Copper (Cu) is an indispensable co-factor of various crucial metabolic enzymes in a great deal of physiological processes, they will flow into metabolic disorders with aberrant Cu homeostasis (Xie et al., 2023). Thus the Cu level in human body must be maintained within a balanced range to ensure the normal cellular activities. Shellfish and organ meats tend to be the most abundant food sources for human absorbing Cu, and the intake of Cu mainly occurs in small intestine epitheliums, which should be recommended 0.8–2.4 mg/day for adult to maintain Cu homeostasis (Bost et al., 2016; Xie et al., 2023). The intracellular Cu level is regulated by a complex network of cuproenzymes, membrane transporters and Cu chaperones, which together coordinate the transport, reservation and intracellular metabolism of Cu, thereby keeping intracellular Cu level within a balanced range to prevent the damages of Cu overload and deficiency. With the assistance of specific Cu chaperones, intracellular Cu(I) are transported to the specific metabolic processes in cytoplasm and nucleus through activating the target enzymes: antioxidant-1 (ATOX1) carries Cu(I) to nucleus (gene regulation) and Golgi apparatus (protein folding), copper chaperone for superoxide dismutase (CCS) carries Cu(I) to superoxide dismutase (SOD) (redox homeostasis), and cytochrome c oxidase 17 (COX17) carries Cu(I) to mitochondria (mitochondrial respiration) (Figure 1) (Chen et al., 2022).

Cu-dependent cell death found in 2022 is a novel non-apoptotic programmed cell death, which accelerates the development of field of Cu homeostasis imbalance. There are few known mammalian lipoylated proteins and most of them are concentrated in tricarboxylic acid (TCA) cycle, while cells with active mitochondrial respiration show elevated lipoylated proteins (Tsvetkov et al., 2022; Chen et al., 2022; Xie et al., 2023). Excess Cu ions aggravate the insoluble aggregation of lipoylated proteins, reduce Fe-S cluster protein, cause proteotoxic stress, elevate heat shock protein 70 (HSP70) and reactive oxygen species (ROS), ultimately leading to cell death effects, named cuproptosis. The discovery of cuproptosis is inseparable from the progress of previous studies on Cu homeostasis and Cu-induced cell death, involving Cu-mediated diseases [Wilson disease (Wilson, 1934), Menkes disease (Danks et al., 1972)], membrane transporters [ATP7A (Vulpe et al., 1993; Chelly et al., 1993; Mercer et al., 1993), ATP7B (Bull et al., 1993; Petrukhin et al., 1993; Tanzi et al., 1993), solute carrier family 31 member 1 (SLC31A1) (Dancis et al., 1994)], anti-cancer agents of Cu complexes [disulfiram (Chen et al., 2006), elesclomol (Nagai et al., 2012)], action mode between elesclomol-Cu(II) and ferredoxin 1 (FDX1) (Tsvetkov et al., 2019) (Figure 2).

Based on the characteristic changes, cuproptosis is a new form of Cu-dependent cell death distinct from traditional cell death including apoptosis, autophagy, ferroptosis, pyroptosis, necrosis, and Cu homeostasis imbalance will lead to some adverse health effects, involving genetic, neurodegenerative, cardiovascular, tumoral diseases (Tsvetkov et al., 2022; Chen et al., 2022). For genetic diseases: Menkes disease and Wilson disease, caused by mutations in ATP7A/7B gene, generalize severe organ Cu deficiency or overload. For neurodegenerative diseases: in Alzheimer’s disease, Cu ions may interplay with the key causative factors like amyloid-β peptides and tau protein; in amyotrophic lateral sclerosis, Cu deficiency is reported to promote the aberrant hydrophobicity of pathogenic mutant SOD1; in Huntington’s disease, Cu overload occurs in the striatum of patients and mouse models. For cardiovascular diseases: excess Cu in relation to atherosclerosis while Cu deficiency in relation to cardiac hypertrophy. For tumoral diseases: Cu is involved in the malignant progression of cancer by facilitating cell proliferation, angiogenesis and metastasis. Hence, comprehensive exploration of potential mechanism of Cu homeostasis in disease development, and in-depth study of Cu homeostasis-associated regulatory pathways under different pathological areas have grand medical value and translational significance.

With the emergency of cuproptosis, scientific communities pay more attention to Cu-induced cell death owing to its enormous potential in disease target therapy. Nowadays, a growing body of studies have revealed the target molecules and metabolic pathways in response to Cu cell death effects. Meanwhile, inducers and inhibitors of cuproptosis have been widely applied in scientific and medical areas. However, the targets and metabolic pathways of Cu-dependent cell death are not totally explained, which limits the development and application of cuproptosis inducers and inhibitors. In this study, we intend to briefly summarize the essential targets and metabolic pathways of cuproptosis, then categorize commonly used and newly discovered inducers and inhibitors with the overview of principle, scientific and medical application, and discuss their future development tendency, hoping to increase the understanding of the inducers and inhibitors in research field and disease target therapy based on cuproptosis.

The metabolic homeostasis of metal elements play a key role in internal environment and normal metabolism. The overload or deficiency of metal elements will disrupt and impair metabolic homeostasis, like iron and ferroptosis (Dixon et al., 2012). Over the past decade, multiple types of ferroptosis inducers and inhibitors have been employed in practice in terms of the targets and metabolic pathways involved in Fe-dependent cell death (Du and Guo, 2022). Similarly, the strategies of cuproptosis inducers and inhibitors have been continuously improved based on the key targets in cuproptosis metabolic pathways (Figure 3): oxidative stress, mitochondrial respiration, ubiquitin-proteasome system (UPS).

The proper cuproptosis inducers should cause intracellular Cu ions overload and lead to Cu-dependent cell death via the characteristic targets and metabolic pathways of cuproptosis mentioned above. With the discovery of cuproptosis, inducers of Cu-dependent cell death receive increasing concerns and novel inducers are constantly invented. Since cells can not suffer Cu overload, it suggests that the accumulation of Cu ions enables to selectively kill the morbid cells. Therefore cuproptosis inducers show promising application values both in scientific and medical areas. And choosing an appropriate inducer to induce Cu-dependent cell death can not divorce from the properties of inducers, targets, relevant studies and diseases. In this section, based on the characteristic targets and metabolic pathways of cuproptosis, the cuproptosis inducers we collect are categorized into small molecule compounds, transcription factors and non-coding RNAs (ncRNAs) (Figure 4; Table 1).

The eligible cuproptosis inhibitors should antagonize the cell death effects of Cu overload and alleviate the cellular damages induced by cuproptosis including oxidative stress, mitochondrial respiration dysfunction and UPS inhibition. Tsvetkov and colleagues verified that the cytotoxicity of Cu ionophore was eliminated by Cu chelator (tetrathiomolybdate) rather than other pathway inhibitors including Z-VAD-FMK, Boc-D-FMK (apoptosis), ferrostatin-1 (ferroptosis) and necrostatin-1 (necroptosis), whereas other metal chelators such as deferoxamine mesylate [high affinity for Fe(III)] and TPEN [high affinity for Zn(II)] had no effect on the killing capacity induced by Cu ionophore (Tsvetkov et al., 2022). Mechanically, the Cu cell death effects depended on mitochondrial respiration and TCA cycle, since treatment with mitochondrial antioxidants, fatty acids, mitochondrial function inhibitors induced distinct effects on Cu ionophore sensitivity; and furthermore treatment with electron transport chain complexes (ETC) inhibitors, mitochondrial pyruvate uptake inhibitors attenuated the cell death; treatment with mitochondrial uncoupler FCCP had no effect on Cu cytotoxicity (Tsvetkov et al., 2022). The scientific and medical communities have continuously developed available drugs for the diseases in Cu overload, and the discovery of cuproptosis significantly accelerates the progress of cuproptosis inhibitors. Based on the targets and metabolic pathways involved, like inducers, the cuproptosis inhibitors we collect can be similarly categorized into small molecule compounds, transcription factors and ncRNAs (Figure 5; Table 2).

Cu homeostasis dysfunction and cuproptosis are closely correlated with the occurrence of some genetic, neurodegenerative, cardiovascular, tumoral diseases, and the medical options have been developed into recovering Cu homeostasis or triggering Cu dysfunction (Chen et al., 2022). For instances, in Wilson disease and Menkes disease, the pathogenic variants of ATP7A/7B disturb the intracellular Cu content and homeostasis, the treatment is to recover normal intracellular Cu content (Wilson, 1934; Danks et al., 1972); in tumoral diseases, mounting evidences suggest that Cu dyshomeostasis plays a prominent role in energy metabolism and angiogenesis, the treatment targets oxidative stress, mitochondrial respiration, UPS and angiogenesis using Cu complexes (Ge et al., 2022). Although the main morphological features of cuproptosis are similar to apoptosis, including cytomembrane/chromatin rupture, endoplasmic reticulum damage and mitochondrial contraction, but its mechanism is varied from other known cell death pathways (Liao et al., 2019; Zhao et al., 2020). Excess Cu ions induce cuproptosis by promoting the insoluble oligomerization of lipoylated proteins in TCA cycle, suppressing Fe-S cluster protein, causing proteotoxic stress and elevating HSP70, ROS (Figure 3) (Tsvetkov et al., 2022). Hence, the discovery of cuproptosis will widen the path for future drug development targeting Cu homeostasis, intensive research of the potential role of cuproptosis in diverse diseases and the molecular mechanism will contribute to exploring novel therapeutic targets and thereby driving drug innovation. Nowadays, the cuproptosis inducers and inhibitors have been continuously improved and employed for scientific studies and medical options, and in this study they are collected and categorized into small molecule compounds, transcription factors and ncRNAs, in terms of the key targets in cuproptosis metabolic pathways.

The discovery of cuproptosis by Tsvetkov P and colleagues emphasizes the regulatory mechanism of positive regulatory factors FDX1, lipoylated proteins (LIPT1, LIAS, DLD), lipoylated targets (DLAT, PDHA1, PDHB), negative regulatory factors GLS, CDKN2A, MTF1 and mitochondrial metabolism (lipoylated pathway, TCA cycle) in Cu-induced cell death (Tsvetkov et al., 2022). The cells using TCA cycle and OXPHOS as their energy production mode exhibit higher sensitivity to Cu stress than the cells relied on glycolysis. In this process, FDX1 plays a critical role in reducing Cu(II) to Cu(I), lipoylating DLAT and destabilizing Fe-S cluster protein to trigger cuproptosis. We pay more attention to small molecule compounds which influence the progress of Cu-induced cell death, rather than transcription factors, ncRNAs or other types of regulators, since they are more readily screened and dosed to produce therapeutic outcome than other types of candidates. For inducers of cuproptosis, to raise intracellular Cu content, we collect some small molecule compounds targeting Cu ionophores: elesclomol, disulfiram, clioquinol, 8-quinolinol, schiff base, pyrithione, zinc metallochaperone, Cu(II)ATSM/Cu(II)GTSM, UM4118, they are capable of delivering Cu ions into cells; Cu membrane transporters: epigallocatechol gallate, β-elemene, CID 2011756, omeprazole, 6-hydroxydopamine hydrobromide, they can upregulate SLC31A1 expression or downregulate ATP7A/7B expression to drive Cu deposition; Cu reservation: 6-hydroxydopamine hydrobromide, actinomycin D, PAPA NONOate, DL-buthionine-(S,R)-sulfoximine, they enable to aggravate the release of intracellular Cu ions; Cu chaperones: DC_AC50, it has the ability to reduce Cu trafficking by inhibiting ATOX1 and CCS. To enhance mitochondrial respiration and TCA cycle, the small molecule compounds phloretin, PX-478, 2-deoxy-D-glucose and galactose can be employed as glycolysis inhibitors to shift the energy metabolism to TCA cycle and OXPHOS. The transcription factors ELF3, Sp1, ATF3/SPI1, facilitating the expression of SLC31A1 and the ncRNAs LINC02362/miR-18a-5p, facilitating the expression of FDX1 are accompanied to enhance the sensitivity to Cu stress beyond small molecule compounds (Figure 4; Table 1). For inhibitors of cuproptosis, to reduce intracellular Cu content, we collect some small molecule compounds targeting Cu chelators: GSH, tetrathiomolybdate, bathocuproinedisulfonic acid, trientine, penicillamine, 2,6-Pyridinedicarboxylic acid, 2,6-bis[2-[(4-carboxyphenyl) methylene] hydrazide], 3,5-di-tert-butylsalicylaldehyde rhodamine hydrazone, captopril, LCC-12, curcumin, they are capable of chelating intracellular Cu ions; Cu reservation: Zinc acetate, it can elevate metallothionein level in intestinal cells. To retard mitochondrial respiration and TCA cycle, the small molecule compounds rotenone, antimycin A, UK5099 can be employed as mitochondrial respiration inhibitors to shift the energy metabolism to glycolysis. To recover oxidative homeostasis, the small molecule compounds coumarin, deferiprone, phenolic acids are helpful to eliminate the ROS derived from Cu Fenton reaction. The transcription factors HIF-1α, facilitating hypoxia, MTF1, facilitating ATP7B and metallothionein expression and the ncRNA miR-21-5p, decreasing FDX1 expression are accompanied to reduce the sensitivity to Cu stress beyond small molecule compounds (Figure 5; Table 2). The strategies to collect cuproptosis inducers originate from raising the intracellular Cu content and the intensity of mitochondrial respiration and TCA cycle, while the strategies to collect cuproptosis inhibitors originate from reducing intracellular Cu content, the intensity of mitochondrial respiration, TCA cycle, oxidative stress and UPS inhibition. Importantly, the medical values of cuproptosis inducers and inhibitors are mainly concentrated in the treatment of genetic, neurodegenerative, cardiovascular and tumoral diseases, achieving by recovering normal Cu homeostasis, inducing Cu overload or deficiency. But to date, we have not collected applicable small molecule compounds for the cuproptosis inhibitors that target Cu membrane transporters, Cu chaperones or UPS inhibition during Cu stress and they require further exploration.

It can be concluded from the studies that cuproptosis inducers and inhibitors have the potential to modulate Cu cell death effects relying on their composition and property, but the action mode may vary from distinct drug candidates and cell types. Some compounds we collect have been applied for the clinical trials of Wilson disease such as tetrathiomolybdate (NCT00004339) (Brewer et al., 2006), trientine (NCT01472874) (Ala et al., 2015), penicillamine (Schilsky et al., 2022), Zinc acetate (NCT00212355) (Shimizu et al., 2010) and reduced Cu accumulation in liver and brain through complex and metallothionein chelation, respectively. Cu overload and deficiency constitute an exploitable dependency in cancer therapy, and some Cu ionophores we collected have been pursued in cancer clinical trials and produced considerable therapeutic outcome such as elesclomol (NCT00084214, NCT00888615) (O’Day et al., 2009; Monk et al., 2018) and disulfiram (NCT01907165) (Huang et al., 2016). Hence, cuproptosis has attracted dramatic interests in providing a new avenue for medical options, and promising compounds that can modulate Cu-induced cell death in preclinical studies have been also collected and displayed in our study. We should dedicate to improve the combination strategies so that the cuproptosis inducers and inhibitors we collect can provide better therapeutic approaches. Firstly, Cu ionophore plus chemotherapy. Clinical trails had revealed encouraging efficacy for elesclomol plus paclitaxel in metastatic melanoma versus paclitaxel alone, and elesclomol had stronger anti-tumor activity in patients with lower LDH level, indicating that elesclomol was more sensitive to the cells harboring intensive mitochondrial respiration and lipoylated TCA enzymes (O’Day et al., 2009; O’Day et al., 2013). Secondly, Cu ionophore plus ferroptosis inducer. The data of Wang W et al. provided the evidence that ferroptosis inducers sorafenib and erastin reduced FDX1 degradation and GSH synthesis to enhance elesclomol-Cu-induced cell death in liver cancer cells, suggesting a link between cuproptosis and ferroptosis in the therapy depending on metal element dysfunction (Wang W. et al., 2023). Thirdly, Cu complexes have great potential use for disease treatment, however, it is important to note that long-term use of Cu complexes can disrupt the base metal homeostasis and cause serious side effects in patients undergoing treatment. This field is in its early stage of development and lack of specificity is a major challenge. The scientific communities have developed innovative nanomedicine delivery vectors to improve the specificity of Cu complexes to morbid cells and obtain more accurate therapeutic outcome. Cui L and colleagues formulated a mitochondria-targeted Cu depleting nanoparticle consisting of Cu chelator, semiconducting polymers and phospholipid-polyethylene glycol, the positive surface charge favored its accumulation in mitochondria and herein reduced the Cu content, which dampened OXPHOS activity and drived oxidative stress to inhibit triple-negative breast cancer cells with minimal side effects (Cui et al., 2021). Therefore, nanocarriers selectively delivering cuproptosis inducers and inhibitors to the lesions can elevate precise therapeutic outcome and reduce toxicity for medical applications based on Cu cell death effects, but the composition, property, cell type of these delivery system, and further clinical trials still necessitate in-depth exploration.

In addition to the small molecule compounds, transcription factors and ncRNAs we collect in this study, we note that other types of cuproptosis-related molecules can also modulate Cu cell death effects. Proteases are such a kind of potential drug target. For instances, the phosphodiesterase 3B has been confirmed to increase the sensitivity of Cu ionophore to bladder cancer cells (Feng et al., 2024); the methyltransferase METTL16 is reported to promote FDX1 mRNA stability via m⁶A modification and FDX1 accumulation to enhance the sensitivity of elesclomol treatment in gastric cancer (Sun et al., 2023). Transcription factors, ncRNAs and proteases have been taken into therapeutic consideration for cuproptosis, however, it remains a big challenge to successfully deliver gene expression vectors or ncRNAs in human body. Chiefly, small molecule compounds are strong candidates in regulating Cu-induced cell death owing to the suitable screen and administration routes. The candidate compounds can be excavated from rounds of verification: bioinformatic analysis, molecular simulation and docking, interaction analysis. In this study, we have collected kinds of cuproptosis inducers and inhibitors based on the key targets and metabolic pathways of Cu-induced cell death, but the combination strategy and drug carrier remain incompletely documented, which may thus be promising tools for curing patients with Cu homeostasis dysfunction. Moreover, in view of the significance of cuproptosis and ferroptosis, the collapse and recovery of other metal ion homeostasis also have definite research value for the exploration of cell death identification, disease mechanisms and therapeutic targets. Jiang JK et al. found that excess manganese ions downregulated the expression of sirtuin1 to induce apoptosis of nerve cells, suggesting that blocking manganese ions-mediated cell death might have a certain prospect in developing therapeutics of neurodegenerative diseases (Zhao et al., 2019). Xu H et al. reported a lysosomal calcium/zinc channel-mediated and zinc-dependent cell death in metastatic melanoma so that targeting zinc dysfunction showed potential value for melanoma therapy (Du et al., 2021). Another metal element calcium signaling activates specific proteins to regulate many physiological processes, and its abnormal transduction is expected to provide a new approach for tumor treatment (Bai et al., 2022). Herein, we will keep our eye on Cu- and other metal elements-mediated cell death, particularly in the field of inducers and inhibitors, which are valuable for identifying cell death pathways, exploring disease mechanisms and therapeutic targets based on metal element dysfunction.

In summary, cuproptosis is a newly discovered pathway of Cu-induced cell death which provides an innovative avenue for medical options. We mainly collect the small molecule compounds that induce or inhibit cuproptosis based on the key targets and metabolic pathways of Cu stress in this study, accompanied by relevant transcription factors and ncRNAs. They have the promising potential to be employed as inducers or inhibitors in the scientific studies and medical options of Cu homeostasis dysfunction. However, the action mode of Cu-induced cell death still requires in-depth investigation to understand the hallmark of drug targets and metabolic pathways. Meanwhile the drug species, combination and delivery strategies of cuproptosis inducers or inhibitors also await further exploration.