Role of copper chelating agents: between old applications and new perspectives in neuroscience

Abstract

The role of copper element has been an increasingly relevant topic in recent years in the fields of human and animal health, for both the study of new drugs and innovative food and feed supplements. This metal plays an important role in the central nervous system, where it is associated with glutamatergic signaling, and it is widely involved in inflammatory processes. Thus, diseases involving copper (II) dyshomeostasis often have neurological symptoms, as exemplified by Alzheimer’s and other diseases (such as Parkinson’s and Wilson’s diseases). Moreover, imbalanced copper ion concentrations have also been associated with diabetes and certain types of cancer, including glioma. In this paper, we propose a comprehensive overview of recent results that show the importance of these metal ions in several pathologies, mainly Alzheimer’s disease, through the lens of the development and use of copper chelators as research compounds and potential therapeutics if included in multi-target hybrid drugs. Seeing how copper homeostasis is important for the well-being of animals as well as humans, we shortly describe the state of the art regarding the effects of copper and its chelators in agriculture, livestock rearing, and aquaculture, as ingredients for the formulation of feed supplements as well as to prevent the effects of pollution on animal productions.

Introduction

Copper is the third most abundant transition metal in the human body (Akatsu et al., 2012), and it is most commonly associated with metabolically active organs, i.e. liver, kidneys, heart, and brain. Only about 5% of total copper is found in serum, where it is mostly bound to ceruloplasmin (Cp) (Bremner, 1998; Baldari et al., 2020). This ion is essential for several physiological processes, for example as a cofactor or structural component of various copper proteins, and its oxidation from Cu⁺ (cuprous ion) to Cu²⁺ (cupric ion) is essential (Agarwal et al., 1989; Gromadzka et al., 2020). Copper has several physiological roles: for example, it is the central metal ion of cytochrome c oxidase, and it is endowed with antioxidant activity as a component of Cu/Zn superoxide dismutase (Kaplan and Maryon, 2016; Latorre et al., 2019; Falcone et al., 2021). Moreover, recently the role of copper as a regulator of some cell signaling pathways has been studied (Grubman and White, 2014; Latorre et al., 2019). In particular, it activates various growth factors and it is involved in several pathways related to processes such as inflammation, fibrosis, and lipogenesis (Latorre et al., 2019).

It is worth noting that an imbalance of copper homeostasis is associated with harmful effects on human health (Gromadzka et al., 2020). In particular, copper cations are involved in genetic diseases such as Wilson’s disease, neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases, diabetes, and some types of cancer (Bremner, 1998; Baldari et al., 2020; Mateo et al., 2023; Wang et al., 2023).

Copper chelators have been developed and used to understand the physiological functioning of copper and copper-binding proteins, but they have also found clinical use in the treatment of diseases linked to alterations in copper metabolism and in the diagnosis of copper metabolic disorders (Figure 1).

Figure 1.

Chelating agents are fundamental for balancing copper dyshomeostasis in medicinal chemistry, agriculture, and animal science.

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The capacity of a compound to form complexes with copper is linked to its chelate denticity, to the presence of suitable donor-binding groups, and to other pharmacophoric requirements that allow it to reach inside the metal binding pocket of the target metalloprotein. Moreover, the two oxidation states of copper are targeted by different donor-binding groups. In particular, Cu⁺ favors tetrahedral, trigonal, or even linear geometries, mostly with relatively soft polarizable ligands such as thioethers, nitriles, cyanide, iodide, and thiolates. Cu²⁺, as a borderline acid, prefers amines, imines, and oxygen donors to form square-planar, distorted square-planar, trigonal-pyramidal, and square-pyramidal geometric conformations (Haas and Franz, 2009; Ding et al., 2011). To this day, the most widely used methods for the determination of a compound’s capacity for copper chelation are potentiometric and UV/Vis titrations, or simple UV/vis spectrophotometric determinations. Other, more specific assays, can help researchers identify the correlation or coexistence of copper chelation and further potentially beneficial effects for various pathological states (Wang et al., 2021; Brunetti et al., 2022a).

This review focuses on the current state of the art in the field of copper chelators, whose clinical field of application would entail the therapy of two important neurodegenerative disorders (NDs), mainly Alzheimer’s disease (AD), as well as chemotherapy of some forms of cancer, including glioma. Furthermore, particular attention will be given to the possible role of chelators in agricultural practices and livestock and aquaculture (Figure 1).

Search Strategy

Publication years of reviewed articles ranged from 2016 to 2023. Article search was performed on the main free abstract and citation databases between November 2022 and October 2023, with some of the following search terms used: “Copper dyshomeostasis,” “Metal dyshomeostasis,” “Copper and neurodegenerative diseases,” “Copper chelators,” “Copper and Alzheimer’s disease,” “Copper and Parkinson’s disease,” “Copper and Wilson’s disease,” “Copper and cancer,” “Copper and agriculture,” “Copper and feed and supplements.”

Neurodegenerative Disorders

NDs are primarily characterized by motor and cognitive dysfunctions (Dugger and Dickson, 2017). The role of copper has been widely studied in the etiopathology of the most prominent NDs, namely AD and PD.

Alzheimer’s disease

Pro-neurodegenerative mechanism of copper

AD is the most common neurodegenerative disorder. It is considered a multifactorial disease, and, most recently, much research has been carried out regarding the role of transition metals, particularly iron (Fe), zinc (Zn), and copper (Cu) in its etiology (Mateo et al., 2023). These metals have been hypothesized to bind to the amyloid-β peptide (Aβ), thus driving Aβ aggregation and promoting the formation of amyloid plaques (Kawahara et al., 2017). Metal ions can also interfere with the physiological processing and function of the amyloid-β precursor protein (APP) and with the phosphorylation of tau, resulting in its aggregation and the subsequent formation of neurofibrillary tangles (Crouch et al., 2009). Dyshomeostasis of Fe and Cu has also been linked to oxidative stress and neuroinflammation in the AD brain (Sayre et al., 2007; Praticò, 2008; Choo et al., 2013; Mathys and White, 2017), and their control can be useful itself or in synergistic mechanisms with other systems (Piemontese, 2017, 2019) in innovative approaches for the treatment of the pathology.

Copper ions represent 7.3% of all metal ions in the brain. Free ionic Cu is released at NMDA-responsive glutamatergic synapses, and Cu efflux is associated with the activation of NMDA receptors, so impairments in Cu regulation could promote the glutamatergic dysfunction that is typically correlated with AD (Ayton et al., 2015; Mathys and White, 2017). Cu also plays a role as a “negative modulator” of GABAergic and AMPAergic neurotransmission and has a sharp inhibitory effect on long-term potentiation mechanisms (Opazo et al., 2014; Mathys and White, 2017). Cu²⁺, along with other metal ions such as Zn²⁺, Fe³⁺, and Al³⁺, is chelated by Aβ, contributing to protein precipitation and plaque formation (Domingo, 2006; Kawahara et al., 2017; Leuci et al., 2020). Aβ aggregation results in the production of reactive oxygen species (ROS) that lead to an increase in intracellular calcium levels, leading to lipid peroxidation, mitochondrial dysfunctions, and neuroinflammation (Atrián-Blasco et al., 2018; Piemontese et al., 2019; Leuci et al., 2020). Notably, clioquinol (CQ), an antibacterial agent with an 8-hydroxyquinoline structure at its core, showed very promising effects in early clinical trials on AD patients: these effects have been linked to its capability to form neutral 2:1 complexes with both zinc and cupric ions (Di Vaira et al., 2004; Leuci et al., 2020).

Copper chelators

For this reason, zinc and cupric ion chelators have seen significant developments in the past decade. Tetradentate chelators (Santos et al., 2016), such as bis-8-aminoquinolines, are capable of forming 1:1 copper complexes, allowing them to exert their beneficial effects at lower doses. One such compound, PA1637 (Figure 2) was tested as an inhibitor of episodic memory loss in a mouse model of AD, where it was able to reverse cognitive deficits, with fewer toxic effects than CQ (Ceccom et al., 2012). PA1637 and two of its analogs (1 and 2; Figure 2) also proved to be very selective copper chelators, as they are not able to chelate Fe and Zn ions. Moreover, their affinity constants to Cu²⁺ are higher than that of CQ, so they could feasibly extract these ions from their complexes with Aβ in the brains of AD patients (Nguyen et al., 2014).

Figure 2.

Structure of bis-8-aminoquinoline ligands and their copper (II) complexes (Ceccom et al., 2012; Nguyen et al., 2014).

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The 8-aminoquinoline scaffold has proven to be quite versatile and useful regarding copper chelation, as shown by the recent development of TetraDentateMonoQuinolines (TDMQ, Figure 3), some of which showed very high LogK_app values for Cu, between 14 and 17. In general, compounds based on this scaffold form 1:1 complexes with copper, and also show an interesting selectivity towards this metal ion concerning Zn. The structures of the Cu and Zn complexes of some of the most interesting compounds in the solid state were elucidated by X-ray diffraction on single crystals. Importantly, one of these compounds, TDMQ22, was able to demethylate Cu-Aβ_1–16 and then transfer Cu to glutathione, making it an attractive candidate for further study as an anti-AD agent (Zhang et al., 2018). In a successive study, another compound of the series, named TDMQ20, was easily oxidated by metalloporphyrin catalysts mimicking cytochrome P450. The resulting metabolites are fully soluble in aqueous solution. These results suggested that TDMQ20 should be probably metabolized in vivo without accumulation in tissues (Nguyen et al., 2023).

Figure 3.

General structure of TDMQ ligands (Zhang et al., 2018).

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Among this class of compounds, it is also important to mention NHHQ, which can compete with Aβ for Cu²⁺ and zinc ions (Hauser-Davis et al., 2015) and prevent memory impairment in a mice model of AD by a mechanism that could involve a lessening in the generation of ROS by the Cu-Aβ_1–16 adduct (De Falco et al., 2020).

It is important to note that in the past decade, a multi-target approach has increasingly been incorporated in the design of new potential anti-AD agents. This has led to the development of various classes of Cu chelators endowed with additional biological targets, the most common of which are cholinesterases, Aβ inhibition, and antioxidant properties (Brunetti et al., 2020).

1-Benzylpyrrolidine-3-amine–based compounds were recently noted for their anti-AD bioactivity profile. In particular, several compounds of this kind have shown activity as inhibitors of butyrylcholinesterase, beta-secretase (BACE-1), Aβ and tau protein aggregation, as well as their activity as antioxidants and as chelators of ten metal ions, namely Al³⁺, Ca²⁺, Co²⁺, Cu²⁺, Fe²⁺, Fe³⁺, Mg²⁺, Ni²⁺, Pb²⁺ and Zn²⁺ (Więckowska et al., 2018). Selective copper chelation, measured by UV/vis spectrometry, was shown by ten compounds (including compound 3 and compound 4; Figure 4). Interestingly, these compounds are all amines with phenyl or benzyl substituents in position 4 of a piperazine or in position 3 of a piperidine ring, while the corresponding amides lacked this activity (Wichur et al., 2020).

Figure 4.

Structure of 3 and 4, two of the most promising 1-benzylpyrrolidine-3-amine-based ligands (Więckowska et al., 2018; Wichur et al., 2020).

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3-benzylidene/benzylphthalide Mannich bases (Figure 5) are also a promising scaffold for multi-target copper chelators, which are active as inhibitors of enzymes AChE, BuChE, and MAOs. In general, the presence of a double bond at 3-position enhanced these activities. Compounds were also assayed for their antioxidant ability and for their capability to inhibit Aβ_1–42 aggregation and induce disaggregation. UV/Vis spectrometry was used to study the metal-chelating property of compound (Z)-5 (Figure 5), the most promising multi-target compound, towards Cu²⁺, Zn²⁺, Fe²⁺, and Al³⁺. Results revealed that it interacted with these biometals, and in particular, the Cu²⁺-(Z)-13c complex had a 1:1 stoichiometry (Cao et al., 2021).

Figure 5.

General structure of 3-benzylidene/benzylphthalide Mannich base derivatives and structure of compound (Z)-5 (Cao et al., 2021).

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Hydroxypyridinones are N-heterocyclic metal chelators used for the development of several new pharmaceutical drugs (Chaves et al., 2018b). Among them, 1-phenyl-3-hydroxy-4-pyridinone scaffold was recently revealed as an unconventional starting point for the development of multi-target anti-AD agents with the ability to chelate copper (Figure 6). Compounds of this class were biologically active as antagonists of H3 receptors and as inhibitors of Aβ self-aggregation. Most compounds of the series showed a similar chelating activity compared to the reference compound deferiprone. The best compound of the series was selected for other assays, including the inhibition of Cu²⁺-induced Aβ aggregation and disaggregation of Cu²⁺-induced Aβ aggregation, using Th-T fluorescence and transmission electron microscopy (TEM) (Sheng et al., 2016).

Figure 6.

General structure of the 1-phenyl-3-hydroxy-4-pyridinone derivatives (Sheng et al., 2016).

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The design of hybrid compounds, which link chemical moieties endowed with specific biological activities to others that mimic clinically used drugs has become a prominent strategy for the development of multi-target–directed ligands for the treatment of AD (Chaves et al., 2021).

In a recent study, a 2-hydroxyphenol-benzimidazole portion (BIM), known for its Zn²⁺ and Cu²⁺ bidentate chelating capacity due to the presence of N and O donor atoms, was linked with fragments mimicking two ChEs inhibitors, tacrine (TAC, an anti-AD drug that was withdrawn from market due to hepatotoxicity) or donepezil (currently used for the treatment of AD), affording two series of hybrid compounds, referred to as TAC-BIM and DNP-BIM respectively. Three of these BIM-hybrids (PP-BIM, PZ-BIM, and TAC-BIM; Figure 7) demonstrated good chelating action towards Cu²⁺ (pM=10.7–11.1) and a moderate one towards Zn²⁺ (pM = 6.3–6.4), forming 1:1 and 1:2 complexes. Compounds of both hybrid series could inhibit self-induced Aβ aggregation, probably through a mechanism of ligand intercalation between β-sheets of Aβ fibrils, but they do not seem to interfere with Cu²⁺-induced Aβ aggregation (Chaves et al., 2018a).

Figure 7.

Structures of hybrids PP-BIM, PZ-BIM, and TAC-BIM (Chaves et al., 2018a; Piemontese et al., 2018a).

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In continuity with this work, the same group carried out the further characterization of some of these compounds, particularly the benzimidazole and benzofuran donepezil hybrids, by synthesizing additional analogs and by broadening the spectrum of biological assays. PP-BIM was confirmed as the most promising compound of this class, with thioflavin T (ThT) assays and TEM highlighting its capability to prevent Cu²⁺-mediated Aβ aggregation (Piemontese et al., 2018a). Moreover, all these compounds exerted neuroprotective effects on SH-SY5Y neuroblastoma cells treated with the Aβ peptide. Simultaneously, these compounds showed promising activities as inhibitors of AChE, while phenol-containing compounds of this series were also moderate antioxidants, making them very promising multi-target agents (Piemontese et al., 2018a). Subsequently, a new sustainable synthetic approach of the hit compound PZ-BIM or PZ1 was studied, using so-called deep eutectic solvents as an alternative to classical volatile organic solvents and leading to a fully green preparation of the hybrid (Piemontese et al., 2020).

A further development of the SAR of these compounds led to PP-BIM-5 (Figure 8), a more selective copper(II) chelator (pM = 14.3), due to the high possibility of the establishment of a tridentate coordination to the metal ion (N,O,O) involving the closer carbonyl oxygen atom and the subsequent high stability of the complex with the metal (Chaves et al., 2020). The structure of this compound was simply an isomer of PP-BIM and this modification was also effective for the Cu²⁺-mediated Aβ aggregation (58.9% vs. 18.6% at 40 μM), but led to a slight loss of efficacy in the inhibition of AChE (9 μM vs. 4.2 μM) (Chaves et al., 2018a, 2020; Piemontese et al., 2018a).

Figure 8.

Structure of hybrid PP-BIM-5 (Chaves et al., 2020).

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A further research effort from the same group afforded a series of twenty more hybrids composed of a donepezil-like moiety (N-benzylpiperidine or N-benzylpiperazine) linked with different aryloxyacetic acids, with the primary goal of inhibiting AChE, BuChE, and endocannabinoid-degrading enzyme fatty acid amide hydrolase. Most compounds of this series were indeed active against cholinesterases and fatty acid amide hydrolase, and the most active compound, N-benzylpiperazine SON38 (Figure 9), was also assayed for copper chelation via pH-potentiometric titrations, which showed a significant change in the compound’s deprotonation profile in the presence of copper, although complex formation kinetics were slower than previous benzimidazole-and benzofuran-donepezil hybrids. The coordination core of SON38 was then elucidated via ¹H-NMR titration of a 1:1 Zn²⁺/SON38 complex, showing that both piperazine nitrogen atoms are involved in metal complexation. The formation of the 1:1 Cu²⁺: SON38 complex was also corroborated by ESI-MS (Brunetti et al., 2022a).

Figure 9.

Structure of donepezil-like compound SON38 (Brunetti et al., 2022a).

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In a study by a different group, the N-benzylpiperazine fragment was instead combined with a pyridine moiety. Compounds showed significant activity towards cholinesterases, showed no relevant cytotoxic effects on NGF-treated, rat pheochromocytoma PC12 cells, which bear a strong resemblance to adrenergic neurons, and were shown to be able to pass the BBB in an in vitro assay. Moreover, these compounds showed promising results from preliminary in silico ADMET studies. The most promising member of this series, compound 6 (Figure 10), was evaluated for its capability to chelate various metal ions, namely Cu²⁺, Zn²⁺, Fe²⁺, Fe³⁺, and Al³⁺. UV–Vis spectroscopy revealed that it was able to form complexes only with Cu, and not with the other tested metal ions (Zhou et al., 2019).

Figure 10.

Structure of the donepezil-like compound 6 (Zhou et al., 2019).

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Rivastigmine, another molecule with current clinical application in the therapy of AD, has also inspired recent attempts at multi-target-directed ligand design. In one such study, a series of ten rivastigmine derivatives was designed, achieving good activities in terms of AChE inhibition and radical scavenger activity. Moreover, one of the best compounds (7; Figure 11) was tested for its capability to chelate copper by UV−Vis spectrometry, to explain its antioxidant activity. Results revealed that, indeed, the radical scavenger activity of 4 relates to its copper chelating properties (Sestito et al., 2019).

Figure 11.

Structure of the rivastigmine-like compound 7 (Sestito et al., 2019).

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In another recent work, seven RIV-BIM hybrids were synthesized and assayed for their capacity to inhibit cholinesterases and Aβ aggregation, as well as for their antioxidant properties. In particular, molecular fluorescence spectroscopy displayed that the inhibition of Cu²⁺-induced Aβ₄₂ aggregation by some compounds was moderate/good (41.2%–60.8%), with values only slightly higher than those obtained for self-aggregation inhibition (39.0%–58.7%), while TEM highlighted that Cu²⁺ enhanced the formation of the fibrils and compounds were less capable to reduced them in presence of Cu²⁺. All these hybrid compounds also showed good activity as MAO-A and MAO-B inhibitors both in silico and in vivo. The copper chelation capabilities of compounds 8 and 9 (Figure 12) were further elucidated via UV/Vis and ¹H-NMR titrations, showing that the position of the BIM moiety strongly influences this interaction, with ortho-substituted BIM hybrids such as 9 being much more potent copper chelators (Vicente-Zurdo et al., 2022, 2023).

Figure 12.

Structures of RIV-BIM hybrids 8 and 9 (Vicente-Zurdo et al., 2022, 2023).

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Another important source of inspiration for the development of new molecules is natural compounds, which are often endowed with multiple bioactivities, and their semisynthetic or synthetic derivatives (Poliseno et al., 2021; Piemontese, 2023). As an example, chalcones are natural flavonoid derivatives that are very common in plants, and whose pharmacological profile includes the capability to inhibit Aβ aggregation, as well as antioxidant and anti-inflammatory effects (Wang et al., 2021). Their molecular scaffold was recently exploited in a design strategy that comprised the replacement of the benzene ring with a pyridine, to obtain compounds that could modulate the interaction between metal ions and Aβ. The resulting derivatives were good inhibitors of Aβ_1–42 self-induced aggregation and antioxidant compounds. UV−Vis spectrophotometry allowed to determine that the most active compound, 10 (Figure 13), was able to selectively form complexes with Cu²⁺, while it did not interact with any other metal ions (Wang et al., 2021). Copper chelation was further confirmed by UV/Vis titration. Moreover, a ThT assay was performed to evaluate the inhibitory activity of the compound on Cu²⁺-induced Aβ_1–42 aggregation, using clioquinol and resveratrol as controls. The presence of CQ, compound 10, and resveratrol led to a reduction in fluorescence intensity by 81.6%, 87.1%, and 70.1%, respectively, confirming this activity. The disaggregation activity of 10 was also evaluated by ThT assay after a first incubation of Aβ_1–42 – Cu²⁺ at 37°C for 24 hours (to form Aβ₁₋₄₂ fibrils), and another one with the addition of 10 in the same conditions. 10 demonstrated a 60.7% disaggregation rate, higher than CQ (45.8% disaggregation rate). TEM assay confirmed the ability of 10 to inhibit the formation of Aβ_1–42 fibrils and to disaggregate Aβ_1–42 fibrils (Wang et al., 2021). These results are particularly interesting considering the very low toxicity of 10 (tested on SH-SY5Y cells) and its strong neuroprotective properties against Aβ_1–42-induced cell injury. These results led researchers to proceed to in vivo studies, in which 10 proved capable to reverse scopolamine-induced memory impairment in mice (Wang et al., 2021).

Figure 13.

Structure of the chalcone derivative 10 (Wang et al., 2021).

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Chelating agents capable of targeting Aβ aggregates have been investigated not only as therapeutic agents, but also as tools for the diagnosis of AD, specifically as imaging probes, although none of them has been approved for clinical use yet (Staderini et al., 2015).

The requirements for the diagnostic use of copper chelators are thus two: capacity for selective targeting of Aβ aggregates (modulating unit) and the presence of a fluorophoric chemical moiety whose fluorescence is altered by its binding with copper and/or with the amyloid peptide (imaging unit). ThT, a 2-arylbenzothiazole, is one such compound, and it has thus been adopted as a probe for the identification of Aβ aggregates (Noël et al., 2013). For this reason, the 2-arylbenzothiazole moiety has been increasingly used by researchers to find a clinically viable fluorescent chelator that could be applied both as an imaging probe and as a therapeutic modulator for metal-induced Aβ aggregation. In a recent work, novel compound TBT (Figure 14), containing the 2-arylbenzothiazole-like 2-phenylbenzothiazole portion linked by a methylene to a cyclen group (capable to attenuate metal-induced Aβ aggregation), and possessing high lipophilicity and affinity to Aβ, was reported (Yang et al., 2016a). Fluorescence titration demonstrated that thanks to its cyclen group, TBT chelated Zn²⁺ and Cu²⁺ with a ratio of 1:1 and with an association constant (K_a) of 4.93 × 10⁶ M^–1 and 6.8 × 10⁷ M^–1, respectively. It was also demonstrated that TBT could interfere with the metal-induced formation of Aβ aggregates and contribute to their disaggregation (Yang et al., 2016a).

Figure 14.

Structures of theranostic fluorescent chelators TBT and BTTA (Yang et al., 2016a, b).

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The same researchers subsequently developed another potential theranostic fluorescent chelator, named BTTA (Figure 14). Like TBT, BTTA is composed of a cyclen group as a modulating unit, while the imaging unit is comprised of a benzothiazole aniline (BTA) group (Yang et al., 2016b). In contrast with TBT, however, these two portions are linked by an amide group which, thanks to its additional length, confers to BTTA a high degree of selectivity towards Cu²⁺, binding to which results in quenching of BTTA’s fluorescence. This was proven by fluorescence titration, which showed that BTTA forms a 1:1 metal complex with Cu²⁺, with a K_a that is probably sufficient to capture Cu²⁺ from Aβ. Competitive experiments demonstrated that BTTA also had a high selectivity for Cu²⁺ compared to other metal ions. ThT fluorescence competition assay, and ESI–MS experiments confirmed BTTA’s high binding affinity towards Cu²⁺, and docking studies suggested a possible interaction of the compounds with Aβ (Yang et al., 2016b). Subsequently, the ability of BTTA to cause disaggregation of Cu²⁺-inducted Aβ aggregates was also proven to occur significantly even with 24-hour incubation times. Lower levels of disaggregation were obtained when coincubating BTTA with Cu²⁺-free Aβ aggregates, meaning that BTTA can specifically attenuate Cu²⁺-induced over Cu²⁺-free Aβ aggregation. The compound’s theranostic potential was corroborated by an experiment on the brain of APPswe/PSEN1 transgenic mice, where the quenching of BTTA fluorescence allowed to monitor the disaggregation of Aβ aggregates (Yang et al., 2016b).

Parkinson’s disease

Pro-neurodegenerative mechanism of copper

PD is a neurodegenerative disorder that compromises the motor abilities of the patient, leading to tremors, uncontrolled movements, and muscle stiffness. In the most advanced stages of the disease, muscles involved in the respiratory process are also affected, leading to death (Poewe et al., 2017). Beyond aging, environment, and genetics, the dyshomeostasis of heavy metals such as iron, copper, and lead can be a possible cause of the onset and progression of the disease (Poewe et al., 2017; Mateo et al., 2023).

The role of copper in the pathological processes of PD is highly controversial: while recently some studies reported lower concentrations of copper in the brains of PD patients, an excess of copper can increase PD risk, too (Beshgetoor and Hambidge, 1998; Bisaglia and Bubacco, 2020).

In particular, it has been hypothesized that decreased copper levels could be linked to an increased risk of developing PD, because copper stimulates the ferroxidase activity of ceruloplasmin (Cp) and participates in iron homeostasis, so low copper levels could indirectly alter iron concentrations (Wittung-Stafshede, 2016; Bisaglia and Bubacco, 2020). Simultaneously, an excess of free copper can lead to indiscriminate inhibition of several enzymes through the binding to cysteine residues of these proteins, and it can lead to higher levels of oxidative stress and dopamine oxidation (Zhou et al., 2023). Moreover, the interaction of copper and other metal ions with α-synuclein (α-syn), has been proposed to stabilize the pathological, partially folded conformation of this protein (Bisaglia and Bubacco, 2020). For these reasons, copper chelators have been recently proposed as possible therapeutical agents against PD.

Copper chelators

The controversial role of copper in PD has led to the design of a Metal–Protein Attenuating Compound, a moderate chelator capable of interfering with abnormal metal–protein interactions, by competing with the metal ion in the binding with α-syn in physiological conditions (Scott and Orvig, 2009). 1-Methyl-1H-imidazole-2-carboxaldehyde isonicotinoyl (X1INH; Figure 15) is a recently reported Metal–Protein Attenuating Compound, whose synthesis occurs in an eco-friendly one-step process with excellent yield. Its hydrochloride salt was also prepared for X-ray crystallography, although with a lower yield. In silico pharmacokinetic studies demonstrated that this compound does not violate Lipinski’s rule of 5. UV–Vis spectrometry confirmed the interaction between Cu²⁺ and X1INH in a 1% DMSO/H₂O solution. Moreover, ¹H-NMR titration suggested that the primary anchoring point for copper (II) ions in X1INH is the 1-methyl-¹H-imidazole moiety. Interestingly, 2D NMR experiments showed that X1INH was able to disrupt Cu⁺–α-syn interaction with a higher affinity than Cu²⁺–α-syn (Cukierman et al., 2020). This preference is important because copper(II) must be reduced to copper(I) before entering cells through a CTR1-mediated transport process and α-syn inclusions are mostly intracellular in PD. X1INH showed no toxicity on H4 human neuroglioma cells by X1INH, in contrast to other structurally similar compounds, and it affected the aggregation of α-syn in a cellular model of synucleinopathy (Cukierman et al., 2020).

Figure 15.

The metal–protein attenuating compound X1INH (Cukierman et al., 2020).

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Another useful strategy for the treatment of PD is the design of synergic chelators for different ions usually involved in this pathology. For example, a new 7,8-dihydroxycoumarin-based Fe²⁺/ Cu²⁺ chelator, named DHC12 (Figure 16), was recently reported as a neuroprotective agent in cell and animal models of PD. DHC12 was tested as a chelator against Fe²⁺, Fe³⁺, Cu²⁺, Zn²⁺, Hg²⁺, Co²⁺, Ca²⁺, Mn²⁺, Mg²⁺, Ni²⁺, Pb²⁺, and Cd²⁺ using a fluorescence-based assay (Aguirre et al., 2017). Results revealed that it was highly selective for Fe²⁺ and Cu²⁺, less active on Zn²⁺ and Fe³⁺, while it possessed no ability to bind the other tested metal ions. The compound showed no cytotoxic effects in SH-SY5Y dopaminergic neuroblastoma cells after 24 hours of incubation at concentrations up to 50 μM, moreover, researchers demonstrated that DHC12 enters cells, concentrates in mitochondria, and chelates both the mitochondrial and the cytoplasmic labile iron pools, reducing lipid peroxidation (Aguirre et al., 2017). Additionally, it inhibited MAO-B in a concentration-dependent manner, and MAO-A more weakly (Aguirre et al., 2017).

Figure 16.

Structure of DHC12 (Aguirre et al., 2017).

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New perspectives about the use of copper chelators in other diseases

Wilson’s disease

The dyshomeostasis of copper is also involved in other pathologies, such as Wilson’s disease and some forms of cancer (Crisponi et al., 2010; Patil et al., 2013). Wilson’s disease is a genetic condition, with autosomal recessive transmission, which is caused by mutations of the ATP7B gene which result in excessive concentrations of copper throughout the whole organism (Bandmann et al., 2015; Shanbhag et al., 2021).

The clinical manifestations of the disease comprise tremors, liver failure, and neurological symptoms (Bull et al., 1993; Brewer, 2000; Roberts and Schilsky, 2003; Baldari et al., 2020). Importantly, the therapy of Wilson’s disease is currently the only clinical application of copper chelators, chiefly D-penicillamine, trientine hydrochloride, and tetrathiomolybdate (Roberts and Schilsky, 2008; Mohr and Weiss, 2019; Baldari et al., 2020), to remove the accumulated copper ions from tissues (de-coppering) and of preventing re-accumulation (maintenance) (Squitti et al., 2018; Baldari et al., 2020).

The clinically approved copper chelators used in the therapy of Wilson’s disease may also possibly be employed as epigenetic drugs for the treatment of diffuse intrinsic pontine glioma (DIPG) (Michniewicz et al., 2022). DIPG is an incurable pediatric brain cancer of the ventral pons characterized by its complex epigenetic profile. Most patients present the H3SHS mutation that leads to a deregulation of the expression of several genes through an aberrant pattern of epigenetic modification. This complicated situation is accompanied by difficulties in the identification of effective therapeutic strategies (Michniewicz et al., 2022). Recently, starting from the hypothesis that copper, while being naturally abundant in the pons and essential for normal brain function, is implicated also in the onset of this pathology, researchers demonstrated that the chelator tetraethylenepentamine (TEPA) can reduce cell growth and induce apoptosis in DIPG cell lines (Michniewicz et al., 2022). In particular, TEPA downregulates SAM, S-adenosyl homocysteine, its demethylation products, and α-ketoglutarate, an important cofactor of lysine demethylase 6B, which is probably involved in DIPG. In vitro, synergistic activity of TEPA in combination with Panobinostat, an HDAC inhibitor, has also been shown in the same work. Moreover, in vivo studies demonstrated that TEPA improved survival in an orthotopic patient-derived xenograft model, also demonstrating total cancer regression in 25% of treated mice (Michniewicz et al., 2022).

The same research group experimented the use of TEPA as a copper chelator also in other types of cancer, by analyzing its effects on disease progression and in particular on the development of metastasis (Poursani et al., 2023).

Cancer

Cancer is one of the most common causes of death in the world. The development of therapies against cancer is slow and it has historically been hindered by the different pathological features of different kinds of cancer, as well as the adaptation mechanisms that cancer cells can adapt to escape from immunity and therapeutic agents (Guo et al., 2023). Interestingly, high levels of copper in the tumor microenvironment have been linked to cancer progression, driving cell proliferation and facilitating metastatic processes via angiogenesis (Babak and Ahn, 2021; Kadu et al., 2021; Guan et al., 2023).

Indeed, the administration of copper chelators could positively impact the tumor microenvironment (Steinbrueck et al., 2020); for example, the capability of some copper chelators to generate ROS can be useful: in fact, Cu²⁺ complexes have been studied for their cytotoxic effects in cancerous cells in a process named cuproptosis (Peña et al., 2021; Cobine and Brady, 2022). According to this hypothesis, three Cu²⁺ salphen-like ligands containing N,N,O-chelating cores and differing for aromatic substitution (H, Cl, or Br) (Figure 17) were recently proposed as copper chelators for the treatment of cancer (Peña et al., 2021). Cu(OAc)₂ was used for the complexation of pure substances, and the resulting powders were not well soluble in common organic solvents. A solid-state complexation study suggested that a 1:1 complex was formed, with the deprotonation of –OH and –NH groups (Peña et al., 2021). Moreover, the generation of ROS was evaluated by cyclic voltammetry experiments. Compounds did not cause redox reactions on their own, while their copper complexes were redox active (Peña et al., 2021). The in vitro antiproliferative activity of the complexes and their free ligands were determined on cancer cell lines (HeLa and MCF7): free ligand 11 displayed poor toxicity, while 12 and 13 were more cytotoxic, likely due to the presence of halogen atoms in their structures. Complexes C11, C12, and C13 exhibited a relevant dose-dependent cytotoxicity in both cancer cells. C11 was less cytotoxic towards normal embryotic fibroblasts (NIH 3T3) non-tumor cell lines compared to cancer cells (Peña et al., 2021). Other experiments revealed that complexes are only slightly capable of cleaving DNA by themselves, but this activity increased with the presence of reductive species, such as ascorbic acid, probably due to the generation of Cu (I) which enhances ROS production (Peña et al., 2021).

Figure 17.

Structure of N,N,O-Chelating Salphen-like ligands and the corresponding Cu²⁺ complexes (Peña et al., 2021).

Created with molsketch.

In another work, the cytotoxic, pro-oxidant, and antibacterial activities of a series of eight halogenated 1,3-disubstituted arylthioureas (Figure 18) were reported (Chrzanowska et al., 2021). The substituents on the phenyl ring were chosen to study the biological effects of electron-withdrawing groups and their positions. The reaction between free thioureas (L) and CuCl₂ led to the formation of a 2:1 ligand: Cu²⁺ complex (Chrzanowska et al., 2021). Some compounds showed toxicity for several cancer cell lines but not for healthy cell lines. The same compounds showed pro-apoptotic activity on cancer cells after 72-hour incubation, and an anti-inflammatory activity, blocking the release of human interleukin-6. Interestingly, some compounds also had antimicrobial activity against selected strains of Staphylococci (Chrzanowska et al., 2021).

Figure 18.

General structure of arylthiourea ligands and the corresponding Cu²⁺ complexes (Chrzanowska et al., 2021).

Created with molsketch.

Treatment with metformin, which is clinically used in the therapy of type 2 diabetes (Rena et al., 2017), has been known to selectively kill persistent cancer cells through a relatively unclear mechanism of action (Müller et al., 2018). A recent work (Figure 19) demonstrated that the anti-cancer activity of metformin could be linked to its capability to form copper complexes, chelating mitochondrial copper and thus halting the mesenchymal-to-epithelial phenotype transition that characterizes aggressive cancer cells (Rusanov et al., 2022). This interesting potential repurposing of the drug was the starting point for new more accurate research. An interesting result was achieved through the development of an alkyne analog of metformin, named Metforminyn (Met), which contained a terminal alkyne which allowed visualizing its intra-cellular site of action. Both metformin and Met were evaluated on human breast cancer cell lines and circulating tumor cells from patients with colorectal cancer, and results revealed that Met was much more potent than Metformin (Müller et al., 2018). Moreover, fluorescence microscopy and flow cytometry analysis revealed that the natural mitochondrial abundance of endogenous copper was sufficient to promote the labeling of Met in native conditions, confirming, as for metformin, that biguanides interact with copper (Müller et al., 2018).

Figure 19.

Structure of Metformin and its analog Metforminyn (Müller et al., 2018).

Created with molsketch.

Finally, about the possible impact of copper in cancer research, another interesting application is copper nanoparticles, which may be used in therapy due to their ability to modulate oxidative stress, the induction of apoptosis and autophagy, and the modulation of immune response. Due to their rapid elimination from the body, copper nanoparticles show lower systemic toxicity and have a low cost compared to nanoparticles based on other metals, such as gold, silver, and palladium (Cazzoli et al., 2023). All things considered, copper chelators may represent a possible new therapeutic approach for some forms of cancer characterized by poor prognosis, low survival rates over time, and limited therapeutic options.

Agriculture, Livestock, and Aquaculture

As reported above, copper is an essential trace element for the functioning of living organisms (Flemming and Trevors, 1989). Thus, it is necessary to introduce it through diet in suitable and different amounts for each animal species. However, being a heavy metal, excessive exposure to it can lead to toxicity, and consequently, its removal from some polluted environments may be necessary (Chen et al., 2022; Lv et al., 2023).

As far as agriculture is concerned, the fungicidal action of copper, often used in combination with sulfur, has been known since the middle of the last century (McCallan, 1949; Ortega et al., 2022) and is expressed through the formation of complexes with proteins which are toxic to the plant pathogen. However, the presence of large quantities of heavy metals, including copper, can be a problem (Izydorczyk et al., 2021). In this case, chelation is a strategy that nature itself has devised through hyperaccumulator plants and their metallothioneins, proteins rich in cysteines, present in varying amounts and different plant tissues and which can be very useful in soil decontamination (Gaetke and Chow, 2003; Sheoran et al., 2010).

Flavonoids, and polyphenols in general, which possess appropriate structural characteristics are also naturally efficient copper chelators (Říha et al., 2014). Natural compounds are increasingly being studied thanks to their easy recovery from food industry waste, and to the possibility to concentrate them in food or feed supplements through several green extraction methods (Brunetti et al., 2022b). Their present and future success is linked to the suggested protective properties against the onset of cardiovascular and neurodegenerative diseases (Leuci et al., 2021; Piemontese et al., 2022), which can also be due, in some cases, to their possible metal chelating capacity (Piemontese et al., 2018b; Poliseno et al., 2021).

Synthetic chelating agents are, instead, essential for numerous phytoremediation techniques developed over the years and are currently used in agriculture (Lingua et al., 2014; Lin et al., 2022).

Copper has been shown to be an essential microelement for animals and plays an important role in many biological processes, even if its safety was recently reconsidered by EFSA and its maximum allowed levels were lowered (EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), 2016). This revision was due to the influence of dietary copper on selected animal microbiota and to the environmental occurrence of bacterial copper tolerance, as well as resistance to a series of antibiotics (EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), 2016). However, low levels of copper impact the development of a wide range of hepatic and neurological disorders, and dietary copper supplements are still commonly used in all livestock productions (Sivertsen and Løvberg, 2014).

On the contrary, the toxic effects of an excess of copper vary among species. Monogastrics appear to tolerate it much better than ruminants, and sheep are particularly susceptible due to their less efficient excretory mechanism (Van Saun, 2023). These animals accumulate copper in their liver more readily than any other livestock species, leading to common occurrences of chronic poisoning (Borobia et al., 2022).

In the field of aquaculture, the speciation of copper strongly affects the ability of the metal to create toxicity (Donat et al., 1994; Tait et al., 2018; Zitoun, 2019; Malhotra et al., 2020). However, copper is also an essential bioactive trace metal in marine environments and an important micronutrient for many aquatic species (Donat et al., 1994; Zitoun, 2019) and in complex forms, copper is less toxic than the free ionic form Cu²⁺ (Donat et al., 1994; Moffett et al., 2012; Zitoun, 2019; Malhotra et al., 2020).

The different oxidation states of copper have been used in the recent past to design nanoparticles with various sets of distinctive properties such as antimicrobial ones (Moffett et al., 2012; Oldham et al., 2014; Malhotra et al., 2020). Accordingly, it becomes necessary to evaluate the release of copper ion forms in the marine environment to analyze the toxicity and bioavailability if accumulated in an environment containing aquatic species (Keller et al., 2017; Malhotra et al., 2020). The similarities of copper metabolism in fish and mammals were studied by Syed and Coombs and the metal was found in different organs of fish, including the brain (Syed and Coombs, 1982; Heath, 1987; Malhotra et al., 2020). Due to a large number of interactions of copper, it is important to maintain a balance of trace minerals without over-supplementing minerals premix in diets (Suttle, 1991).

Environmental pollution from heavy metals, including copper, is an problem for aquatic organisms and consequently for fishing and aquaculture (Cao et al., 2022), and it can transfer into the human food chain, creating health problems (Rocha et al., 2016; Gopal et al., 2023). Microbial resistance to metals, as well as resistance to antibiotics, is increasingly common in fish and seafood grown in different parts of the world (Veena et al., 2020) and it is an ever-increasing food safety problem (Rocha et al., 2016; Veena et al., 2020; Puvača et al., 2023). The addition of plants producing bioactive substances including copper chelating agents to the natural environment could be a valuable solution in specific situations. Moreover, it can be an interesting option to improve the adverse effects of these pollutants on aquatic organisms (Li et al., 2023).

Conclusions

Copper ions play a crucial role in several physiological mechanisms, and their dysregulation is involved in a multitude of pathologies ranging from AD to several kinds of cancer, including glioma. These metal cations are also of great importance in agriculture and zootechnics, even if they can be dangerous if present in large quantities, becoming food and/or environmental contaminants. The development of copper chelators has been the focus of a great volume of research work in the last few years, yielding promising results in terms of research compounds with potential applications in the therapy of copper-related pathologies, as well as in the field of formulation of feed additives or decontamination of environment or food. In particular, the development of multi-target-directed ligands with copper chelating ability for the treatment of NDs is a highly active field of research, with new series of compounds being developed continuously. Copper chelators in the field of cancer chemotherapy have attracted comparatively less interest in the past five years, but their development has been nonetheless significant.

Overall, the main challenge regarding copper chelating agents is tailoring them to therapeutic targets that are specific to the pathology of interest, to keep the most beneficial effects while curbing adverse reactions. This refinement of these compounds’ therapeutic profile thus remains a fascinating and promising field of research, whose development is poised to grow further in the coming years.

Data availability statement:

Not applicable.