# A cocktail of two chelators is not better than one ligand in excess to halt the Cu,Zn-(amyloid-β)induced ROS production in the context of Alzheimer's disease

Marie Bagrel, Christelle Hureau, Enrico Falcone

► To cite this version:

Marie Bagrel, Christelle Hureau, Enrico Falcone. A cocktail of two chelators is not better than one ligand in excess to halt the Cu,Zn-(amyloid-β)induced ROS production in the context of Alzheimer's disease. Inorganic Chemistry, 2025, 64 (42), pp.20875-20880. 10.1021/acs.inorgchem.5c03352. hal-05332720

HAL Id: hal-05332720

https://hal.science/hal-05332720v1

Submitted on 27 Oct 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
A cocktail of two chelators is not better than one
ligand in excess to halt the Cu,Zn-(amyloid-β)-
induced ROS production in the context of
Alzheimer's disease

Marie Bagrel, Christelle Hureau, * Enrico Falcone, *

LCC-CNRS, Université de Toulouse, CNRS, Toulouse, France.

**ABSTRACT.** In this study, we demonstrate that an excess of the non-selective chelator EDTA can effectively extract both copper(II) and zinc(II) from the amyloid-β (Aβ) peptide and halt Cu(Aβ)-catalyzed ROS production in the context of Alzheimer's disease. These findings raise questions about the relevance of a recently reported “cocktail” approach employing two different chelators that target Cu and Zn, respectively.
The accumulation of the amyloid-β (Aβ) peptide,¹,² copper (Cu) dyshomeostasis³⁻⁵ and oxidative stress⁶,⁷ are well-known hallmarks of Alzheimer's disease (AD).⁸⁻¹⁰ In this context, the Cu-Aβ complex has been shown to catalyse efficiently the formation of reactive oxygen species (ROS),¹¹ and hence potentially contribute to the oxidative stress observed in AD.¹²,¹³ Therefore, Cu chelation has emerged as a strategy to counteract the pro-oxidant activity of Cu-Aβ¹³ and the Cu-promoted formation of Aβ oligomers,¹⁴,¹⁵ regarded as key neurotoxic intermediates. To this end, various chelators have been developed to extract Cu from Aβ and halt the ROS production.¹⁶⁻²⁰ However, the efficiency of most chelators may be compromised in the presence of zinc(II) (Zn),²¹,²² which is a physiological neuromodulator,²³⁻²⁵ attains higher concentration than Cu in the brain,²⁶⁻²⁸ and binds to Aβ.⁹,²⁹,³⁰ Indeed, most ligands, including the common chelator EDTA (ethylenediaminetetraacetic acid), have lower Cu-vs-Zn selectivity (S, see Equation 1, where $K_L^M$ is the binding constant of the ligand L for the metal M) than Aβ (see Table 1).

$$S = \log(K_L^{Cu}) - \log(K_L^{Zn}) \qquad \text{Eq. 1}$$

<table><caption>**Table 1.** Binding constants and Cu-vs-Zn selectivity (S) of Aβ and EDTA.</caption><thead><tr><th></th><th>log K<sup>Cu</sup></th><th>log K<sup>Zn</sup></th><th>S</th></tr></thead><tbody><tr><td>Aβ<sup>a</sup></td><td>10<sup>31</sup></td><td>5<sup>32</sup></td><td>5</td></tr><tr><td>EDTA<sup>b</sup></td><td>15.9<sup>33</sup></td><td>13.5<sup>33</sup></td><td>2.4</td></tr></tbody></table>

Hence, a specific design must be carried out to obtain a chelator with a suitable selectivity to retrieve Cu from Aβ and stop the associated Cu(Aβ)-catalysed ROS production in the presence of excess Zn (Scheme 1, top).¹⁶,²¹,³⁴ To tackle this challenge, a few selective ligands have been developed over the last decade.³⁵⁻⁴²

<footnote>a Apparent binding constant at pH 7.4</footnote>

<footnote>b Conditional binding constant at pH 7.4</footnote>
In this context, an alternative strategy has been recently proposed that relies on the use of a so-called “cocktail” of chelators, specifically two peptoids, the first one able to retrieve Cu(II) and stop ROS formation, and the second one targeting Zn (Scheme 1, middle).<sup>43</sup>

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

**Scheme 1.** Schematic representation of different strategies to halt the Cu(Aβ)-catalyzed ROS production in the presence of Zn: *top*) Cu-selective chelation, in which a chelator (*yellow*) with higher Cu-vs-Zn selectivity than Aβ retrieves Cu selectively from Cu,Zn-Aβ; *middle*) “cocktail” approach, in which two different chelators are employed to retrieve selectively Cu (*blue*) and Zn (*grey*) from Cu,Zn-Aβ; *bottom*) the use of an excess of a non-selective chelator with higher affinity for both Cu and Zn than Aβ (e.g. EDTA, *red*) that retrieves both Cu and Zn from Cu,Zn-Aβ. In all cases, the chelator forms a redox-inert complex and hence stops the Cu(Aβ)-catalyzed ROS production.
To prove the relevance of the “cocktail” approach, the authors reported a control experiment in which Cu(Aβ)-induced ROS production in the presence of one equivalent of Zn was not stopped by adding 2 equivalents of the non-selective chelator EDTA. However, this finding is inconsistent with (i) the higher affinity of EDTA for both Cu and Zn relative to Aβ (Table 1) and (ii) the reported ability of EDTA to stop Cu(Aβ)-induced ROS production in the absence of Zn.43,44 Indeed, based on these established properties, EDTA is anticipated to quantitatively retrieve both metals when added in two-fold excess and stop associated ROS generation (Scheme 1, bottom) by Cu(II) bound to full-length Aβ (Aβ₁-x) as well as N-terminally truncated Aβ variants.45-48

In this work, we aim to address this inconsistency and provide solid evidence that 2 equivalents of EDTA are actually able to remove both Cu and Zn from Cu,Zn-Aβ and to stop associated ROS production by Cu,Zn-Aβ. This leads in turn to questioning the utility and relevance of the proposed “cocktail” approach.

We first evaluated the impact of EDTA on the ROS formation catalyzed by Cu-Aβ₁₆ (the latter being an established model for Cu-binding to full-length Aβ)49-51 in the presence of one equivalent of Zn, noted Cu,Zn-Aβ, by measuring the kinetics of ascorbate (Asc) oxidation, which is directly linked to ROS production,22,52 by UV-vis absorption (see SI).

First, control experiments in the absence of Zn (Figure 1.A) showed that, in line with previous studies, free Cu (green) and, to a lesser extent, Cu-Aβ₁₆ (black) efficiently catalyze Asc oxidation. On the contrary, consistently with its very low reduction potential (~ -1.5 V vs SCE, Figure S1) and as previously reported,43,53,54 Cu-EDTA (orange) did not significantly change the rate of metal-free Asc auto-oxidation (grey), and hence it does not produce ROS by itself. As a consequence, the addition of 1 equivalent (relative to Aβ₁₆) of EDTA to Cu-Aβ₁₆ (purple)
completely stops Asc oxidation, commensurate with the higher Cu-affinity of EDTA relative to
Aβ₁₆ (Table 1) and fast Cu transfer (vide infra).

<figure><img src="image_3.png" /><figcaption>**Figure 1.** Kinetics of Asc oxidation in the absence (Ctrl, *grey*) or in the presence of: Cu (A, *green*), Cu-Aβ₁₆ (A, B, *black*), Cu-EDTA (A, *orange*), Cu-Aβ₁₆ + 1 eq. EDTA (A, *purple*), Cu,Zn-Aβ₁₆ (B, *blue*), Cu,Zn-Aβ₁₆ + 1 eq. EDTA (B, *pink*) and Cu,Zn-Aβ₁₆ + 2 eq. EDTA (B, *red*). Conditions: [Asc] = 100 µM, [Cu] = 9 µM, [Aβ] = 10 µM, [Zn] = 10 µM, [EDTA] = 10 (1 eq.) or 20 µM (2 eq.), [HEPES] = 50 mM, pH = 7.4. Aβ₁₆, Cu, Zn, and EDTA were mixed within 100 s and incubated for 500 s before adding Asc (at ca. 600 s).</figcaption></figure>

As previously reported, no significant difference is observed between the Asc consumption in the
presence of Cu-Aβ₁₆ (**Figure 1.B**, *black*) and Cu,Zn-Aβ₁₆ (**Figure 1.B**, *blue*).²²,⁵² The addition of
1 equivalent of EDTA to Cu,Zn-Aβ₁₆ (**Figure 1.B**, *pink*) weakly lessens the rate of Asc oxidation,
confirming that the lower selectivity of EDTA relative to Aβ₁₆ (**Table 1**) impairs its efficiency in
halting Cu(Aβ)-induced ROS production in the presence of Zn. In contrast, when 2 equivalents of
EDTA are added to Cu,Zn-Aβ₁₆ (**Figure 1.B**, *red*), no Cu-Aβ catalysed Asc oxidation was
observed, in line with the anticipated ability of EDTA to retrieve both Cu and Zn from Aβ₁₆ and
prevent ROS formation.

Of note, we also performed analogous measurements using the full-length Aβ (Aβ40) peptide
(Figure S2), which gave similar results as those detailed above for the Aβ16 peptide.
Overall, these observations are consistent with the previously reported Cu- and Zn-binding constants of Aβ and EDTA (Table 1), and the resistance of Cu-EDTA to reduction by Asc.

In order to directly assess such thermodynamic predictions, the metal exchange between Aβ₁₆ and EDTA was also investigated by means of UV-vis absorption, electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spectroscopies.

UV-vis spectra (Figure 2.A) showed distinct d-d bands for Cu,Zn-Aβ₁₆ (black, λmax ~625 nm) and Cu-EDTA (orange, λmax ~730 nm). Upon the addition of 1 equivalent of EDTA to Cu,Zn-Aβ₁₆, a broader d-d band at intermediate λmax was observed (pink), suggesting the partial formation of Cu-EDTA. Indeed, this spectrum could be well reproduced by a linear combination of Cu,Zn-Aβ₁₆ and Cu-EDTA spectra at a ~60:40 ratio (dashed black), meaning that 1 equivalent of EDTA can retrieve about 40% Cu from Cu,Zn-Aβ₁₆. No significant evolution of this band was observed during 500 s, proving that the metal exchange equilibrium is reached within the mixing time (Figure 2.B).

In line with this Cu distribution, the Asc oxidation measured upon the addition of 1 equivalent of EDTA to Cu,Zn-Aβ₁₆ corresponds approximately to that of 6 µM Cu-Aβ₁₆ (Figure S3). Upon further addition of a 2nd equivalent of EDTA, the observed d-d band (Figure 2.A, red) matches that of Cu-EDTA, indicating that EDTA can completely extract Cu from Cu,Zn-Aβ₁₆.
<figure><img src="image_4.png" /><figcaption>**Figure 2.** Metal exchange between Cu,Zn-Aβ₁₆ and EDTA followed by UV-vis absorption (A, B), EPR (C) and NMR (D) spectroscopies. A) Visible spectra of Cu,Zn-Aβ₁₆ (blue), Cu-EDTA (orange), Cu,Zn-Aβ₁₆ + 1 eq. EDTA after 500 s incubation (pink), Cu,Zn-Aβ₁₆ + 2 eq. EDTA (red) and linear combination of Cu,Zn-Aβ₁₆ and Cu-EDTA spectra at 60:40 ratio (dashed black); B) Kinetic evolution of Cu,Zn-Aβ₁₆ (625 nm, dashed blue) and Cu-EDTA (730 nm, solid) d-d bands upon the addition of 1 (pink) and 2 eq. (red) EDTA to Cu,Zn-Aβ₁₆; C) X-band EPR spectra (T = 110 K) of Cu,Zn-Aβ₁₆ (blue), Cu,Zn-Aβ₁₆ + 1 eq. EDTA (pink), Cu,Zn-Aβ₁₆ + 2 eq. EDTA (red), and Cu-Aβ₁₆ + EDTA (black). D) NMR spectra (3.5-2.5 ppm region, v = 600 MHz, T = 298 K) of Cu,Zn-Aβ₁₆ (blue), Cu,Zn-Aβ₁₆ + 1 eq. EDTA (pink), Cu,Zn-Aβ₁₆ + 2 eq. EDTA (red) and Zn-EDTA (grey); impurities (< 2%) present in HEPES-d₁₈ buffer are indicated by *. Conditions: [Cu] = 180 µM, [Aβ₁₆] = 200 µM, [Zn] = 200 µM, [EDTA] = 200 (1 eq.) or 400 µM (2 eq.), [HEPES/HEPES-d₁₈] = 50 mM, pH = 7.4. EPR samples were prepared using isotopically pure ⁶⁵Cu and supplemented with 10% glycerol. NMR samples were prepared in HEPES-d₁₈ buffer and H₂O:D₂O 90:10.</figcaption></figure>
Cu transfer between Aβ₁₆ and EDTA was also monitored through low-temperature (110 K) continuous-wave X-band EPR spectroscopy (Figure 2.C and S6-7; see Table S1 for spin-Hamiltonian EPR parameters). As previously described, the EPR spectrum of Cu,Zn-Aβ₁₆ shows the presence of two components (Aβ-I and Aβ-II, Table S1).²²,⁵⁰,⁵²,⁵⁵ Upon addition of 2 equivalents of EDTA, the EPR spectrum shows one predominant species, which is virtually identical to that obtained by the addition of EDTA to Cu-Aβ₁₆ or by the addition of Aβ₁₆ to Cu-EDTA (Figure S6-7 and Table S1). In the presence of 1 equivalent of EDTA, an intermediate spectrum is observed which can be approximately reproduced by a linear combination of the spectra of Cu-Aβ₁₆ and Cu,Zn-Aβ₁₆ + 2 eq. EDTA at ~65:35 ratio (Figure S5.B), as supported by EPR simulations (Figure S7 and Table S1) and in agreement with UV-vis (Figure 2.A) and Asc oxidation measurements (Figure S2). EPR experiments also showed that Cu-EDTA and Aβ₁₆ form a ternary species where Aβ₁₆ binds to Cu-EDTA via an imidazole (Im) group from the His residues (Figure S6-7 and Table S1). The formation of such a ternary (CuEDTA)-Im complex, which has been previously reported,⁵⁶,⁵⁷ was not detected by UV-vis spectroscopy since the addition of either 1 equivalent Im or Aβ₁₆ to Cu-EDTA does not significantly shift its d-d band (Figure S8). Moreover, it is worth noting that the rate of Asc oxidation by Cu-EDTA is not altered upon addition of 1 equivalent of Aβ₁₆ or even 10 equivalents of Im (Figure S5). This indicates that the amount of the ternary ImAβ-Cu-EDTA is negligible at 10 µM, commensurate with a low stability constant for (CuEDTA)-Im ternary complex,⁵⁶,⁵⁷ and/or that it does not catalyze ROS production on its own.

Finally, ¹H-NMR spectra (Figures 2.D and S9-10) were also recorded to detect the concomitant formation of Zn-EDTA. Indeed, upon Zn binding, the signals of EDTA (Figure S9) are shifted upfield, and the methylene protons of the acetyl moieties become diastereotopic (couple doublets
at 3.3 ppm, **Figure 2.D**) due to the formation of the metal chelate.⁵⁸⁻⁶⁰ In the presence of 1 (*pink*) or 2 (*red*) equivalents of EDTA to Cu,Zn-Aβ₁₆, the NMR spectra clearly show signals at 3.3 (coupled doublets from methylene protons of the acetyl groups) and 2.8 ppm (singlet from the methylene protons of the ethylenediamine moiety) corresponding to Zn-EDTA (*grey*). Moreover, the large broadening of Aβ₁₆ signals caused by the binding to the paramagnetic Cu is counteracted by the addition of EDTA (**Figures S9 and S10**). Noteworthy, a partial broadening of the His signals (Hδ at 6.85-6.95 and Hε at 7.0-7.75 ppm) is observed even in the presence of 2 equivalents of EDTA (**Figure S10**, *red*), further corroborating their partial binding to Cu-EDTA.

In summary, our data show that 1 equivalent of EDTA can partially retrieve Cu and Zn from Aβ (about 40% of Cu and 60% of Zn) and decrease the associated ROS production, while the complete removal of both Cu and Zn and the concomitant halt of ROS production is obtained, as anticipated, upon addition of 2 equivalents of EDTA (**Scheme 2**).

<figure><img src="image_5.png" /><figcaption>**Scheme 2.** Schematic representation of the metal transfer between Aβ and EDTA and consequent halt of ROS production.</figcaption></figure>

This shows that putting two equivalents of a non-specific ligand would have the very same effect on the inhibition of Cu(Aβ)-induced ROS as putting a cocktail of two ligands targeting Cu and Zn, respectively. Noteworthy, in the cocktail approach, the two different ligands should have the same biodistribution and pharmacokinetics, which is very challenging to achieve. In addition,
considering the biologically relevant excess (10-100-fold) of Zn vs Cu in the brain, in either approach, a high excess of ligands should be administered, which could lead to the undesired perturbation of Zn homeostasis. As a consequence, the use of a highly selective Cu chelator forming a redox-inert complex remains the most promising translational approach.

In conclusion, our findings and considerations question the utility and convenience of a “cocktail” of chelators, as well as of a non-selective chelator in excess, to halt the ROS production by Cu-Aβ in the presence of Zn and rather draw attention to the actual need for suitably Cu-selective chelators in the context of AD.

## ASSOCIATED CONTENT

**Supporting Information.** Experimental details, CV, ascorbate oxidation assays, EPR spectra, UV-vis spectra, NMR spectra (PDF).

## AUTHOR INFORMATION

### Corresponding Authors

Enrico Falcone - LCC-CNRS, Université de Toulouse, CNRS, 31077 Toulouse, France;

ORCID: https://orcid.org/0000-0002-1944-3552; email: enrico.falcone@lcc-toulouse.fr

Christelle Hureau - LCC-CNRS, Université de Toulouse, CNRS, 31077 Toulouse, France;

ORCID: https://orcid.org/0000-0003-3339-0239; email: christelle.hureau@lcc-toulouse.fr

### Author
## Author Contributions

**M.B.:** Investigation, Formal analysis, Visualization, Writing – original draft; **C.H.:** Conceptualization, Funding acquisition, Supervision, Validation, Writing – review & editing; **E.F.:** Conceptualization, Investigation, Formal analysis, Visualization, Supervision, Validation, Writing – original draft. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

## Funding Sources

ANR-22-CE29-0026; ERC-StG-aLzINK-638712

## ACKNOWLEDGMENTS

E.F. acknowledges financial support from the ANR-22-CE29-0026 project. C.H. acknowledges financial support from ERC-StG-aLzINK-638712. The authors thank Prof. Peter Faller (University of Strasbourg) for fruitful discussions, Dr. Charlène Esmieu (LCC-CNRS, Toulouse) for help with CV measurements, Dr. Christian Bijani (LCC-CNRS, Toulouse) for help with NMR measurements, and Dr. Melanie Rossotti (LCC-CNRS, Toulouse) for help with EPR simulations.

## ABBREVIATIONS

$A\beta$, amyloid-β; AD, Alzheimer's disease; Asc, ascorbate; EDTA, ethylenediaminetetraacetic acid; EPR, electron paramagnetic resonance; NMR, nuclear magnetic resonance; ROS, reactive oxygen species; UV-vis, ultraviolet-visible.
## REFERENCES

(1) Murphy, M. P.; LeVine, H. Alzheimer's Disease and the Amyloid-Beta Peptide. *J Alzheimers Dis* **2010**, *19* (1), 311-323. https://doi.org/10.3233/JAD-2010-1221.

(2) Sun, X.; Chen, W.-D.; Wang, Y.-D. β-Amyloid: The Key Peptide in the Pathogenesis of Alzheimer's Disease. *Front Pharmacol* **2015**, *6*, 221. https://doi.org/10.3389/fphar.2015.00221.

(3) Squitti, R.; Faller, P.; Hureau, C.; Granzotto, A.; White, A. R.; Kepp, K. P. Copper Imbalance in Alzheimer's Disease and Its Link with the Amyloid Hypothesis: Towards a Combined Clinical, Chemical, and Genetic Etiology. *Journal of Alzheimer's Disease* **2021**, *83* (1), 23-41. https://doi.org/10.3233/JAD-201556.

(4) Kepp, K. P.; Squitti, R. Copper Imbalance in Alzheimer's Disease: Convergence of the Chemistry and the Clinic. *Coordination Chemistry Reviews* **2019**, *397*, 168-187. https://doi.org/10.1016/j.ccr.2019.06.018.

(5) Gromadzka, G.; Tarnacka, B.; Flaga, A.; Adamczyk, A. Copper Dyshomeostasis in Neurodegenerative Diseases-Therapeutic Implications. *Int J Mol Sci* **2020**, *21* (23), 9259. https://doi.org/10.3390/ijms21239259.

(6) Chen, Z.; Zhong, C. Oxidative Stress in Alzheimer's Disease. *Neurosci Bull* **2014**, *30* (2), 271-281. https://doi.org/10.1007/s12264-013-1423-y.

(7) Bai, R.; Guo, J.; Ye, X.-Y.; Xie, Y.; Xie, T. Oxidative Stress: The Core Pathogenesis and Mechanism of Alzheimer's Disease. *Ageing Res Rev* **2022**, *77*, 101619. https://doi.org/10.1016/j.arr.2022.101619.

(8) Yi, Y.; Lim, M. H. Current Understanding of Metal-Dependent Amyloid-β Aggregation and Toxicity. *RSC Chem. Biol.* **2023**, *4* (2), 121-131. https://doi.org/10.1039/D2CB00208F.

(9) Atrián-Blasco, E.; Gonzalez, P.; Santoro, A.; Alies, B.; Faller, P.; Hureau, C. Cu and Zn Coordination to Amyloid Peptides: From Fascinating Chemistry to Debated Pathological Relevance. *Coordination Chemistry Reviews* **2018**, *371*, 38-55. https://doi.org/10.1016/j.ccr.2018.04.007.

(10) Selkoe, D. J.; Hardy, J. The Amyloid Hypothesis of Alzheimer's Disease at 25 Years. *EMBO Molecular Medicine* **2016**, *8* (6), 595-608. https://doi.org/10.15252/emmm.201606210.

(11) Falcone, E.; Hureau, C. Redox Processes in Cu-Binding Proteins: The “in-between” States in Intrinsically Disordered Peptides. *Chemical Society Reviews* **2023**, *52* (19), 6595-6600. https://doi.org/10.1039/D3CS00443K.

(12) Eskici, G.; Axelsen, P. H. Copper and Oxidative Stress in the Pathogenesis of Alzheimer's Disease. *Biochemistry* **2012**, *51* (32), 6289-6311. https://doi.org/10.1021/bi3006169.

(13) Cheignon, C.; Tomas, M.; Bonnefont-Rousselot, D.; Faller, P.; Hureau, C.; Collin, F. Oxidative Stress and the Amyloid Beta Peptide in Alzheimer's Disease. *Redox Biol* **2018**, *14*, 450-464. https://doi.org/10.1016/j.redox.2017.10.014.

(14) Weibull, M. G. M.; Simonsen, S.; Oksbjerg, C. R.; Tiwari, M. K.; Hemmingsen, L. Effects of Cu(II) on the Aggregation of Amyloid-β. *J Biol Inorg Chem* **2019**, *24* (8), 1197-1215. https://doi.org/10.1007/s00775-019-01727-5.

(15) Oliveri, V. Unveiling the Effects of Copper Ions in the Aggregation of Amyloidogenic Proteins. *Molecules* **2023**, *28* (18), 6446. https://doi.org/10.3390/molecules28186446.

(16) Esmieu, C.; Guettas, D.; Conte-Daban, A.; Sabater, L.; Faller, P.; Hureau, C. Copper-Targeting Approaches in Alzheimer's Disease: How To Improve the Fallouts Obtained from
in Vitro Studies. *Inorg. Chem.* **2019**, *58* (20), 13509–13527.
https://doi.org/10.1021/acs.inorgchem.9b00995.

(17) Singh, S. K.; Balendra, V.; Obaid, A. A.; Esposto, J.; Tikhonova, M. A.; Gautam, N. K.; Poeggeler, B. Copper-Mediated β-Amyloid Toxicity and Its Chelation Therapy in Alzheimer's Disease. *Metallomics* **2022**, *14* (6), mfac018.
https://doi.org/10.1093/mtomcs/mfac018.

(18) Fasae, K. D.; Abolaji, A. O.; Faloye, T. R.; Odunsi, A. Y.; Oyetayo, B. O.; Enya, J. I.; Rotimi, J. A.; Akinyemi, R. O.; Whitworth, A. J.; Aschner, M. Metallobiology and Therapeutic Chelation of Biometals (Copper, Zinc and Iron) in Alzheimer's Disease: Limitations, and Current and Future Perspectives. *J Trace Elem Med Biol* **2021**, *67*, 126779.
https://doi.org/10.1016/j.jtemb.2021.126779.

(19) Thakur, R.; Karwasra, R.; Umar, T. Understanding Alzheimer's Disease and Its Metal Chelation Therapeutics: A Narrative Review. *Curr Pharm Des* **2023**, *29* (30), 2377–2386.
https://doi.org/10.2174/0113816128263992231012113847.

(20) Savelieff, M. G.; Nam, G.; Kang, J.; Lee, H. J.; Lee, M.; Lim, M. H. Development of Multifunctional Molecules as Potential Therapeutic Candidates for Alzheimer's Disease, Parkinson's Disease, and Amyotrophic Lateral Sclerosis in the Last Decade. *Chem. Rev.* **2019**, *119* (2), 1221–1322.
https://doi.org/10.1021/acs.chemrev.8b00138.

(21) Atrián-Blasco, E.; Conte-Daban, A.; Hureau, C. Mutual Interference of Cu and Zn Ions in Alzheimer's Disease: Perspectives at the Molecular Level. *Dalton Trans.* **2017**, *46* (38), 12750–12759.
https://doi.org/10.1039/C7DT01344B.

(22) Conte-Daban, A.; Day, A.; Faller, P.; Hureau, C. How Zn Can Impede Cu Detoxification by Chelating Agents in Alzheimer's Disease: A Proof-of-Concept Study. *Dalton Transactions* **2016**, *45* (39), 15671–15678.
https://doi.org/10.1039/C6DT02308H.

(23) Tóth, K. Zinc in Neurotransmission. *Annu Rev Nutr* **2011**, *31*, 139–153.
https://doi.org/10.1146/annurev-nutr-072610-145218.

(24) Krall, R. F.; Tzounopoulos, T.; Aizenman, E. The Function and Regulation of Zinc in the Brain. *Neuroscience* **2021**, *457*, 235–258.
https://doi.org/10.1016/j.neuroscience.2021.01.010.

(25) Bizup, B.; Tzounopoulos, T. On the Genesis and Unique Functions of Zinc Neuromodulation. *J Neurophysiol* **2024**, *132* (4), 1241–1254.
https://doi.org/10.1152/jn.00285.2024.

(26) Kozlowski, H.; Luczkowski, M.; Remelli, M.; Valensin, D. Copper, Zinc and Iron in Neurodegenerative Diseases (Alzheimer's, Parkinson's and Prion Diseases). *Coordination Chemistry Reviews* **2012**, *256* (19), 2129–2141.
https://doi.org/10.1016/j.ccr.2012.03.013.

(27) Trapani, G.; Satriano, C.; Mendola, D. L. Peptides and Their Metal Complexes in Neurodegenerative Diseases: From Structural Studies to Nanomedicine Prospects. *Current Medicinal Chemistry* **25** (6), 715–747.
https://doi.org/10.2174/0929867324666171026163144.

(28) Wang, L.; Yin, Y.-L.; Liu, X.-Z.; Shen, P.; Zheng, Y.-G.; Lan, X.-R.; Lu, C.-B.; Wang, J.-Z. Current Understanding of Metal Ions in the Pathogenesis of Alzheimer's Disease. *Translational Neurodegeneration* **2020**, *9* (1), 10. https://doi.org/10.1186/s40035-020-00189-z.

(29) Arena, G.; Rizzarelli, E. Zn2+ Interaction with Amyloid-B: Affinity and Speciation. *Molecules* **2019**, *24* (15), 2796. https://doi.org/10.3390/molecules24152796.
(30) Kozin, S. A. Role of Interaction between Zinc and Amyloid Beta in Pathogenesis of Alzheimer's Disease. *Biochemistry* **2023**, 88 (1), S75-S87. https://doi.org/10.1134/S0006297923140055.

(31) Alies, B.; Renaglia, E.; Rózga, M.; Bal, W.; Faller, P.; Hureau, C. Cu(II) Affinity for the Alzheimer's Peptide: Tyrosine Fluorescence Studies Revisited. *Anal. Chem.* **2013**, 85 (3), 1501-1508. https://doi.org/10.1021/ac302629u.

(32) Talmard, C.; Bouzan, A.; Faller, P. Zinc Binding to Amyloid-β: Isothermal Titration Calorimetry and Zn Competition Experiments with Zn Sensors. *Biochemistry* **2007**, 46 (47), 13658-13666. https://doi.org/10.1021/bi701355j.

(33) Falcone, E.; Faller, P. Thermodynamics-Based Rules of Thumb to Evaluate the Interaction of Chelators and Kinetically-Labile Metal Ions in Blood Serum and Plasma. *Dalton Trans.* **2023**, 52 (8), 2197-2208. https://doi.org/10.1039/D2DT03875G.

(34) Liu, Y.; Nguyen, M.; Robert, A.; Meunier, B. Metal Ions in Alzheimer's Disease: A Key Role or Not? *Acc. Chem. Res.* **2019**, 52 (7), 2026-2035. https://doi.org/10.1021/acs.accounts.9b00248.

(35) Gonzalez, P.; Sabater, L.; Mathieu, E.; Faller, P.; Hureau, C. Why the Ala-His-His Peptide Is an Appropriate Scaffold to Remove and Redox Silence Copper Ions from the Alzheimer's-Related Aβ Peptide. *Biomolecules* **2022**, 12 (10), 1327. https://doi.org/10.3390/biom12101327.

(36) Lefèvre, M.; Malikidogo, K. P.; Esmieu, C.; Hureau, C. Sequence-Activity Relationship of ATCUN Peptides in the Context of Alzheimer's Disease. *Molecules* **2022**, 27 (22), 7903. https://doi.org/10.3390/molecules27227903.

(37) Lefèvre, M.; Lantigner, L.; Andolfo, L.; Vanucci-Bacqué, C.; Benoist, E.; Esmieu, C.; Bedos-Belval, F.; Hureau, C. Reduced Schiff-base derivatives to stop reactive oxygen species production by the Cu(Abeta) species: a structure-activity relationship. *Comptes Rendus. Chimie* **2023**, 26 (S3), 67-77. https://doi.org/10.5802/crchim.255.

(38) Hu, X.; Zhang, Q.; Wang, W.; Yuan, Z.; Zhu, X.; Chen, B.; Chen, X. Tripeptide GGH as the Inhibitor of Copper-Amyloid-β-Mediated Redox Reaction and Toxicity. *ACS Chem. Neurosci.* **2016**, 7 (9), 1255-1263. https://doi.org/10.1021/acschemneuro.6b00145.

(39) Behar, A. E.; Sabater, L.; Baskin, M.; Hureau, C.; Maayan, G. A Water-Soluble Peptoid Chelator That Can Remove Cu2+ from Amyloid-β Peptides and Stop the Formation of Reactive Oxygen Species Associated with Alzheimer's Disease. *Angewandte Chemie International Edition* **2021**, 60 (46), 24588-24597. https://doi.org/10.1002/anie.202109758.

(40) Esmieu, C.; Balderrama-Martínez-Sotomayor, R.; Conte-Daban, A.; Iranzo, O.; Hureau, C. Unexpected Trends in Copper Removal from Aβ Peptide: When Less Ligand Is Better and Zn Helps. *Inorg. Chem.* **2021**, 60 (2), 1248-1256. https://doi.org/10.1021/acs.inorgchem.0c03407.

(41) Zhang, W.; Huang, D.; Huang, M.; Huang, J.; Wang, D.; Liu, X.; Nguyen, M.; Vendier, L.; Mazères, S.; Robert, A.; Liu, Y.; Meunier, B. Preparation of Tetradentate Copper Chelators as Potential Anti-Alzheimer Agents. *ChemMedChem* **2018**, 13 (7), 684-704. https://doi.org/10.1002/cmdc.201700734.

(42) Zhang, W.; Liu, Y.; Hureau, C.; Robert, A.; Meunier, B. N4-Tetradentate Chelators Efficiently Regulate Copper Homeostasis and Prevent ROS Production Induced by Copper-Amyloid-B1-16. *Chemistry – A European Journal* **2018**, 24 (31), 7825-7829. https://doi.org/10.1002/chem.201801387.
(43) Behar, A. E.; Maayan, G. A Cocktail of Cu2+- and Zn2+-Peptoid-Based Chelators Can Stop ROS Formation for Alzheimer's Disease Therapy. *Chem. Sci.* **2024**, *15* (45), 18855–18864. https://doi.org/10.1039/D4SC04313H.

(44) Buettner, G. R.; and Czapski, Prof. G. Ascorbate Autoxidation in the Presence of Iron and Copper Chelates. *Free Radical Research Communications* **1986**, *1* (6), 349–353. https://doi.org/10.3109/10715768609051638.

(45) Mital, M.; Wezynfeld, N. E.; Frączyk, T.; Wiloch, M. Z.; Wawrzyniak, U. E.; Bonna, A.; Tumpach, C.; Barnham, K. J.; Haigh, C. L.; Bal, W.; Drew, S. C. A Functional Role for Aβ in Metal Homeostasis? N-Truncation and High-Affinity Copper Binding. *Angewandte Chemie International Edition* **2015**, *54* (36), 10460–10464. https://doi.org/10.1002/anie.201502644.

(46) Esmieu, C.; Ferrand, G.; Borghesani, V.; Hureau, C. Impact of N-Truncated Aβ Peptides on Cu- and Cu(Aβ)-Generated ROS: CuI Matters! *Chemistry – A European Journal* **2021**, *27* (5), 1777–1786. https://doi.org/10.1002/chem.202003949.

(47) Wezynfeld, N. E.; Tobolska, A.; Mital, M.; Wawrzyniak, U. E.; Wiloch, M. Z.; Płonka, D.; Bossak-Ahmad, K.; Wróblewski, W.; Bal, W. Aβ5-x Peptides: N-Terminal Truncation Yields Tunable Cu(II) Complexes. *Inorg. Chem.* **2020**, *59* (19), 14000–14011. https://doi.org/10.1021/acs.inorgchem.0c01773.

(48) Sudzik, D.; Bal, W.; Wezynfeld, N. E. Oxidative Activity of Copper Ions in Ternary Systems With N-Truncated Amyloid Beta Peptides and Low Molecular Weight Substances. *Journal of Neurochemistry* **2025**, *169* (7), e70145. https://doi.org/10.1111/jnc.70145.

(49) Kowalik-Jankowska, T.; Ruta, M.; Wiśniewska, K.; Łankiewicz, L. Coordination Abilities of the 1–16 and 1–28 Fragments of β-Amyloid Peptide towards Copper(II) Ions: A Combined Potentiometric and Spectroscopic Study. *Journal of Inorganic Biochemistry* **2003**, *95* (4), 270–282. https://doi.org/10.1016/S0162-0134(03)00128-4.

(50) Syme, C. D.; Nadal, R. C.; Rigby, S. E. J.; Viles, J. H. Copper Binding to the Amyloid-β (Aβ) Peptide Associated with Alzheimer's Disease: FOLDING, COORDINATION GEOMETRY, pH DEPENDENCE, STOICHIOMETRY, AND AFFINITY OF Aβ-(1-28): INSIGHTS FROM A RANGE OF COMPLEMENTARY SPECTROSCOPIC TECHNIQUES*. *Journal of Biological Chemistry* **2004**, *279* (18), 18169–18177. https://doi.org/10.1074/jbc.M313572200.

(51) Karr, J. W.; Akintoye, H.; Kaupp, L. J.; Szalai, V. A. N-Terminal Deletions Modify the Cu2+ Binding Site in Amyloid-β. *Biochemistry* **2005**, *44* (14), 5478–5487. https://doi.org/10.1021/bi047611e.

(52) Alies, B.; Sasaki, I.; Proux, O.; Sayen, S.; Guillon, E.; Faller, P.; Hureau, C. Zn Impacts Cu Coordination to Amyloid-β, the Alzheimer's Peptide, but Not the ROS Production and the Associated Cell Toxicity. *Chem. Commun.* **2013**, *49* (12), 1214–1216. https://doi.org/10.1039/C2CC38236A.

(53) Buettner, G. R.; and Czapski, Prof. G. Ascorbate Autoxidation in the Presence of Iron and Copper Chelates. *Free Radical Research Communications* **1986**, *1* (6), 349–353. https://doi.org/10.3109/10715768609051638.

(54) Rao, M. V. L.; Sastry, L. V. L.; Srinivasan, M.; Subrahmanyan, V. Inhibition of Oxidation of Ascorbic Acid by EDTA. *Journal of the Science of Food and Agriculture* **1959**, *10* (8), 436–441. https://doi.org/10.1002/jsfa.2740100807.

(55) Alies, B.; Eury, H.; Bijani, C.; Rechignat, L.; Faller, P.; Hureau, C. pH-Dependent Cu(II) Coordination to Amyloid-β Peptide: Impact of Sequence Alterations, Including the H6R and
D7N Familial Mutations. *Inorg. Chem.* **2011**, *50* (21), 11192–11201.
https://doi.org/10.1021/ic201739n.

(56) Israeli, J.; Saulnier, H. Mixed Complexes Containing Imidazole. *Inorganica Chimica Acta* **1968**, *2*, 479–481. https://doi.org/10.1016/S0020-1693(00)87090-5.

(57) Pyreu, D.; Bazanova, M. Thermodynamics of Mixed-Ligand Complex Formation of Metal (II) Complexonates with Imidazole in Aqueous Solution. *Inorganica Chimica Acta* **2016**, *453*, 687–691. https://doi.org/10.1016/j.ica.2016.09.027.

(58) Ćendić, M.; Deeth, R. J.; Meetsma, A.; Garribba, E.; Sanna, D.; Matović, Z. D. Chelating Properties of EDTA-Type Ligands Containing Six-Membered Backbone Ring toward Copper Ion: Structure, EPR and TD-DFT Evaluation. *Polyhedron* **2017**, *124*, 215–228. https://doi.org/10.1016/j.poly.2016.12.025.

(59) Mathuthu, E.; Janse van Rensburg, A.; Du Plessis, D.; Mason, S. EDTA as a Chelating Agent in Quantitative 1H-NMR of Biologically Important Ions. *Biochem. Cell Biol.* **2021**, *99* (4), 465–475. https://doi.org/10.1139/bcb-2020-0543.

(60) Santoro, A.; Wezynfeld, N. E.; Stefaniak, E.; Pomorski, A.; Płonka, D.; Krężel, A.; Bal, W.; Faller, P. Cu Transfer from Amyloid-B4-16 to Metallothionein-3: The Role of the Neurotransmitter Glutamate and Metallothionein-3 Zn(Ii)-Load States. *Chem Commun* (*Camb*) **2018**, *54* (89), 12634–12637. https://doi.org/10.1039/c8cc06221h.

**For Table of Contents Only.**

<figure><img src="image_6.png" /></figure>

A two-fold excess of a chelator can be as efficient as a “cocktail” of two different chelators in halting the ROS production catalyzed by Cu,Zn-(amyloid-β).