# Cu(I) chelators: Useful tools to reveal and control Cu(I) homeostasis and toxicity

Charlène Esmieu, Sarah Hostachy, Christelle Hureau

► To cite this version:

Charlène Esmieu, Sarah Hostachy, Christelle Hureau. Cu(I) chelators: Useful tools to reveal and control Cu(I) homeostasis and toxicity. Coordination Chemistry Reviews, 2025, 539, pp.216684. 10.1016/j.ccr.2025.216684. hal-05085144

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Submitted on 26 May 2025

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# Cu(I) chelators: Useful tools to reveal and control Cu(I) homeostasis and toxicity ☆

Charlène Esmieu<sup>a, *,</sup> , Sarah Hostachy<sup>b</sup> , Christelle Hureau<sup>a</sup>

<figure><img src="image_2.png" /></figure>

ARTICLE INFO

Keywords:

Copper metabolism

Cu(I) affinity/stability constant

Cu(I) fluorescent probes

Cu(I) chelation therapy

A B S T R A C T

Copper (Cu) is an essential trace element for numerous biological processes, including energy production, aer-
obic respiration, and antioxidant defense. Despite its importance, disruptions in Cu homeostasis can lead to
severe pathological conditions, such as Wilson's disease (WD), Menkes disease (MD), Alzheimer's disease (AD),
and cancer. The present review focuses on the role of Cu(I) probes and chelators as molecular tools for under-
standing and managing copper-related toxicity and misregulation. It provides an overview of Cu(I) coordination
chemistry, trafficking pathways, and the dual toxicity mechanisms of Cu (both ROS-dependent and ROS-
independent). Key experimental approaches to study Cu(I), including chromophoric and fluorescent probes,
are highlighted, with a focus on their design, applications, and limitations. Finally, the therapeutic potential of
Cu(I)-targeting ligands is discussed, showcasing their role in fighting oxidative stress and restoring metal ho-
meostasis in diseases. Hence, this review offers a comprehensive perspective on Cu(I) chelation's role in
advancing both fundamental understanding and therapeutic strategies against copper-related disorders.

1. Introduction

1.1. *Cu as biological metal: A brief introduction*

Copper (Cu) is a transition metal ion that is essential for living or-
ganisms and is obtained through dietary intake. Approximately 100 mg
Cu is found in an average human adult [1]. Cu plays a central role as an
enzyme cofactor, it supports crucial enzymatic functions and is involved
in numerous biological processes, including energy production, iron

metabolism, and antioxidant defense. It is involved in many essential
reactions of electron transfer due to its ability to cycle between two bio-
accessible redox states, Cu(I) and Cu(II) [2]. In the following, the no-
tation Cu will refer to either Cu redox state. When the redox state is well-
defined, then Cu(I) or Cu(II) will be used. Cu is for instance involved as a
metal catalytic center in vital enzymes, such as superoxide dismutase
(SOD), tyrosinase cytochrome C oxidase (COX), lysyl oxidase (LOX),
dopamine β-hydroxylase (DBH), ascorbate oxidase (AAO), peptidylgly-
cine- and α-amidating monooxygenase (PAM) [2–4]. It also has non-

**Abbreviations:** AD, Alzheimer's disease; ATCUN motif, Amino-Terminal Cu and Ni-binding motif; Aβ, Amyloid-β; APP, Amyloid Precursor Protein; AAO, Ascorbate Oxidase; ATOX1, Antioxidant Protein 1; BC, Bathucoproine; BCA, Bicinchoninate anion; BCS, Bathucoproine disulfonate; BBB, Blood Brain Barrier; BODIPY, Boron Dipyromethene; BSA, Bovine Serum Albumin; BAL, British Anti-Lewisite; Cp, Ceruloplasmin; CT, Charge Transfer; Ctr1, Copper transporter 1; Cu, Copper; CCS, Copper Chaperone for superoxide dismutase; ATP7A, Copper Transporting Alpha P-type ATPases; ATP7B, Copper Transporting beta P-type ATPases; Cys, Cysteine; DTDP, 4,4'-dithiodipyridine; D-Pen, D-penicillamine; DMSA, dimercaptosuccinate; DMPS, Dimercaptopropane sulfonate; DBH, dopamine β-hydroxylase; Fs, Ferene; Fz, Ferrozine; GSH, Glutathione; His, Histidine; HSAB, Hard and Soft Acids and Bases; ICP-MS, Inductively Coupled Plasma Mass Spectrometry; ITC, Isothermal Titration Calorimetry; LOX, lysyl oxidase; MMPs, Matrix Metalloproteases; MD, Menkes Disease; MTs, Metallothioneins; Met, Methionine; MCL, Monovalent Copper Ligands; nano-SIMS, nano-Secondary Ion Mass Spectrometry; NIR, Near-Infrared Region; NC, Neocuproine; NTA, Nitrilotriacetic Acid; PEMEA, 2-Pyridylethylbis(2-methylthioethyl)amine; PAM, Peptidylglycine- and α-Amidating Monooxygenase; PET, Photo-induced Electron Transfer; PTA, 1,3,5-triaza-7-phosphaadamantane; ROS, Reactive Oxygen Species; SIMS, Secondary Ion Mass Spectrometry; Steap, six-transmembrane epithelial antigen of the prostate; SLC31A1, solute carrier family 31 member 1; SOD, Superoxide Dismutase; XAS, Synchrotron-based X-ray Absorption Spectroscopy; TTM, Tetrathiomolybdate; TETA, Triethylenetetramine; Trp, Tryptophane; Tyr, Tyrosine; COX, Tyrosinase Cytochrome C Oxidase; UV, Ultraviolet; WD, Wilson's Disease; XFM, X-ray Fluorescence Microscopy; Zn, Zinc; Zn7-MT-3, Zn-loaded Metallothionein 3; Zn7-MT-2A, Zn-loaded Metallothionein 2A.

<footnote><sup>a</sup> LCC-CNRS, Université de Toulouse, CNRS, Toulouse, France</footnote>

<footnote><sup>b</sup> IRIG, SyMMES, Univ. Grenoble Alpes, CEA, CNRS, Grenoble INP, 38000 Grenoble, France</footnote>

<footnote>* This article is part of a Special issue entitled: ‘50th anniversary of LCC’ published in Coordination Chemistry Reviews.</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: charlene.esmieu@lcc-toulouse.fr (C. Esmieu).</footnote>
enzymatic functions, such as structural, signaling and regulatory roles [5]. Because of its crucial functions and redox ability, Cu regulation is essential to the proper functioning of cells and, more generally, of organisms. Indeed, if misregulated, and despite the vital role Cu plays in the functioning of living organisms, it can lead to harmful effects. Among the most well-known and studied pathological conditions are Wilson's disease (WD), Menkes disease (MD), Alzheimer's disease (AD), and certain cancers.

## 1.2. Cu homeostasis in the body

### 1.2.1. Coordination features of the Cu ions

To prevent potential Cu toxicity and to ensure that Cu reaches its destination (specific organs, cuproenzymes ...), Cu is handled by a specific machinery throughout its journey in the body. The two main redox states of Cu found in the biological systems are Cu(II) and Cu(I). They both have different electronic configurations and are classified differently according to the Hard and Soft Acids and Bases (HSAB) principle [6,7]. This implies a huge difference in terms of coordination preferences. Cu(II) is a paramagnetic cation with a 3d⁹ electronic configuration. It is stabilized within distorted octahedral geometries, a phenomenon known as the Jahn-Teller effect, minimizing the global energy of the complex. Depending on the distortion, Cu(II) can adopt principally square planar, square pyramidal or octahedral and trigonal bipyramidal coordination geometries. According to the HSAB principle, Cu(II) shows an intermediate to high Lewis acid character. It thus prefers N and O atom donors that include amines, carboxyl and imidazole groups of side chains of amino-acid residues present in proteins. The Cu(I) cation exhibits a d¹⁰ configuration with d orbitals full occupancy, implying that Cu(I) complexes do not undergo ligand field stabilization upon coordination. The coordination number observed in Cu(I) complexes is frequently low, with 2, 3, or 4 coordinates in linear, trigonal planar, or tetrahedral geometries [8,9]. Moreover, Cu(I) being a soft acid according to the HSAB principle, it exhibits a preference for soft bases ligands. This results in a tendency to interact with molecules that contain sulfur donor atoms, such as thiolate or thioether chemical functions [8]. In proteins, Cu(I) is thus mainly coordinated by side chains of cysteine (Cys) and methionine (Met) residues but also by the imidazole of histidine (His) residues. More information about the binding of biomolecules with Cu(I) can be found in references [8, 9].

### 1.2.2. Cu trafficking

After being absorbed in the intestine, Cu(II) is transported to the liver, where it is then exported to other organs. Cu is secreted extracellularly as protein-bound Cu(II) [10]. In the bloodstream, Cu(II) is bound to carriers including the ferroxidase ceruloplasmin (Cp, between 70 % and 85 % [11]) depending on the study), albumin (6 %) and transcuprein [12,13]. Cp is metalated before its excretion from the liver with six Cu(II) ions. Three Cu(II) are in Type 1-like sites (coordinated by the side chains of two His and one Cys residues, with an additional ligand being either a Met or a Leu side chain), while the three additional Cu(II) are in a trinuclear arrangement [14,15]. Albumin binds Cu(II) *in vitro* through the Amino-Terminal Cu and Ni-binding (ATCUN) motif H₂N–XXX–Zzz–His [16], while the coordination site of transcuprein for Cu(II) is still unknown. Cu(II) binding facilitates its transport to tissues without causing toxicity [17,18]. Cu can also be found bound to amino acids, or to small peptides such as GHK (9 %) [5]. The excess of Cu is excreted from the liver cells to the bile and eliminated with feces. Readers interested in Cu(II) carriers or its homeostasis in living organisms are referred to references [8, 10, 17, 19]. Cu enters the cell from the bloodstream *via* the passive Cu transporter Ctr1 (for copper transporter 1 or SLC31A1 for solute carrier family 31 member 1) (Fig. 1) [20], which is a homo-trimeric membrane protein with extracellular N-terminal domain rich in Met and His residues (Fig. 2) [21]. It is not clearly defined yet whether the Cu(II) is reduced by a metalloreductase such as Steap (for six-transmembrane epithelial antigen of the prostate) to facilitate its uptake [22], reduced directly by the N-terminal domain of Ctr1 [23,24] or reduced by glutathione (GSH) intracellularly (Fig. 1). Additionally, research on the HSA (human serum albumin) protein, *in vitro*, suggests that the Cu(II)HSA could directly transfer Cu(II) to the N-terminal domain of Ctr1 [25] or that Cu(II)HSA complex could be reduced by ascorbate extracellularly to Cu(I)HSA complex, where Cu(I) is bound in a coordination site that has yet to be identified. This complex, with an apparent affinity constant estimated to log Kapp = 14.0 [26] may play a role in the delivery of Cu(I) to Ctr1 [26,27]. Subsequently, Cu(I) is transported to different cell compartments and specific targets by Cu(I) chaperones *via* protein-protein interactions [28]. In eukaryotic cells, under normal conditions, it is believed that there is no free Cu [29,30]; Cu would be bound to small molecules or proteins. Indeed, when not bound to cuproenzymes or chaperones, Cu is bound by GSH or metal-lothioneins (MTs). The role of the three latter actors is described here after.

<table><caption>**Table 1**

Association constants of main proteins involved in Cu homeostasis and the methods that enabled their determination. In hCtr1 “h” stands for human. X represents any amino acid.</caption><thead><tr><th rowspan="3">Protein</th><th colspan="6">pH</th></tr><tr><th colspan="2">6.5</th><th colspan="2">7.0</th><th colspan="2">7.5</th></tr><tr><th>Log K</th><th>Method (ref)</th><th>Log K</th><th>Method (ref)</th><th>Log K</th><th>Method (ref)</th></tr></thead><tbody><tr><td rowspan="2">CCS</td><td>16.6</td><td>BCS ([42])</td><td></td><td></td><td>17.7</td><td>BCS ([42])</td></tr><tr><td>17.0</td><td>BCA ([42])</td><td></td><td></td><td></td><td></td></tr><tr><td>CCS-1<br>CXXC</td><td>16.7</td><td>BCS ([42])</td><td></td><td></td><td>17.7</td><td>BCS ([42])</td></tr><tr><td>CCS-3<br>CXC</td><td>15.6</td><td>BCA ([42])</td><td></td><td></td><td>16.8</td><td>BCA ([42])</td></tr><tr><td rowspan="2">ATOX1</td><td></td><td></td><td>17.7</td><td>BCS ([43])</td><td rowspan="2">10.5</td><td rowspan="2">BCA (ITC, log β<sub>2</sub> = 14.7, [44])</td></tr><tr><td></td><td></td><td>17.4</td><td>BCS ([45])</td></tr><tr><td>ATP7A-1</td><td></td><td></td><td>17.6</td><td>BCS ([43])</td><td></td><td></td></tr><tr><td>ATP7B(5–6)</td><td></td><td></td><td rowspan="2">17.6</td><td rowspan="2">BCS ([46])</td><td rowspan="2">10.3–10.8</td><td rowspan="2">BCA (ITC, log β<sub>2</sub> = 14.7, [44])</td></tr><tr><td>ATP7B(1–6)</td><td></td><td></td></tr><tr><td>hCtr1_HCH motif</td><td></td><td></td><td></td><td></td><td>13.4</td><td>BCA (log β<sub>2</sub> = 17.2, [47])</td></tr><tr><td>KKAVVVDITEHCH</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>hCtr1_N-ter</td><td></td><td></td><td></td><td></td><td>12.8</td><td>BCA (10.4 if considering Cu(BCA) species ([48])</td></tr><tr><td>DHSHHMGMSYMDS</td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Schematic representation of Cu trafficking in cells showing the pathways for Cu entrance and exit from eukaryotic cells, and for Cu distribution inside the cell. For clarity the clusters that may form between GSH and Cu(I) are not represented. Only the 1:1 interaction is represented. The main association constant of the proteins involved in Cu(I) homeostasis are listed in Table 1.</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Example of Cu(I) binding sites found in proteins and small molecules in living organisms, where the three main coordination geometries (linear, trigonal and tetrahedral) of Cu(I) are encountered. Cu(I)₈-MT cluster was drawn from the crystal structure of *S. cerevisiae* Cu(I)₈-MT (PDB ID: 1RJU) [41].</figcaption></figure>

The pseudo tripeptide glutathione (γ-L-glutamyl-L-cysteinylglycine, GSH) (Fig. 1 and Fig. 2) plays several essential roles in cellular processes, such as reductant and radical scavenger. It is present at millimolar concentration in cells. In the context of Cu metabolism, GSH can tightly but reversibly bind Cu(I) ions in a metal-cluster arrangement (Fig. 2), aiding in their transport and storage within cells. This helps regulating

the availability of bioactive Cu(I) and preventing Cu-induced oxidative damage. It has been proposed that Cu(I) binds to GSH before binding to chaperones or to the MTs (vide infra) [31,32]. Nevertheless, additional research has indicated that intracellularly Cu(I) is buffered around 1 aM, which is below the threshold required for Cu-GSH ([Cu₄(GS)₆]⁻) cluster assembly, under standard cellular conditions [30]. It does not preclude
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Schematic representation of the main mechanisms of Cu toxicity.</figcaption></figure>

that GSH binds transiently to Cu(I) and serves as a carrier, facilitating the transfer of Cu(I) released from Ctr1 to other proteins or enzymes with greater affinity. As the typical volume of a cell is in a range of picolitres, the quantity of free Cu(I) per cell is negligible [29,30]. GSH might also be a very important biomolecule to consider when thinking of the speciation of Cu complexes used for therapeutic purpose. It has been recently proposed that GSH could interact in the cell with exogenously added Cu-complexes such as bis(phenanthroline)copper and Cu-thiosemicarbazone complexes and modulate their reactivity through the formation of ternary species [33,34]. In general, the reductive dissociation of Cu(II) complexes within cells by GSH generating Cu(I)-GSH clusters may markedly diminish the capacity of the Cu complexes to generate ROS and exert their cytotoxic effects (when used as anti-cancer drug) [34]. Nevertheless, the formation of ternary species GS-Cu-ligand might help in keeping a pro-oxidant activity of Cu as very recently highlighted for the Cu-phen₂ complex, which evolves in solution to form a ternary complex at low pH (at lysosome relevant pH) probably accountable for the cytotoxic activity of this compound [34].

Metallothioneins (MTs) are small, cysteine-rich proteins that play a critical role in Cu(I) and zinc (Zn) metabolisms. They bind metal ions in clusters (Fig. 2, up to twelve Cu(I) ions) [35,36] through the thiolate groups of their Cys residues, and help eliminate the excess of Cu and other heavy metals, thereby preventing potential toxic effects such as oxidative stress and cellular damage (vide infra) [36]. They act as radical scavengers or antioxidants through their thiolate reactivity. By binding Cu(I) ions, MTs maintain intracellular Cu levels and could provide a reserve of bioavailable Cu(I). The function of MTs in Cu(I) homeostasis is still a field of research and more evidence are needed to determine whether MTs could be involved in the scavenging, storage, or buffering of Cu(I) [36].

Chaperones are another very important Cu(I) intracellular carrier. They are cytosolic proteins that facilitate the secure transfer of Cu(I) to

specific cellular targets, including SOD [37], cytochrome c oxidase in mitochondria, and components of the secretory pathway (Fig. 1). They exhibit a sequence motif, Cys-(Xxx)n-Cys (n = 0–3), which enables them to bind Cu(I) in a linear geometry. Some of them have an additional His residue nearby allowing a T-shape geometry [8]. The well-studied chaperones ATOX1 (Antioxidant Protein 1) [38] and CCS (copper chaperone for superoxide dismutase) [39] (Fig. 1) bind Cu(I) via a Cys-Xxx-Zzz-Cys sequence motif (Fig. 2) [8]. While CCS delivers Cu(I) to Cu/Zn SOD1 in the cytoplasm, ATOX1 transfers Cu(I) to P-type ATPases ATP7A and ATP7B on the trans-Golgi network, where the metalation of cuproenzymes occurs. The cuproenzymes are then excreted from the cells (Fig. 1) [5].

In excess of Cu(I) in the cell, ATP7A/B can also export Cu(I) directly at the membrane and/or by loading vesicles that will excrete Cu(I) (Fig. 1) [40]. ATP7B represents a principal Cu efflux system within the liver, while ATP7A is a primary Cu-efflux system in the majority of other tissues. Additionally, ATP7A plays a pivotal role in the transfer of Cu across the blood brain barrier (BBB) into the brain [5].

When the organisms, tissues, cells are not able to handle Cu properly anymore, due to overload, deficiency or misregulation, acute toxicity can be observed. The next section describes the currently documented Cu toxicity in cells.

### 1.3. Cu toxicity

The production of reactive oxygen species (ROS) by Cu has been demonstrated to induce a variety of cell death pathways including apoptotic and non-apoptotic processes [49]. Furthermore, Cu has been demonstrated to induce toxicity through pathways that are independent of ROS production [50–55]. This is detailed hereafter.
### 1.3.1. Toxicity related to ROS production

It was first established that the toxicity of excess Cu was due to its redox ability. In the presence of dioxygen, Cu(I) can be oxidized to Cu(II) while partially reducing O₂ to form ROS, including superoxide (O₂⁻), hydrogen peroxide (H₂O₂) and hydroxyl radical (HO•) (Fig. 3). The generated Cu(II) can then be reduced again by a biological reductant, leading to the catalytic production of ROS. O₂⁻ can rapidly dismutates to O₂ and H₂O₂ in presence of SOD. O₂⁻ can additionally react with nitric oxide (NO) to form the peroxynitrite (ONOO⁻) anion which can participate in oxidation of biomolecules [56]. ONOO⁻ can be involved in both cellular apoptosis and necrosis [56]. Though hydrogen peroxide (H₂O₂) is less reactive than HO•, it is still highly toxic as it can diffuse and act as a precursor for other ROS, including HO• via the Fenton reaction [57]. H₂O₂ can also directly oxidize specific biomolecules, contributing to oxidative stress. HO• can damage surrounding biomolecules, including proteins, membrane lipids, and nucleic acids, leading to aberrant cell function and death.

It is likely that proteins represent a significant site of oxidation, with the potential for oxidation of aliphatic side chains (leucine, isoleucine, valine, and proline), formation of crosslinked products RS-SR, Trp-Trp, Tyr-Tyr, or cleavage of the peptide backbone [58]. The interaction between membrane phospholipids and ROS results in lipid peroxidation, whereas the interaction between DNA and ROS gives rise to DNA base oxidation [59,60] (Fig. 3).

### 1.3.2. ROS-independent toxicity

#### 1.3.2.1. Proteotoxic stress.
It was recently proposed that Cu overload could induce a proteotoxic stress that leads to cell death independently of ROS production. It was suggested that Cu(I) may react directly with proteins, resulting in their misfolding and aggregation. Binding of Cu(I) to GSH would prevent the deleterious interaction between Cu and proteins by forming an inactive complex [53]. In the context of Cu as an antibacterial agent, previous researches demonstrated that Cu overload under anaerobic conditions can lead to a widespread increase in protein aggregation, which is a central mechanism of Cu toxicity in *Escherichia coli* (Fig. 3) [61].

#### 1.3.2.2. Alteration of iron-sulfur clusters.
Due to its thiophilic nature, Cu (I) has the ability to interact with cysteine-containing proteins, interfering with their functions. In bacteria, it was shown that Cu(I) could alter the Fe/S metabolism either by impeding the cluster formation and/or by displacing Fe [62–64]. For example, in *Escherichia coli*, it was shown that Cu(I) ions could inactivate deshydratase enzymes by replacing Fe ions [55]. Further *in vitro* studies proposed that the maturation of mitochondrial [4Fe-4S]-containing proteins is impaired by Cu (I). Indeed, Cu(I) was shown to be able to interact with the cysteine residues of two key proteins in the maturation of mitochondrial proteins, preventing them to transfer the cluster to the final apo-protein. Thus, a misregulation of Cu homeostasis could damage mitochondrial protein maturation [54]. Investigations into the effects of Cu(I) on the Fe/S biogenesis in eukaryotic mitochondria have been conducted on both cells and mouse models of WD and demonstrate that Cu(I) is also responsible for the impairment of Fe/S assembly process [65].

#### 1.3.2.3. Cuproptosis.
Very recently, a pathway of Cu-induced toxicity has been reported, which relies on (i) lipoylated protein aggregation and (ii) Fe/S cluster containing protein loss leading to proteotoxic stress and cell death. The use of ionophores (Elesclomol) capable of importing Cu into cells facilitated the elucidation of a novel pathway of cell death, distinct from other known mechanisms, named cuproptosis. This mechanism of Cu toxicity is linked to mitochondrial respiration. In the tricarboxylic acid cycle (TCA), Cu can bind to lipoylated proteins, thereby inducing their aggregation. Additionally, a reduction of Fe/S clusters in proteins and the induction of 70-kDa heat shock proteins

(HSP70) were observed in Cu-overloaded cells that rely on mitochondrial respiration. The same biomarkers were observed in the liver of mice models of WD, which accumulate Cu intracellularly. This indicates that Cu overload in WD could have the same cellular effects as those induced by Cu ionophores, namely cuproptosis [51].

### 1.4. Main Cu-related diseases

Dysfunction of Cu trafficking in link with the previously mentioned toxicities can simultaneously impair several biochemical functions, resulting in severe adverse outcomes. This is exemplified by the two most prevalent Cu-related disorders: Menkes and Wilson diseases. These are caused by mutations in the membrane transporters ATP7A and ATP7B, respectively. Besides these two genetic diseases, Cu homeostasis misregulation was also identified in cancer and several neurodegenerative diseases such as Alzheimer's disease, amyotrophic lateral sclerosis and Parkinson's disease [5,66].

#### 1.4.1. Cu deficiency

Menkes disease (MD) is caused by a mutation in the ATP7A gene [67], which results in reduced Cu influx into the blood due to decreased functional capacity for Cu uptake in the intestine [68]. The marked Cu deficiency results in a notable decline in the activity of cuproenzymes leading to the death of the patients. In 2020, it was demonstrated that Elesclomol, a Cu ionophore, has the potential to elevate the Cu level in mitochondria and enhance the cytochrome c oxidase level in brain tissues in a murine model of severe MD [69]. This has the beneficial effect of preventing detrimental neurodegenerative changes and improving the survival of the animals [69,70].

#### 1.4.2. Cu overload

Wilson disease is a disorder that results from mutations in the ATP7B gene, leading to the accumulation of Cu in liver and brain tissues [3,71]. Current treatment consists in using chelators to reduce the Cu level content (vide infra).

#### 1.4.3. Cu misregulation

The involvement of Cu misregulation in cancer and in Alzheimer's disease (AD) is less straightforward than in the other diseases mentioned above, Menkes or Wilson disease. However, a body of evidence supports its role in cancer development and in AD [72–74].

##### 1.4.3.1. Cancer.
Cu has been linked to cancer development and progression and can facilitate cell proliferation, angiogenesis and metastasis [75–77]. The accumulation of Cu and the up-regulation of Cu-transporters/chaperones (e.g. Ctr1, ATOX1, CCS, COX17-cytochrome c oxidase copper chaperone-, SCO1- synthesis of cytochrome c oxidase 1-, as well as ATP7A/B) in cancer cells have been documented in a multitude of tumor types [76]. The serum level of Cu are also increased in patients [76]. In this context, two therapeutic strategies are the subject of current investigations. The first one involves the use of a Cu chelator to deplete Cu within cells to stop the tumor growth [17,78,79], while the second employs Cu ionophores to facilitate the delivery of Cu into cells, thereby increasing intracellular levels and triggering cell death [5,51].

##### 1.4.3.2. Alzheimer's disease.
Although the main characteristic of AD is the formation of protein-based deposits, extracellular senile plaques made of the amyloid-β (Aβ) peptides extracellularly and intracellularly Tau tangles built on hyperphosphorylated Tau proteins, a disruption in Cu homeostasis has also been identified as a key process [80–82]. Depending on the biological compartment (extra- or intracellular) the Cu levels are either up-regulated or down-regulated [83–85]. A high Cu concentration is found in the plaques (extracellularly) where Cu has the ability to bind Aβ. The CuAβ complex has been linked to the increase of the oxidative stress in AD through its ability to produce ROS
<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Scheme of Cu(I) ligands used to determine Cu(I) affinity. BCS = bathucuproine disulfonate; BCA = bicinchoninate anion; Fz = ferrozine; Fs = ferene; PEMEA = 2-Pyridylethylbis(2-methylthioethyl)amine and MCL = monovalent copper ligands.</figcaption></figure>

extracellularly [86,87] while a decrease in intra-cellular Cu levels results in a decline in the activity of copper-related enzymes that are crucial for antioxidant defenses (SOD) and for ATP synthesis. This, in turn, leads to an increase in ROS levels. Moreover, the hypothesis that increasing the intracellular Cu content would trigger extracellular Aβ degradation *via* matrix metalloproteases (MMPs) has been proposed [82,88]. More generally research has demonstrated that the majority of proteins that play a pivotal role in the development of AD, including the amyloid precursor protein (APP), the Aβs, the secretases, and the MMPs that are primarily responsible for degrading Aβ, possess Cu binding sites [81,82]. Furthermore, researchers have demonstrated that the mean blood level of loosely bound Cu is elevated in individuals with AD compared to healthy controls [89,90]. Cu has thus been identified as a potential therapeutic target; ligands able to remove Cu from CuAβ complex have mainly been studied [66,83].

Given that Cu is toxic when not properly regulated, it has become a therapeutic target of choice. Hence, chelating molecules that bind Cu(I) have emerged as a potential treatment for a number of diseases related to Cu metabolism, including WD, cancer, and AD. In order to design effective molecules, it is imperative to gain a thorough view of Cu(I) metabolism in living organisms and thus, to develop appropriate tools to study its speciation. The present review reports an overview of the tools that have been designed for the study of Cu(I) *in vitro* and *in vivo* as well as small Cu(I) chelating molecules that have been developed for the treatment of Cu(I)-related diseases (AD, WD and cancer).

## 2. Tools to evaluate Cu(I) affinity *in vitro* by UV-Vis

Affinity of ligands and proteins for Cu(I) is a key piece of information to understand Cu speciation in complex biological environments, and to design Cu(I) chelators of appropriate affinity able to compete against biomolecular ligands. For guidelines to determine metal-protein affinities, the reader is referred to the excellent reviews by Xiao et al. [45,96]. Affinity constants are usually determined *in vitro*, with the assumption that their relative values will remain the same in complex biological media [45]. Determining stability constants of Cu(I) complexes presents its own challenges, including oxygen sensitivity of the Cu(I) ion, risk of disproportionation to Cu(0) and Cu(II) at low pH, or precipitation to Cu₂O at neutral and high pH [92]. For small-molecule ligands, a number of affinity constants were initially determined using the stability constant of the Cu(II) complex, its reduction potential and Nernst Eq. [92]. However, the accurate determination of redox potentials requires the Cu(I)/Cu(II) couple to be reversible, which is not the case for all complexes.

This triggered the development of affinity standards that could be

used in competition experiments to determine binding constants of ligands of interest (e.g. proteins or small molecules). Wedd et al. characterized a series of water-soluble, chromophoric probes for Cu(I) affinity (Fig. 4, top): bathucuproine disulfonate (BCS) and bicinchoninate anion (BCA) [91,97]. These ligands form 1:2 (M:L, M for metal and L for ligand) complexes with Cu(I) that exhibit intense absorption bands in the visible region (Table 2), thereby providing an easy readout in competition assays (the free ligands do not absorb in this region). The formation constant (log β₂) of the complex formed between BCS and Cu (II) has been determined using Nernst equation (Table 2). Once determined the value for BCS, that for BCA was deduced by competition experiments. Later probes of weaker affinities (ferrozine, Fz and ferene, Fs) were studied with the aim to evaluate Cu(I) binding affinity to the Aβ peptides [94,95]. Their formation constants were deduced from competition with Cu(I) proteins, from which the values were first deduced from competition with BCA. Hence, a first series of values for Cu(I) indicators all come from the original BCS values of log β₂ = 19.8 (Table 2). However, for the weaker probes Fz and Fs, direct competition can be applied, although it has some limitations [92,95]. This in part explains the discrepancies reported between values (Table 2), while no method seems ideal. In the former case, the BCS value was determined based on a poorly reversible Cu(I)/Cu(II) that prevents the use of the Nernst equation. In the latter case, direct titration experiments may be biased by the formation of Cu₂O or by dismutation if not properly determined. Besides, it should be noted that the values obtained by titration are apparent values (Kapp) (i.e. at a given pH, taking into account the competition with buffer or / and exogenous ligand) and hence are systematically lower than the absolute values.

In brief, when using these chromophoric probes, the reference value of log β₂ used to determine affinities of proteins or ligands for Cu(I) should always be clearly stated. In the following, the values given are Kapp and unless otherwise stated, were obtained by competition with BCS or derivatives, with log β₂ (BCS) = 19.8. Once precaution is taken into account and the appropriate probe chosen, affinities between ~10⁷ to 10²⁰ can be evaluated, which covers most protein affinities.

Furthermore, some proteins can form ternary complexes with these ligands, thereby presenting a risk to misestimate the affinity for Cu(I) [98]. Control experiments should always be conducted, including careful examination of the solution spectra, metal titration of the ligand in presence and absence of protein, examining the effect of varying concentrations on the affinity constants, etc.

To overcome the formation of ternary complexes, Fahrni and coworkers developed a set of ligands (6–8, MCL1–3, for monovalent copper ligands 1–3, Fig. 4) forming stoichiometric complexes with Cu(I).
<caption>**Table 2**</caption>

Thermodynamic parameters of ligands shown in Fig. 4. The values shown are apparent association constants, and experimental conditions in which they were determined are given. pCu values were reported at pH 7.0 for total Cu(I) and L concentrations of 1 and 10 µM, respectively.

<table><thead><tr><th>L</th><th>ε x 10<sup>3</sup> M<sup>-1</sup>·cm<sup>-1</sup> (λ<sub>max</sub> (nm)) of [Cu(I)<sub>L</sub>]<sup>3-</sup></th><th>log K<sub>Cu(I)</sub><sub>L</sub></th><th>log β<sub>2</sub><sup>APP</sup></th><th>Method</th><th>pCu</th><th>Ref</th></tr></thead><tbody><tr><td rowspan="3">BCS</td><td rowspan="3">13.3 (483)</td><td rowspan="3">-</td><td>19.8</td><td>from log β<sub>2</sub> ([Cu(II)BCS<sub>2</sub>]<sup>2-</sup>) and E<sup>0</sup>([CuBCS<sub>2</sub>]<sup>3-/-2</sup>) in 0.01 M acetate or 2-(N-morpholino)-ethanesulfonic acid buffer and ionic strength 0.1</td><td rowspan="3">15.6<sup>b</sup></td><td rowspan="3">[46,91]</td></tr><tr><td>20.8</td><td>from log β<sub>2</sub> ([Cu(II)BCS<sub>2</sub>]<sup>2-</sup>) and E<sup>0</sup>([CuBCS<sub>2</sub>]<sup>3-/-2</sup>)</td></tr><tr><td>22.1</td><td>pH 5.0 buffer (10 mM PIPBS, 0.1 M KCIO<sub>4</sub> Competition with MCL-1 Competition with cyanide, pH 6.0</td><td>16.6</td><td>[92]</td></tr><tr><td rowspan="4">BCA</td><td rowspan="4">7.9 (562)</td><td rowspan="4">-</td><td>14.7</td><td>Calorimetry – From Cu(I) under anaerobic conditions – HEPES 50 mM, 7.5, 0.2 M NaCl</td><td rowspan="4">13.5</td><td rowspan="4">[92]</td></tr><tr><td>17.2</td><td>Competition with BCS</td></tr><tr><td>17.7</td><td>Competition with MCL-1</td></tr><tr><td>11.4</td><td>Direct metal titration (pH 6.0)</td></tr><tr><td rowspan="3">Fz</td><td rowspan="3">4.32 (470)</td><td rowspan="3">-</td><td>11.6</td><td>Direct metal titration (UV) – from Cu(I), anaerobic conditions, HEPES 0.1 M pH 7.4</td><td rowspan="3">10.9<sup>b</sup></td><td rowspan="3">[94,95]</td></tr><tr><td>>12.5</td><td>Direct metal titration – from Cu(II) in Mops 50 mM, 7.0, NH<sub>2</sub>OH 2 mM, Asc 1.0 mM</td></tr><tr><td>15.1</td><td>Ligand competition (PcoC, CopK)</td></tr><tr><td rowspan="2">Fs</td><td rowspan="2">6.7 (484)</td><td rowspan="2">-</td><td>11.0</td><td>Direct metal titration – from Cu(II) in Mops 50 mM, 7.0, NH<sub>2</sub>OH 2 mM, Asc 1.0 mM</td><td rowspan="2">9.5<sup>b</sup></td><td rowspan="2">[95]</td></tr><tr><td>13.7</td><td>Ligand competition (CopK)</td></tr><tr><td>PEMEA</td><td>-</td><td>15.7</td><td>-</td><td>from log K([Cu(II)L]<sup>-</sup>) and E<sup>0</sup>([Cu(II)L/Cu(I)L]<sup>-/-2</sup>) pH 5.0, 10 mM PIPBS, 0.1 M KCIO<sub>4</sub></td><td>16.2</td><td>[92]</td></tr><tr><td>MCL-1</td><td>-</td><td>16.3</td><td>-</td><td>from log K([Cu(II)L]<sup>-</sup>) and E<sup>0</sup>([Cu(II)L/Cu(I)L]<sup>-/-2</sup>) pH 5.0, 10 mM PIPBS, 0.1 M KCIO<sub>4</sub></td><td>16.9</td><td>[92]</td></tr><tr><td>MCL-2</td><td>-</td><td>13.1<sup>a</sup></td><td>-</td><td>Competition with BCA</td><td>11.9</td><td>[92]</td></tr><tr><td>MCL-3</td><td>-</td><td>13.8<sup>a</sup></td><td>-</td><td>Competition with BCA and BCS</td><td>14.8</td><td>[92]</td></tr></tbody></table>

The stability constants of the resulting complexes ranged from 13.1 for MCL-2 to 16.3 for MCL-1. These values of log K<sub>Cu(I)L</sub>, obtained for 1:1 complexes (M:L), cannot be directly compared with log β₂ obtained for the 1:2 (M:L) complexes. Nevertheless, the capacity of buffering Cu(I)aq of these ligands can be compared using pCu = -log [Cu(I)aq], for given total concentrations of Cu(I) and ligand. Examples are shown in Table 2. The MCL ligand series was capable of buffering Cu(I) concentrations between 10<sup>-10</sup> and 10<sup>-17</sup> M at pH 7. These ligands are optically inactive, and can thus complement the chromophoric probes when the ligand of interest has a spectroscopic signature overlapping the one of the probes. MCL ligands can also be used in fluorescence assays, e.g. to determine the affinities of fluorescent Cu(I) probes. It was also used to determine the affinity of CusF (a periplasmic protein involved in the detoxification of copper and silver ions in E. coli) using tryptophan fluorescence of the protein, quenched upon Cu(I) binding [92].

### 3. Tools to localize and track the Cu labile pools in biological systems by fluorescence

Due to its critical role in a number of physiological functions, it is important to develop tools to better understand the fundamental interactions of Cu(I) with the biological environment and its speciation both in cells and in vivo. Several techniques have been developed to localize Cu(I) in cells, such as inductively coupled plasma mass spectrometry (ICP-MS) [99], secondary ion mass spectrometry (SIMS); nano-secondary ion mass spectrometry (nano-SIMS) [100], synchrotron-based X-ray absorption spectroscopy (XAS), X-ray fluorescence microscopy (XFM) [101–103]. However, while these techniques are very informative for Cu(I) mapping, they are not easy to implement and, in most cases, do not allow for real-time tracking of Cu(I) exchange in a living organism during exogenous stimuli or cellular events.

In this context, researchers have designed small-molecule fluorescent probes able to selectively respond to Cu(I) with spatial and temporal resolution [9,19,104–108]. Fluorescence detection appeared as an appropriate technique as it is nondestructive, sensitive and compatible with visualization in living specimens. These probes are based on the covalent attachment of a binding cavity to a fluorescent moiety. Most of these sensors are intensity-based sensors but some ratiometric sensors are also described [19]. Intensity-based turn-on fluorescent sensors, respond reversibly to Cu(I) with an increased in their fluorescence intensity mostly through a photo-induced electron transfer (PET) [109,110] or an intramolecular charge transfer (CT) process [19,105]. For ratiometric sensors the emission ratio between two distinct wavelengths is measured. This approach normalizes the sensor's response to Cu(I), thereby minimizing the influence of probe concentration, for instance, compared to intensity-based probes [111]. Key examples of both families of Cu(I) sensors (intensity-based and ratiometric sensors) and their use will be described in the following sections. Note that activity-based sensors as well as engineered fluorescent proteins for Cu(I) sensing are out of the scope of this review, see references [19, 107, 112–115].

#### 3.1. Key parameters in the design of the probes

To meet its target (i.e. loosely-bound Cu(I)), it is imperative that the probes possess sufficient selectivity and sensitivity to detect the physiological concentration of Cu(I) in a complex cellular environment, while simultaneously avoiding any disruption to the native function of metalloproteins through the removal of their metal cofactor, which could potentially generate toxicity [105]. Given that the probes are designed to be internalized in cells, it is essential that a suitable balance between solubility and lipophilicity is reached in order to facilitate their passive crossing of cell membranes. The photophysical properties of the probe

<footnote><sup>a</sup> Reference [92] used log β₂ (BCA) = 17.7 and log β₂ (BCS) = 20.8.</footnote>

<footnote><sup>b</sup> Calculated for the conditions described in the caption.</footnote>
<figure><img src="image_7.png" /><figcaption>**Fig. 5.** Scheme of classical fluorescent moieties, violet arrows indicate possible position where the Cu(I) binding cavity can be attached. The fluorescent part of the probe **NCP1** is not shown for clarity as the rhodol is not aromatic. The names of the full probes (binding cavity + fluorescent moieties) incorporating the fluorescent moieties described in the manuscript are indicated in bold under the structure, when they have a meaning, they are given here after, Dns-LCC = dansyl-LeuLeuCys; **CSR1** = Cu Silicon Rhodol 1; **NCP1** = NIR copper probe-1; **CS1,3** = Cu sensor-1,3; **CTAP-1-3** = Cu-responsive triarylpyrazoline-1-3; **Crisp-17** = Cu ratiometric indicator with stabilized phosphines. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

are of great importance too, including brightness, which is linked to the quantum yield and the molar extinction coefficient [116]. As the brightness of the compound increases, the concentration of the probe required decreases, thus affecting the homeostasis of Cu(I) to a lesser

extent. The wavelength of excitation and emission is also a key parameter, as many biomolecules absorb light in the ultraviolet (UV) and visible regions. Furthermore, low-energy, high-wavelength light excitation generates less phototoxicity than high-energy excitation

<figure><img src="image_8.png" /><figcaption>**Fig. 6.** Scheme of most used binding cavities. In bold and brackets are the names of the probes described in the manuscript that incorporate this motif. BETA = bis[2-[2-(2-ethylthio)ethylthio]ethyl]amine, designating this specific binding unit.</figcaption></figure>
[104,105]. The change in fluorescence upon Cu(I) binding, and the photostability are important too. All these parameters have to be adjusted by selecting the appropriate fluorescent moieties and binding cavities.

## 3.2. Fluorescent moieties

A variety of fluorescent moieties have been employed in the construction of Cu(I) fluorescent probes, including for example, naphthalene, pyrazoline, boron dipyrrromethene (BODIPY), rhodol and cyanine derivatives (Fig. 5). Among these families the most prevalent are BODIPY and rhodol derivatives. Both exhibit appropriate excitation and emission wavelengths within the visible region for in cells applications. The photophysical properties of the construct are highly dependent on the chemical modification of the dye core, as these dyes are both highly tunable and synthetically modular [117–119]. The use of cyanine dyes has permitted the synthesis of probes with excitation and emission in the near-infrared region (NIR).

## 3.3. Binding cavities

The binding sites linked to the fluorescent probes designed to target Cu(I) selectively rely predominantly on sulfur-rich ligands. Indeed, as previously stated, according to the HSAB principle, Cu(I), which is a soft acid, will preferentially bind to soft atoms, such as sulfur [6,7]. The inherent propensity of Cys and Met residues to bind Cu(I) in biological

<caption>**Table 3**</caption>

Key characteristics of the fluorescent probes under discussion in this section including the coordinating set of atoms, the apparent dissociation constant and the excitation and emission wavelengths.

<table><thead><tr><th>Sensor name</th><th>Binding cavity</th><th>LogK<sup>Cu(I)</sup> (conditions of determination)</th><th>λ exc/ λ emission (nm)</th><th>Ref</th></tr></thead><tbody><tr><td>CTAP-1</td><td>1N4S<sub>cyclic</sub>, 9</td><td>10.4 (PIPES pH 7.2)<sup>a</sup></td><td>365/480</td><td>[102]</td></tr><tr><td>CTAP-2</td><td>1N3S<sub>cyclic</sub>, 10</td><td>11.4 (PIPBS, pH 5)<sup>b</sup></td><td>396/508</td><td>[128]</td></tr><tr><td>CTAP-3</td><td>1(NH)<sub>3</sub>3S<sub>acyclic</sub>, 11</td><td>10.3 (MES, pH 6.0)<sup>c</sup></td><td>365/455</td><td>[129]</td></tr><tr><td>CS1</td><td>1N4S<sub>acyclic</sub> (BETA), 13</td><td>11.4 (HEPES, pH 7.0)<sup>d</sup></td><td>540/561</td><td>[130]</td></tr><tr><td>CS3</td><td>1N4S<sub>acyclic</sub> (BETA), 13</td><td>13.1 (HEPES, pH 7.0)<sup>d</sup></td><td>540/561</td><td>[131]</td></tr><tr><td>CSR1</td><td>1N4S<sub>acyclic</sub> (BETA), 13</td><td>12.7 (HEPES, pH 7.0)<sup>d</sup></td><td>608/631</td><td>[132]</td></tr><tr><td>ACu1</td><td>1N4S<sub>acyclic</sub> (BETA), 13</td><td>10.7 (HEPES, pH 7.0)<sup>d</sup></td><td>365/492</td><td>[123]</td></tr><tr><td>CS790</td><td>1(NH)<sub>3</sub>3S<sub>acyclic</sub>, 12</td><td>10.5 (HEPES, pH 7.0)<sup>d</sup></td><td>760/790</td><td>[127]</td></tr><tr><td>BDPS1</td><td>1N4S<sub>acyclic</sub> (BETA), 13</td><td>11.3 (HEPES, pH 7.470 % DMF)<sup>d</sup></td><td>600/710</td><td>[133]</td></tr><tr><td>NCP1</td><td>1N4S<sub>acyclic</sub> (BETA), 13</td><td>11.3</td><td>640/704</td><td>[134]</td></tr><tr><td>Dns-LLC</td><td>LeuLeuCys, 14</td><td>13.9 (PBS, pH 7.4)<sup>e</sup></td><td>400/500</td><td>[125]</td></tr><tr><td>Crisp-17</td><td>3(P=S), 15</td><td>17.0 in (4:1) DMPC-DMPG liposomes, PIPES pH 7.0)<sup>f</sup></td><td>450/555</td><td>[30]</td></tr></tbody></table>

systems in addition to the intensive studies conducted on the coordination properties of Cu(I)/(II) in cyclic and acyclic thia-aza-macrocyclic ligands [9,120–122] may have contributed to this choice. Furthermore, in the construct that relies on a PET, an amine function acts as the PET switch. The PET process is inhibited upon binding of the Cu(I) by the lone pair of the amine, thereby enabling the fluorescence to turn-on [110]. The acyclic bis[2-[2-(2-ethylthio)ethylthio]ethyl]amine (1N4S, **BETA**) binding cavity (Fig. 6) has been intensively used as Cu(I) receptor [19]. Depending of the final molecule and the conditions of determination, the apparent binding constants have been shown to span from log K<sub>app</sub> of 10.7 [123] to 13.7 [124] for the same cavity. Other cyclic and acyclic thia-aza ligands have been described with a similar range of affinity values (log K<sub>app</sub> = 10–11.5) [104,106]. Additionally, receptors with higher affinity have been described, one of which relies on a leucine-leucine-cysteine (LeuLeuCys) tripeptide with a log K<sub>app</sub> of 13.9 (Fig. 6, 14) [125] while a receptor based on stabilized phosphines exhibits an apparent affinity as high as a log K<sub>app</sub> of 17.1 (Fig. 6, 15) [30].

## 3.4. Illustrative examples of the use of the probes

A variety of exogenous fluorescent probes are capable of detecting alterations in the concentration of Cu(I) within cells induced by the addition of a Cu(I) chelator to reduce its concentration or when a Cu(II) salt is added to the medium to elevate its concentration [19,123,126,127] or upon an endogenous cellular event. Additionally, spatial information can be obtained regarding the localization of Cu in cells. The following section presents key probes and their respective applications from pioneering work to more recent studies. Physicochemical properties of the probes are gathered in Table 3. For a more exhaustive and up-to-date analysis of this subject, please refer to the following references [19, 107].

### 3.4.1. CTAP family

In a study conducted by Fahrni and colleagues, Cu(I) fluorescent sensors were developed based on a thioether-rich macrocycle (azatetrathiacrown, 1N4S, Fig. 6, 9) as the binding site and a triarylpyrazoline moiety (Fig. 5) as the dye. The initial probe, designated **CTAP-1** for Cu-responsive triarylpyrazoline-1, exhibits a logK<sub>app</sub>Cu(I) of 10.4, and displays a 4.6-fold fluorescence turn-on enhancement upon Cu(I) binding [102]. Following Cu(II) supplementation, the sensor enables the observation of Cu(I) accumulation in the mitochondria and Golgi apparatus of fixed NIH 3T3 fibroblasts. The fluorescence of the probe exhibited a notable degree of colocalization with the Cu mapped by XFM [102].

The optimization of **CTAP-1** resulted in **CTAP-2** and **CTAP-3** (Fig. 6, 9–11), which demonstrated enhanced solubility in pure aqueous solution. Modifications have been made to both the cavity moiety, through the addition of alcohol groups for a better solubility in water, and to the triarylpyrazoline moiety, through the addition of sulfonate groups (Fig. 5). **CTAP-2** contains an azatrichiacrown (1N3S, Fig. 6, 10) chelating group while **CTPA-3** contains an acyclic 1N3S binding motif (Fig. 6, 11) [129]. **CTAP-3** exhibits a 180-fold increase in fluorescence upon Cu(I) binding, with a high quantum yield of 41 % and a complex stability constant of logK<sub>app</sub> = 10.3 [129], in comparison to a 65-fold emission enhancement upon Cu(I) binding, an 8 % quantum yield, and a logK<sub>app</sub> of 11.4 for **CTAP-2** [128]. The injection of [Cu(I)(**CTAP-3**)] complex into live BSC-1 (green monkey kidney) cells results in a loss of fluorescence supportive of a rapid demetallation that hampers its further applicability in living biological systems [30].

### 3.4.2. CS family

The group of Chang developed a Cu(I) sensor based on a BODIPY fluorophore linked to the acyclic 1N4S, **BETA** receptor (Fig. 6, 13). The initial molecule of this series has been designated **CS1** which stands for Cu sensor-1. **CS1** exhibits a 10-fold increase in fluorescence upon Cu(I) binding, a logK<sub>app</sub> of 11.4, a quantum yield of 13 %, and is excitable in the visible region. **CS1** enables the visualization of dynamic changes in

<footnote>a Determined by competitive binding with CH₃CN, using the stability constants for aqueous Cu(I)-CH₃CN system [135] used in nonlinear least-squares data analysis.</footnote>

<footnote>b Determined from the formal potentials of the free and bound Cu(II)/I) couples and the Cu(II) affinity of **CTAP-2** determined by UV–vis titration.</footnote>

<footnote>c Determined by competition with **MCL-2** followed by fluorescence.</footnote>

<footnote>d Determined using thiourea as competitor ligand to provide buffered Cu(I) solutions. Stability constants for thiourea binding were taken from the literature: $β_{12} = 2.0 \times 10^{12}$, $β_{13} = 2.0 \times 10^{14}$, $β_{14} = 3.4 \times 10^{15}$ [136].</footnote>

<footnote>e Determined by competition with **BCS** followed by UV–vis (association constant $10^{19.8}$ M).</footnote>

<footnote>f Determined by competition with **MCL-1**; DMPC–DMPG (4:1) 100 µM final lipids, DMPC = 2-dimyristoyl-*sn*-glycero-3-phosphocholin and DMPG = 2-dimyristoyl-*sn*-glycero-3-phospho-(1'-rac-glycerol).</footnote>
Cu pools in HEK 293T (Human embryonic kidney) cells under conditions of Cu overload [130]. **CS1** has been used as a tool to image Cu uptake and fluctuations in bacteria [137,138], yeast [139–141], plant [142], and mammalian systems [143]. However, **CS1** has demonstrated limitations in its application, as no increase in fluorescence was observed in specific M17 neuronal or U87MG glial cells following Cu supplementation [144].

The improvement of **CS1** resulted **CS3**, which exhibits enhanced optical brightness and a more pronounced turn-on response. The replacement of fluorine by methoxy substituents on the boron atom of the BODIPY core (Fig. 5, R₃) resulted in an enhancement of the quantum yield to 40 % by increasing the electron density over the fluorophore (Fig. 5). Moreover, the fluorescence enhancement upon Cu(I) binding has been markedly increased, reaching a 75-fold enhancement [131]. Despite the identical binding cavity as that present in **CS1** (Fig. 6, 13), the logK<sub>app</sub> value also exhibits an improvement, reaching 13.1. **CS3** has also been applied as a tool for screening the basal pool of Cu in different cell types and to visualize Cu(I) dynamically [19,103]. Notably, **CS3** has enabled the visualization of Cu re-localization in rat hippocampal neurons from somatic cell bodies to peripheral neurons upon depolarization [131]. Furthermore, it has been employed to investigate the correlation between Ctr2 (copper transporter 2) expression and cisplatin resistance [145], as well as the mechanism of migration of the transporter ATP7B in Cu overload conditions into lysosomes and the subsequent apical exocytosis, which permits the elimination of excess Cu from liver cells [146].

It is important to note that dynamic light scattering (DLS) studies on both **CS1** and **CS3** in aqueous buffer at a concentration of 5 µM have indicated the formation of nanoparticles with average sizes of 49 and 67 nm, respectively [128]. Hence, a more photostable and hydrophilic silicon rhodol platform (Fig. 5) was employed to engineer a sensor named **CSR1** (based on the same binding cavity) for Cu Silicon Rhodol 1 which enabled real-time monitoring of Cu(I) movement on cells. The **CSR1** sensor exhibits an affinity for Cu(I) of logK<sub>app</sub> of 12.7. Upon binding, a 12-fold increase in fluorescence is observed, with a quantum yield of 21 %. The treatment of the 3T3-L1 adipocyte cells, a model for white adipose tissue, with isoproterenol (a lipolysis inducer) resulted in a decrease in **CSR1** fluorescence, which was monitored in real time. This finding helped to link Cu homeostasis and lipolysis [132].

The **BETA** cavity (Fig. 6, 13) has also been bound to a derivative of the 2-methylamino-6-acetylnaphthalene (Fig. 5, ACu1) fluorophore. The probe allows Cu to be imaged in tissue from rat hippocampal slices at depths of up to 200 µm. This probe named ACu1 which is a two-photon probe, showed a 4-fold fluorescence enhancement in response to Cu(I) with a logK<sub>app</sub> of 10.7 [123].

### 3.4.3. *In vivo* imaging

The development of a novel probe with excitation/emission (760 and 790 nm respectively) in the near-infrared (NIR) region has enabled the monitoring of fluctuations in exchangeable Cu stored in living HEK 293T cells and in SKH-1 mice and mouse models of WD under basal conditions, as well as in situations of Cu overload or deficiency. The probe designated **CS790** (Coppersensor 790), has been constructed from a cyanine 7 dye (Fig. 5) and a 1N3S binding cavity (Fig. 6, 12). The carboxy groups on the cyanine moiety have been esterified in order to facilitate accumulation of the probe within the cell. Once internalized, the de-esterification occurs leading to **CS790**. The probe binds Cu(I) with a logK<sub>app</sub> of 10.5 and undergoes a 15-fold emission increase upon saturation with Cu(I) with a quantum yield of 7 %. The intraperitoneal injection of **CS790** allows for the measurement of the basal level of Cu(I) in mice and the observation of the dynamic fluctuations in Cu levels upon the addition or depletion of Cu with the use of a chelator (ATN-224, bis(choline)tetrathiomolybdate). Furthermore, the imaging of ATP7B<sup>-/-</sup> mice enables the detection of Cu accumulation in comparison to wild type (WT) [127].

A ratiometric NIR fluorescent sensor based on a BODIPY coupled to

the **BETA** (Fig. 6) through a styrene linker in position 2 (Fig. 5, P₂) named **BDPS1** has recently been described as a tool for tracking labile Cu in mice [133]. **BDPS1** displays a Cu(I)-specific enhancement of both emission (F<sub>ex</sub>670/F<sub>ex</sub>581) and excitation (F<sub>exc</sub>600/F<sub>exc</sub>660) ratios and a logK<sub>app</sub> of 11.3. In mice injected with a Cu(His)₂, a Cu level increase was observed in the upper belly. However, it is important to note that the physico-chemical properties for this probe were reported in a mixture of HEPES buffer pH 7.4 and DMF 70 % (v/v) and that the animal injection were made with 5 % to 10 % DMSO (v/v) probably due to solubility issue in aqueous solution.

A sensor named **NCP1**, based on a rhodol-like fluorophore scaffold, in which a methylindole has been introduced, was linked to **BETA** in position 2 (Fig. 6, P₂), has recently been used to report *in vivo* data. Cu induces an 18-fold increase of fluorescence of **NCP1** and is bound with a logK<sub>app</sub> of 11.3. Prior to its administration to the animals, the probe was encapsulated in bovine serum albumin (BSA), the particles obtained are designated as **NCP1**@BSA. **NCP1**@BSA allows to evaluate the efficacy of four ionophores in facilitating the transport of exogenously added Cu (II) to the tumor site, through the measurement of fluorescent intensities in 4T1-tumor-bearing mice. Three hours following the injection of the ionophore and the CuSO₄ (**NCP1**@BSA injected 12 h before), a higher level of fluorescence was observed in the tumor treated with Elesclomol in comparison to 8-hydroxyquinoline, while minimal enhancement was noted for tetramethylthiuram disulfide and disulfiram. This result might be ascribed to the different affinity of the ionophores for Cu(II) [134]. The impact of the BSA which has a high affinity binding site for Cu(II) is not discussed.

### 3.4.4. Other probes not relying on thioether binding cavity

A dansyl fluorophore (Dns, Fig. 5) has been linked to a tripeptide Leu-Leu-Cys (LLC, Fig. 6, 14) through the N-term with amidated C-term (Dns-LLC). The probe binds Cu(I) with an apparent affinity of 13.9 and with a turn-on in fluorescence of 12. Dns-LLC enters into RKO human colorectal cells and the fluorescence observed upon Cu(I) binding was co-localize in the Golgi [125].

A new type of binding cavity relying on stabilized phosphine (PSP, Fig. 6, 15) with a very high affinity for Cu(I) (logK<sub>app</sub> = 17.0), coupled to a julolidine chromophore (Fig. 5), has been described with the idea of being in the same affinity range than the GSH buffering window. This probe named **Crisp-17** for Cu ratiometric indicator with stabilized phosphines is a ratiometric two photons probe [30]. It has been employed to follow dynamic exchange of Cu in NIH 3T3 mouse fibroblasts, where the fluorescence is localized in the cytoplasm. Addition of thiosemicarbazone, CuGTSM is accompanied by an increase of ratio intensity in the cytoplasm of the cells corresponding to the increase of free Cu(I), the use of **PSP-2** chelator allows to reverse this process. Upon growth of the cell on supplementation of Cu(II), no increase of the fluorescence ratio was observed despite the increase of total Cu in the cells (two-fold). Conversely, an increase was quickly observed after the addition of a thiol-selective oxidant DTDP (4,4'-dithiodipyridine), suggesting that Cu(I) was coordinated to thiol functions of protein of high affinity [30].

Fluorescent probes hold an immense potential for advancing our understanding of Cu biology. While some probes have shown promising results, several limitations remain to be addressed, including issues with photostability, nanoparticle formation, and inadequate responses in certain cell types or organelles. These challenges highlight the need for improved designs to enable more robust and reliable *in vivo* applications.

## 4. Cu(I) ligands for therapy

### 4.1. Alzheimer's disease

#### 4.1.1. Key facts

The regulation of Cu metabolism is disrupted in AD contributing to pathological mechanisms of the disease [81]. More precisely, amyloid
beta (Aβ) peptide, a primary marker of the disease, forms senile plaques in the extracellular space, through complex aggregation processes, that contain elevated levels of d-transition metal ions, including Cu, Fe, and Zn [147–149]. The loosely bound Cu to Aβ maintains its capacity to redox cycle and may contribute to the overall increase in oxidative stress and resulting damage observed in AD brains [87,150]. In AD brains, this oxidative damage is significantly elevated and is considered a key factor in the neurodegenerative process. Moreover, the interaction between Aβ and Cu ions may also influence the aggregation and toxicity of Aβ [151], further exacerbating neuronal damage and cognitive decline. Furthermore, dysregulation of Cu and other metal ions in the brain can affect various biological pathways, including synaptic function, mitochondrial activity, and enzyme regulation. These disruptions may collectively contribute to the progression of the cognitive decline and neuronal loss characteristic of AD, highlighting the importance of metal ion homeostasis in maintaining brain health.

### 4.1.2. Cu chelation-based therapy

The aforementioned information has jointly prompted the development of Cu chelators and ionophores [66,73,83,152–154], which are designed to alleviate Cu toxicity by removing it from Aβ and to redox silent it. They require an appropriate selectivity toward other metal such as Zn in order to maintain their capacity to target Cu in a complex environment [155,156]. An impressive amount on molecules has been described in this context, most of them targeting the Cu(II) state [66,83]. In contrast, the biologically relevant Cu(I) state has been poorly studied despite the lack of knowledge regarding the Cu oxidation state in the synaptic cleft. Cu(I) may be a pertinent target as there is a substantial body of evidence indicating that ascorbate is present in non negligible amount up to 200 µM in the extracellular space in the brain [157–159], in addition to the fact that the Cu(I) state is the one that reacts with O₂ [160]. Furthermore, the synapse is a region with a high concentration of Zn, and targeting Cu(I) may be an effective method for developing Cu-selective ligands since the coordination (HSAB and coordination number) properties of Zn(II) and Cu(I) are more different than those of Zn(II) and Cu(II) [83].

#### 4.1.2.1. Cu(I) chelators in the AD context.
The following section presents a summary of the development of small chelators designed to target Cu(I) and silence its redox activity within the context of AD.

A brief overview of the literature reveals that the Zn-loaded metallothionein 3 (Zn7-MT-3) [161–163] and metallothionein 2A (Zn7-MT-2A) [164] has the potential to remove Cu(II) from Aβ and halt ROS production. The reduction of Cu(II) to Cu(I), occurs concomitantly with the oxidation of thiolate groups into disulfide bonds, effectively reducing its redox ability [35,156].

In the early 2000s, the bathucoproine disulfonate (BCS, Fig. 4) was proposed to possess promising properties, with the potential to serve as a model for AD therapy. It is proposed that the effectiveness of BCS in solubilizing brain Aβ deposit is due to a combination of direct

interaction with the Aβ peptide with the Phe4, Tyr10, and Phe19 amino acid residues and the chelating properties of this compound. However, neither the Cu oxidation state nor the description of the specific chelating ability of the molecule is clearly defined in this work [165]. BCS has also been shown to reduce the Cu toxicity by decreasing the neuronal damage score when Cu(II) and super-stoichiometric amount of chelator (2:5 respectively) were co-administered intracerebrally in rats [166]. This study did not focus on AD.

In 2015, the 1,3,5-triaza-7-phosphaadamantane (PTA, 16 Fig. 7) ligand was successfully employed as a Cu(I)/Cu(II) targeting molecule [167]. PTA has been demonstrated to effectively remove Cu(I) and Cu(II) from Aβ. PTA has the capacity to reduce Cu(II) to Cu(I) and to form a Cu(I) complex stable in the presence of O₂ and ascorbate, thereby arresting most of the Cu(Aβ)-induced-ROS production until the ligand is oxidized since the reduction of Cu(II) is accompanied by the oxidation of the ligand to the corresponding phosphine oxide. Nevertheless, the effectiveness of the process was contingent upon the utilization of an excess of PTA. It is noteworthy that PTA retained its capacity to mitigate Cu(Aβ)-induced ROS production in the presence of 1 equivalent of Zn(II) [168] thus showing an appropriate selectivity versus Zn(II).

A tristhistine pseudopeptide (Fig. 7, NTA-His₃, 17) derived from a nitrilotriacetic acid scaffold (NTA), functionalized with three His amino acid residues, has been described to target both Cu(I) and Cu(II) redox states, with the potential to reduce Cu(Aβ)-induced ROS production. 17 is capable of binding Cu(I) in a tetrahedral geometry and Cu(II) in a square planar geometry with an affinity constants that are slightly higher than that of Aβ. The redox cycling between the two redox states of the Cu(I) and Cu(II) centers, when coordinated by the ligand, is not completely halted; however, it is markedly slowed due to the different binding geometry of the two redox states. This significantly reduces ROS production in the presence of Aβ [169].

Recently a dithiol containing ligand (Fig. 7, LH₂, 18) inspired from MTs chemistry [161] was reported to remove Cu(I) and Cu(II) from Aβ and to inhibit the Cu(Aβ)-associated ROS production [170]. 18 is capable of forming a stable complex with Cu(I) in an air-stable manner, thus preventing its redox cycling and the subsequent production of ROS. Interestingly it has been shown that 18 was very selective for Cu(I) toward Zn(II), up to 250 equivalent and that it can thus be used as the pro-drug Zn-bound ligand instead of the free ligand susceptible to oxidation [170].

#### 4.1.2.2. Cu(I) chelators in other contexts.
Other Cu(I) ligands have been described as having the potential to mitigate Cu-mediated oxidative stress, although this has not been explored in the context of AD. Neocuproine (NC) and bathucoproine (BC) have been shown to reduce Cu(II)-ascorbate-induced carbonylation of BSA. This observation would account to the ability of these ligands to bind Cu(I) and to redox silence it in the conditions of the experiment [171]. Tripodal scaffolds based on NTA with an appended imidazole thione on each arm was employed as a Cu(I) chelator. The study demonstrated that the Cu-catalyzed oxidation of ascorbate in the presence of O₂ was inhibited by the chelator, as was the protein BSA carbonylation induced by Cu-Asc-O₂. The formation of redox-inert Cu(I) complexes was identified as the mechanism behind this inhibition [172].

To date, there have been few developments in the field of Cu(I) chelators for the treatment of AD. Nevertheless, the results obtained in vitro are highly promising and would benefit from further investigations in cells and in model animals.

## 4.2. Wilson disease

### 4.2.1. Key facts

WD is a rare autosomal disorder in which Cu accumulates in liver, brain and other organs [3,70,173]. This results from an impaired function of ATP7B, a Cu-ATPase responsible for Cu export from cells. The malfunction of ATP7B may be caused by a variety of mutations of the

<figure><img src="image_9.png" /><figcaption>**Fig. 7.** Cu(I) chelators described for chelation therapy in AD. PTA = 1,3,5-triaza-7-phosphaadamantane.</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>**Fig. 8.** Structures of Cu chelators used in the treatment of Wilson disease. D-phen = D-penicillamine; TETA = triethylenetetramine; TTM = tetrathiomolybdate; BAL = British anti-Lewisite; DMPS = dimercaptosuccinate; DMSA = dimercaptopropane sulfonate.</figcaption></figure>

corresponding gene (>500 are known). WD being a genetic disease, treatment needs to be lifelong. Treatments aim at maintaining Cu levels at a non-toxic level in the body, either (i) by reducing dietary Cu absorption, (ii) by promoting Cu excretion from tissues and elimination through the digestive or urinary tracks, or (iii) by a combination of both mechanisms [174]. Currently used therapies involve Zn(II) salts and Cu chelators. Zn(II) salts act primarily by limiting Cu uptake in the intestine: Zn(II) ion stimulates the production of MTs by enterocytes, which bind Cu and facilitate its elimination in feces [174]. On the other hand, Cu chelation therapies can act through either or both of the above-mentioned mechanisms. It can be expected that Cu(II) chelators would rather target extracellular Cu(II) ions in the digestive track and prevent Cu absorption, while Cu(I) chelators would target intracellular Cu(I), in competition with Cu proteins, and promote its elimination. Some chelation treatments act through both mechanisms. In the following parts, we will review approved and experimental Cu chelators in the context of WD, depending on their mechanism(s) of action.

### 4.2.2. Reduction of Cu absorption and enhancement of Cu elimination

Some treatments rely on chelators that can bind both Cu(I) and Cu(II), like D-penicillamine (D-Pen) [175], or dimercaptosuccinate (DMSA) and dimercaptopropane sulfonate (DMPS), both of which are derivatives of British anti-Lewisite (BAL), the first treatment for WD (Fig. 8). Triethylenetetramine (TETA), also known as trientine, was approved for treatment of WD and was shown to reduce Cu absorption and increase urinary

Cu elimination [174,176]. Given the much higher association constant of TETA with Cu(II) than with Cu(I) [177], and its relatively modest affinity for Cu(I) as compared to Cu proteins [177,178], it can be assumed that TETA would preferentially target Cu(II) ions [177,179,180]. Tetrathiomolybdate (TTM, ammonium or bis-choline salts) can be used to reduce dietary Cu absorption, but also to promote its excretion [181]. TTM forms stable complexes and clusters with Cu(I), and prevents its nefarious redox cycling. In addition, TTM can form multipartner complexes with Cu(II) and proteins in cells, which are then excreted. In the blood, it forms such ternary complexes with albumin, thereby promoting Cu excretion [182–184]. Nonetheless, administration of 25 and 50 mg/kg of TTM to model rats of WD has indicated that the use of such high doses may lead to the precipitation of insoluble Cu–Mo species in the liver while lower doses helped eliminate excess Cu [185].

### 4.2.3. Targeting Cu(I) to enhance Cu excretion

#### 4.2.3.1. Methanobactins.
Methanobactin (Fig. 9) is a family of small Cu chelating molecules with a peptide-like structure, used by methanotrophs to acquire Cu [186,187]. The structure of methanobactin and the nature of the coordinating moieties depend on the organism, but all form a 1:1 complex with Cu(I), exhibiting a 2N2S coordination, and high affinities (log K<sub>app</sub> = 18–20 in 20 mM HEPES pH 7.5, by BCS competition experiments) [188]. Interestingly, methanobactin from *Methylosinus trichosporium* OB3b was found to promote Cu excretion in the bile in a rat model of WD [189].

#### 4.2.3.2. Bioinspired Cu(I)-targeted chelators.
One major issue of these systemic treatments is that they are distributed across the whole organism, therefore requiring high doses to reach sufficient concentrations in the relevant organs, and entailing side effects. Targeting the chelator to the relevant organ(s) (here, liver) is thus an attractive strategy. In addition, Cu is present as Cu(I) inside cells, often tightly bound to proteins. In order to detoxify these cells, high affinity Cu(I) chelators should thus be developed.

##### 4.2.3.2.1. Cyclic peptide ligands.
About 20 years ago, Delangle and collaborators developed two series of bio-inspired Cu(I)-targeted chelators. The first series was based on a cyclodecapeptide structure (Fig. 10). This structure exhibits two “sides”, enabling to arrange the desired binding side chains on one side and to modulate the properties of the peptide using the other side. The initial peptide, P<sup>C</sup>, was inspired from the conserved binding motif Met-XXX-Cys-XXX-XXX-Cys of Cu

<figure><img src="image_11.png" /><figcaption>**Fig. 9.** Examples of structures of methanobactin in various organisms. The structure of the coordinating oxazolone, imidazolone and pyrazinediol are shown in dark, the coordinating atoms are in bold.</figcaption></figure>
<figure><img src="image_12.png" /><figcaption>Fig. 10. Bio-inspired cyclodecapeptides with 10<sup>15</sup>-10<sup>18</sup> affinities for Cu(I).</figcaption></figure>

<table><caption>**Table 4**

Formation constant of Cu(I) complexes with bioinspired chelators.</caption><thead><tr><th></th><th>Log K<sub>app</sub><sup>a</sup></th><th></th></tr></thead><tbody><tr><td>25(P<sup>c</sup>)</td><td>17.4</td><td>[190,191]</td></tr><tr><td>26(P<sup>1</sup>)</td><td>16.7</td><td>[192,193]</td></tr><tr><td>27(P<sup>2</sup>)</td><td>15.5</td><td>[192,193]</td></tr><tr><td>29(L<sup>1</sup>)</td><td>19.2</td><td>[195]</td></tr><tr><td>30(L<sup>2</sup>)</td><td>18.8</td><td>[195]</td></tr><tr><td>31(L<sup>3</sup>)</td><td>N.D.</td><td>[195]</td></tr><tr><td>32(L<sup>4</sup>)</td><td>17.3<sup>b</sup></td><td>[198]</td></tr><tr><td>33(L<sup>5</sup>)</td><td>16.2</td><td>[198]</td></tr></tbody></table>

<sup>a</sup> K<sub>app</sub> determined by competition with BCS in phosphate buffer, 20 mM, pH 7.4, data were fitted with the program specifit.

<sup>b</sup> Putative complex, only polymetallic species were found.

chaperones such as ATOX1 or CuATPases ATP7A/B. It was found to form a CuS<sub>2</sub> complex by binding Cu(I) through both Cys, with a log K = 16.6 at pH 7 [190,191]. Since Met did not seem to be involved in Cu(I) coordination in P<sup>c</sup>, two peptides showing only two Cys were further developed: P<sup>1</sup> and P<sup>2</sup> [192,193]. P<sup>1</sup> exhibited an affinity similar to P<sup>c</sup> log K = 16.7 at pH 7.4, Table 4), while P<sup>2</sup> showed a slight decrease in affinity (log K = 15.5 at pH 7.4, Table 4). This was attributed to the increased rigidity of the peptide scaffold of P<sup>1</sup> and P<sup>2</sup>, with two β-turns. The other “face” of these cyclic peptides was then further functionalized in order to target hepatocytes, resulting in **Chel1** derivatives. Glyco-conjugates of **Chel1** were designed and synthesized to promote the ASGPR-mediated uptake (asialoglycoprotein receptor) of these potential prodrugs in hepatocytes [193]. Fluorescently-labelled derivatives were also synthesized to assess internalization. Fluorescence microscopy and flow cytometry experiments showed that these derivatives were well internalized in hepatocytes. In addition, incubation of polarized hepatocytes with **Chel1** prodrug in presence of high Cu levels restored the phenotype of normal Cu levels, therefore indicating that it efficiently chelated Cu(I) inside cells.

#### 4.2.3.2.2. NTA-based pseudo-peptide ligands.
Bioinspired ligands enabling CuS<sub>3</sub> coordination were also developed [194–199]. These ligands are based on NTA scaffold, which was decorated with Cys or D-Pen units (Fig. 11). Due to the presence of negatively charged carboxylates near the binding site, they form polymetallic species of low stability. The other ligands formed either 1:1 complexes, or, in presence of excess Cu(I), (Cu<sub>2</sub>L)<sub>x</sub> metal clusters. The Cys tripod showed the highest affinity for Cu(I) and was used to design a liver-targeted Cu(I) prochelator in further studies. Indeed, Cys thiolates can be masked as disulfide bonds that will be reduced once the compound is internalized into cells. This strategy also enables to target hepatocytes by adding GalNAc (N-Acetylgalactosamine) units on the other part of the disulfide bond. Interestingly, this strategy was used not only in hepatocytes [193], but also in a murine model of WD [193,200]. As for **Chel1**, **Chel2** prodrugs were efficiently internalized in hepatocytes (although slightly less efficiently than for **Chel1**). In presence of high Cu levels, incubation of polarized hepatocytes with **Chel2** prodrug also restored the “low Cu” phenotype. ATP7B<sup>-/-</sup> mice treated with **Chel2** showed an increase of Cu excretion in their feces, which indicates that **Chel2** treatment probably restores Cu excretion through the bile in this WD model.

Copper chelation therapy for WD currently relies on simple, untargeted chelators that prevent Cu(I) intake or promote its excretion. Over the years, more elaborate, bioinspired Cu(I)-selective chelators have been developed, that compete with the affinities of Cu(I) proteins. These ligands can be functionalized to target liver cells, and promote Cu(I) excretion from hepatocytes. The potential of these chelators as drugs for WD still remains to be tested in clinical trials.

### 4.3. Cancer

#### 4.3.1. Key facts

Elevated Cu levels have been reported in the serum and tumor of patients suffering from distinct cancer types, e.g. colorectal cancer, breast cancer, liver cancer, etc [76]. The role of Cu in cancer development has thus been investigated over the last decades [77]. Cu was

<figure><img src="image_13.png" /><figcaption>Fig. 11. Bio-inspired pseudopeptides based on a NTA scaffold as Cu(I) chelators.</figcaption></figure>
<figure><img src="image_14.png" /><figcaption>**Fig. 12.** Ligands based on phosphine sulfide-stabilized phosphine (PSP) scaffolds; phenPS = phenylene bridge; naphPS = naphthylene bridge, bithipPS = bithiophene; thipPS = thiophene bridge.</figcaption></figure>

shown to stimulate a number of mechanisms promoting tumor growth, such as cell migration, angiogenesis, cell cycle, apoptosis and ribosome biogenesis [201]. The higher Cu demand of cancer cells makes them more vulnerable to Cu deficiency, and the potential of Cu chelators as anticancer drugs has been considered. It should be noted that another fully opposite strategy consists in utilizing Cu(I) toxicity to kill cancer cells: this strategy relies on small molecules able to address high levels of Cu(I) to cancer cells specifically, ultimately resulting in cell death. These ionophores often present high affinities for Cu(II), to be able to capture Cu outside cells, and lower affinity for Cu(I), in order to release it inside cells. These ionophores are thus outside the scope of the present review,

and the reader is thus referred to reviews that specifically address this question [17,75,79,202,203]. Cu complexes based on pyridine derivative-chelating ligands are also an intensive field of research. For interested readers, please see references [204–207]. In the rest of this part, we will focus on Cu(I) chelators used as cancer-targeting drugs.

### 4.3.2. Repurposed Cu(I) chelators

Efficient copper chelators that had been developed to treat WD were re-investigated as cancer targeting compounds [201,208–210]. Tetra-thiomolybdate (TTM, Fig. 8), for instance, has an anti-tumor effect by inhibiting angiogenesis [181,211,212]. It was submitted to clinical trials, sometimes in combination with other anticancer drugs, for treating breast cancer, colorectal cancer, papillary thyroid cancer, melanoma [213–216]. A derivative, **ATN-224** (bis-choline-tetrathiomolybdate), was also investigated in phase I trials for patients with solid tumors [217]. **D-Pen** (Fig. 8) also exhibits an antiangiogenesis effect, and was used in combination with radiation and low Cu diet to treat glioblastoma in phase II clinical trials [218].

### 4.3.3. Cancer-targeting Cu(I) chelators

New high affinity Cu chelators are also currently in development, and are now directly tested for their effect against cancer rather than in WD. Fahrni and co-workers developed a series of Cu(I) ligands based on phosphine sulfide-stabilized phosphine scaffolds (PSP, Fig. 12) [219–221]. The first ligand of the series, **PSP-1**, formed an air-stable 1:1 complex with Cu(I) with a binding constant of log K<sub>app</sub> = 15.1 at pH 7 (competition with **BCS**, log β<sub>2</sub> = 20.9). The EDTA-like structure of **PSP-2** enabled a significant gain in affinity, with log K<sub>app</sub> = 20.0 (competition with **BCS**, log β<sub>2</sub> = 20.9) [219]. Interestingly, **PSP-2** was able to deplete Cu levels in cells without affecting the levels of other metals. It showed significant anti-angiogenic effects, although no antiproliferative effect could be observed in the initial study [222]. In a later study, lung tumor grafts were implanted on the chorioallantoic membrane of the chicken embryo. **PSP-2**-treated samples showed a reduced angiogenesis and

<figure><img src="image_15.png" /><figcaption>**Fig. 13.** Affinity ranges of Cu(I) chelators compared to proteins involved in Cu homeostasis. Cu(I) chelators forming stoichiometric complexes with Cu(I) and described in this review are represented on the top: thioether-based ligands in blue, thiolate-based (pseudo)peptide ligands in orange, thiophene-based ligands in green, and the methanobactins in red. At the bottom of the figure, the affinities of major Cu(I)-proteins (pink to red diamonds) are reported, as described in Table 1. Most binding constants were determined for mononuclear Cu(I) sites in proteins using competitions assays with **BCS** (log β<sub>2</sub> = 19.8) or **BCA**. When binding constants were determined for distinct binding sites (Ctr1, CCS), each is represented as a diamond. Values for Ctr1 and the lowest value for CCS should be compared cautiously to others, since they rely on distinct values for log β<sub>2</sub>(**BCA**). For metallothioneins, sequential binding constants of 20 Cu(I) were determined using MS [224]. GSH, a key molecule in Cu(I) homeostasis, forms Cu₄(GS)₆ clusters in solution and is not represented here (log β₄₆ = 84.9, logβ11 was fitted to 15.42 (χ² = 7.885)) [225]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
tumor weight, although the mechanism underlying tumor weight reduction remained unclear [223]. Rigidifying the bridge between the phosphines of **PSP-2** with phenylene and naphtylene (**phenPS** and **naphPS** respectively, Fig. 12) units resulted in a further improvement in affinity (log K<sub>app</sub> = 21.1–21.2, competition with **BCS**, log β<sub>2</sub> = 20.9) [220]), which could be attributed to the pre-organization of both ligands. By contrast, the ligand with a rigid, hinged backbone **thipPS** has a slightly lower affinity (log K<sub>app</sub> = 19.5) than **PSP-2**, while **bithipPS** has a slightly higher affinity (log K<sub>app</sub> = 20.4 [221]). Nevertheless, they have the same efficacy as **PSP-2** in depleting Cu levels in mouse fibroblasts.

Cu chelators, used in combination with other treatments (radiation, anticancer drugs), could thus be an effective strategy for cancer therapy. Cu chelators developed for WD or AD could be re-evaluated in the context of cancer therapy, and derivatized to target cancer cells, thereby expanding the medical toolbox.

# 5. Discussion

## 5.1. Key facts

Cu imbalance is involved in numerous pathological processes, making it an important pharmacological target. The pathologies linked to Cu dyshomeostasis under focus in this review include Wilson disease, Alzheimer's disease and cancer. In order to treat these diseases efficiently, it is important to understand the mechanisms and the biomolecular actors involved at the molecular level, prior to design chelators as a key therapeutic strategy.

## 5.2. Binding affinity of Cu(I) proteins and ligands: A key parameter

A significant area of research focused on determining affinities of proteins (or peptide models thereof) for Cu(I), in order to assess the biological competitors of potential molecular Cu(I) chelators. Due to their ease of experimental implementation, UV-active probes of known affinities for Cu(I) have been a popular choice to evaluate binding constants of proteins and ligands for Cu(I) in competition experiments. Nevertheless, several precautions and controls should be taken when performing those experiments, to determine binding constants reliably. To avoid propagating and amplifying experimental errors, concentrations of competitor and metal should be well calibrated and the response should be within 10–90 % of the control response (without competitor). These UV probes form 2:1 (L:M) complexes with Cu(I), and the absence of ternary complex formation with the competitor should also be controlled. This also means that the constants determined for 1:1 complexes cannot be directly compared with the stability constants β₂ of the UV probes. In addition, it should be noted that the values of these stability constants are not fully consistent in the literature. When comparing protein affinities for Cu(I) determined using these probes, it is crucial to ensure that the same standard values were used.

## 5.3. Monitoring Cu(I) trafficking

While these *in vitro* experiments provide useful insights into Cu(I) homeostasis, it is desirable to be able to detect Cu(I) in live cells or even *in vivo*. To that purpose Cu(I) fluorescent probes have been developed.

Numerous intensity-based Cu(I) fluorescent probes have been described, though only a few have been applied *in vivo*, and even fewer have been employed to monitor Cu(I) dynamics in real-time using confocal microscopy. Developing effective *in vivo* tools presents significant challenges. Beyond fluorescence-related parameters such as quantum yield, photostability, phototoxicity, and fluorescence enhancement factor, the key determinant of a probe's efficacy is its affinity for Cu(I). Most probes described in the literature rely on thioether functional groups and exhibit affinities in the range of log K<sub>app</sub> = 10–13, which is markedly lower than those of natural Cu(I) chelators like chaperones and MTs (Fig. 13). Despite this, such probes have been successfully used to monitor basal Cu(I) levels

and their fluctuations upon external stimuli (such as addition of Cu(II) and/or of Cu(I) chelators) in specific cell types. However, it has been demonstrated that no single probe can be considered universal. Some probes exhibit fluorescence enhancement in certain cell types but not in others, emphasizing the need for careful probe selection and to control the cell environment. Conversely, it was put forth that Cu(I) is buffered within the attomolar concentration range, necessitating the use of a higher-affinity probe for the detection of Cu within a normal cellular context. Moreover, intensity-based probes, while straightforward to develop, are susceptible to interference from factors other than Cu(I) levels. For example, fluorescence signals may vary with the probe's local concentration or changes in pH implying that the results need to be interpreted with caution. These issues can be mitigated using ratiometric probe, where the emission ratio from two distinct colour channels is measured. This method standardizes the probe's response to Cu and eliminates variations due to probe concentration, offering a more robust and quantitative approach to Cu(I) detection. These advancements highlight the potential of combining ratiometric strategies with high-affinity, biocompatible probes to improve the reliability and precision of Cu(I) imaging in complex biological systems. The use of DFT calculations may have the potential to address all the aforementioned requirements, thereby facilitating the development of novel solutions for *in vivo* imaging.

## 5.4. Cu(I) chelators

Cu ligands were initially used as treatments for the rare WD, but are now the subject of a renewed interest to treat more frequent diseases like cancer or AD. In the context of chelation therapy in WD and cancers, a key requirement for the ligand is to be able to compete with Cu(I) homeostasis proteins, and in particular with Cu(I) storage pools like MTs, to be able to remove efficiently the excess of Cu (Fig. 13). Currently used treatments (e.g. *TTM*) rely on relatively simple structures and are mostly untargeted, therefore requiring high doses to reach the desired concentration at the organ of interest, and potentially causing undesired side effects. Nevertheless, investigating the molecular mechanisms of Cu homeostasis and relevant biological Cu chelators (e.g. proteins, small ligands) enabled the identification of key parameters (e.g. affinity, nature of the ligands, *etc*) to inspire more elaborate Cu(I) chelators (e.g. 28, 34, 36) with higher affinity and selectivity for Cu(I). Although such ligands are often synthetically more demanding than the currently used treatments, one main advantage is that they can be functionalized to target the desired cell types or organs, and would therefore require lower doses. In addition, it would be possible to adapt the functionalization to the disease (and thus the organ) to treat. Although, to our knowledge, these targeted ligands have not been submitted to clinical trials, they show a potential for targeted therapies against Cu overload-linked diseases, in combination with other drugs when necessary.

The innovative potential of Cu chelators and ionophores, combined with cutting-edge tools for studying Cu dynamics in cells and living systems, is paving the way for groundbreaking advances in copper-related research and therapeutic innovation.

# Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

# Acknowledgments

C.E. thanks the ANR project Copperation (ANR-20-CE07-0009), S.H. gratefully acknowledges the support of this work by the French National Agency for Research in the framework of the "Investissements d'avenir" Program, (ANR-15-IDEX-02), the Labex ARCANE (ANR-11-LABX-003), and the CBH-EUR-GS (ANR-17-EURE-0003).
# Data availability

Data will be made available on request.

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