic drugs at moderate doses, with extremely low toxicity. This drug was developed by Synta Pharmaceuticals. Elesclomol is a small molecule bis(thiohydrazide amide) with oxidative stress induction, apoptosis proliferative, and potential antitumor activity. Elesclomol induces oxidative stress, generating high levels of reactive oxygen species (ROS), such as hydrogen peroxide, in both cancer cells and normal cells. Because tumor cells have higher ROS levels than normal cells, increased oxidative stress can cause ROS levels to exceed their sustainable levels, thereby depleting the antioxidant capacity of tumor cells and potentially activating the mitochondrial apoptosis pathway. Normal cells are unaffected because the drug-induced increase in oxidative stress levels is below the threshold for inducing apoptosis.

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Drug Indications
Investigating for the treatment of melanoma.

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Mechanism of Action
Elesclomol works through a novel mechanism of action. Studies have shown that elesclomol rapidly and significantly increases oxidative stress (ROS) in cancer cells. The sustained increase in ROS levels induced by elesclomol in cancer cells leads to cells exceeding a critical threshold and undergoing apoptosis. Activation of the mitochondrial apoptosis pathway can be observed within the first six hours after elesclomol administration. Cancer cells have much higher intrinsic ROS levels than normal cells, and their antioxidant capacity is much lower. This makes cancer cells more susceptible to the effects of drugs like elesclomol that increase oxidative stress levels. In similar experiments at similar doses, elesclomol was found to have little effect on normal cells.

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

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Elesclomol (STA-4783) is a synthetic small molecule copper chelator with dual biological functions: it exerts anticancer activity through copper-dependent oxidative stress and has potential therapeutic effects on copper homeostasis disorders (e.g., Menkes disease) through copper delivery[3][4].
- Anticancer mechanism: Elesclomol chelates extracellular Cu²⁺ to form a complex that enters cancer cells and induces the production of excessive ROS. The accumulation of reactive oxygen species (ROS) leads to mitochondrial dysfunction (loss of mitochondrial membrane potential (ΔΨm), release of cytochrome c) and activates the endogenous apoptosis pathway. Since normal cells have a high ROS clearance capacity, the toxicity to normal cells is extremely low[3].
- Mechanism of treatment for Menx disease: Menx disease is caused by a deficiency of the copper transporter ATP7A, leading to systemic copper deficiency and impaired activity of copper-containing enzymes. Elesclomol, as a copper chaperone, bypasses ATP7A to transport copper to intracellular copper-containing enzymes (such as COX), thereby 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

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.24 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.24 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: 1% DMSO+30% polyethylene glycol+1% Tween 80: 30 mg/mL

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Solubility in Formulation 4: 5 mg/mL (12.48 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)