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Alzheimer's disease and Zinc: the missing link ?

Periodic Reporting for period 4 - aLzINK (Alzheimer's disease and Zinc: the missing link ?)

Período documentado: 2019-09-01 hasta 2021-08-31

In aLzINK we have adressed how metal ions, mainly copper and zinc, affect the aetiology of Alzheimer’s disease (AD). AD is a neurodegenerative disorder responsible for more than 60% of all dementia cases. Besides the constantly growing number of AD cases, AD more than many other diseases has important and long-lasting fallouts on the caregivers.

In the aLzINK project, the objective is double: (i) understand how metal ions, although crucial for many biological processes, trigger the disease and (ii) define and probe new therapeutic strategies. With respect to role of metal ions in AD, we want to disentangle (i) how metal ions (copper or/and zinc) bind to the Alzheimer-related amyloid-β (Aβ) peptide; (ii) the role played by the metal ions in Aβ self-assembly and in the production of reactive oxygen species (ROS) that contributes to oxidative stress, where Aβ self-assembly and oxidative stress are two hallmarks of AD; (iii) how cell toxicity is mediated by metal ions and the Aβ peptide. On the therapeutic side, we want to (i) design and probe in vitro and on cells, new ligands able to remove the copper ions from the Aβ peptide and stop aberrant interaction responsible for Aβ self-assembly and ROS production (ii) counteract the effect of zinc ions that can prevent copper ions detoxification and (iii) ultimately, trigger copper removal from Aβ using zinc ions.

During the aLzINK project, most of the objectives were reached and the results obtained have opened new researches lines. Results include the identification of the environnement of copper ions in the Cu(Aβ) complex producing ROS, the evaluation of the effects of Cu and Zn on the self-assembly of the Aβ peptide, the design of a wide series of ligands able to have a positive effect of Cu(Aβ)-mediated toxicity and the elucidation of their modes of action using a wide range of physico-chemical and analytic methods.
During aLzINK project, we have worked on the study at the molecular level of the processes at play between copper, zinc and the Aβ peptides in link with AD, with the final aim to deliver guidelines for the design of new sucessful drugs.

First, the description of the binding sites of metal ions (copper or zinc) to Aβ peptides of biologically relevant sequences (familial or murine mutants, N-term truncated peptides) have been completed. Elucidation of the zinc sites to the human and murine Aβ and as a function of pH were solved (Alies, CEJ 2018 & IC 2016). Evaluation of Cu(II) binding affinity to Aβ was performed using a new method (Conte-Daban, AC 2017). A review (Atrian, CCR 2018) and a book chapter (del Barrio, 2017) were written to recapitulate the state-of-the art of metal ions binding to amyloid-forming peptides (including Aβ). In addition, a review on the biological importance of N-term truncated Aβ was delivered (Borghesani, EJIC 2017).

A second part focused on the Cu(Aβ) complex responsible for ROS production. To perfectly describe this state, now called "in-between state", several studies were necessary (Cheignon, DT 2016 & Metallomics, 2016 & CS 2017; Atrian-Blasco, AC 2018). This is a key result because the way the Cu(Aβ) complex is able to produce ROS is likely to be the same for other intrinsically disordered peptides involved in pathologies. The elucidation of the paths by which Cu(Aβ) produces ROS evidenced that in addition to the stable Cu(Aβ) complexes (either in +I or +II redox states) the "in-between state" is also an important therapeutic target. This ROS production mechanism as well as other processes linking Aβ to oxidative stress in AD has been reviewed in Cheignon, RB 2018.

A third part was dedicated to investigations on the role of metal ions in the modulation of the Aβ self-assembly ability. A huge effort was necessary to get reproducible assembly data. This was reviewed in Faller, Front. Chem. 2021. While research papers on the impact of Cu and/or Zn on Aβ self-assembly are currently in the writting process, we have studied the co-assembly of the Aβ peptide with its N-term truncated counterpart, in presence of Cu(II) (Stefaniak, Chem. Eur. J., 2021) or Zn(II). This is the first paper where the co-assembly of two amyloid-forming peptides in presence of metal ions, known as modulators of the self-assembly, was reported. In addition, we have also worked to new Ln-based probes of the assembly (Madjoub, Chem. Eur.J. 2021) and review new possible lines of research with respect to assembly of amyloid-forming peptides in Ayala, ACN 2019. The Aβ-related toxicity in line with its participation to oxidative stress and formation of amyloid deposits was reviewed in a book chapter (Hureau, 2022).

The last part aimed at the design of new drug canditates with respect to chelation-based therapies. In this part, we have obtained many results. We have illustrated the importance of Zinc into the removal of Copper form the Aβ peptide (Conte-Daban, DT 2016, Atrian-Blasco, DT 2017, Zhang, CEJ 2018, Atrian-Blasco, Metallomics, 2019). That was one of the main objectives of aLzINK. In addition, we demonstrated that targeting Cu(I) is a very interesting option, although overlooked in the literature (Atrian, Metallomics, 2015 & 2019, Conte-Daban, CEJ 2017, Atrian-Blasco, CSR 2017), while Cu(I) could interfere in the detoxification of Cu(II) by Cu(II)-specific chelators (Esmieu, CEJ 2021 & IC 2021, Drommi, Molecules, 2022). We have also shown that kinetic aspects must be taken into account in the design of Cu(II) chelator, beyond the classical thermodynamic-only considerations of the equilibria at play (Conte-Daban, CEJ 2018). New kind of ligands (peptoid and polyoxometallates) were also used for the first time to remove Cu(II) from Aβ and stop the associated ROS formation (Behar, ANIE 2021 & Atrian, CC 2022) while the benefit of using Mn-based prodrug (Conte-Daban, CEJ 2017) was also proven. More concepts have to be published, where peptide ligands with interesting binding features (Gonzales, CEJ 2017, CC 2019, Beuning, IC 2021) are under focus. These chelating concepts and others from the literature were rewieved in Esmieu, IC 2019), while a global review on the metal ions/Aβ/chelator-based drug candidates was published in Hureau, EICB, 2018.

In addition to dissemination of the results towards an academic audience, a special input has been given to dissemination to a wide general audience.
The main scientific breakthrough of aLzINKis to participate in making the current paradigm about Copper targeting in AD move towards a more rationale view. Indeed while the impact of metal ions in AD is recognized, their role is far from being understood. Hence, first therapeutic approaches have targeted Cu(II) ions but without taking care of many crucial parameters and the first molecules tested failed to pass clinical trials. This has recently led to a disinterest of a part of the scientific community. With aLzINK, we aimed at counter-balancing such very radical thought by providing new proofs of concept regarding the targeting of copper ions.

There are still several pending research and review articles in the pipeline that should be delivered in the two coming years. They include the report on new concepts of Cu(II) chelation (8 papers - WP3), co-assembly data of two peptides from the Aβ families (3 papers - WP1), and on toxicity associated with the various species present during the self-assembly of Aβ in presence fo Cu(II) and/or Zn(II) (1 paper - WP2), and two reviews.
related to (vi) - structure of the in-between Cu-Aβ state responsible for ROS production
related to (iv) - showing the interference of Zn in Cu(II) targeting by ligands
Related to (i) - Zn binding site in Aβ and related characterizations
Related to (xiii) - Link between oxidative stress, ROS, metal ions and Aβ
related to (ix) - showing the ligand used to target both Cu(I) and Cu(II) and the principe of action
Related to (xi) - scheme showing the interconnection between MTs, metals, Aβ
Related to (ii) - showing the double face of ascorbate in AD
Related to (viii) - showing the double face of Aβ peptide depedening on their length
related to (v) - implement UV-Vis method to evaluate Cu(II) affinity to biomolecules
Related to (vii) - Showing the increasing complexity of metal ions - Aβ system
Related to (iii) - showing the reorganization fo the Cusite after Aβ oxidation