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Multitarget compounds with therapeutic potential against Alzheimer disease: Design and in vitro studies

Periodic Reporting for period 1 - CHELALZ (Multitarget compounds with therapeutic potential against Alzheimer disease: Design and in vitro studies)

Reporting period: 2016-01-01 to 2017-12-31

Aβ protein has been so far the main therapeutic target of choice to develop anti-Alzheimer drugs. However, therapies aiming to disrupt the amyloid cascade have reached Phase III clinical trials and have eventually failed. This is a key evidence of the multifactorial aetiology of the disease.
CHELALZ project aims to explore a novel synthetic approach that targets multiple hallmarks of AD: Aβ aggregation, copper imbalance, and decrease of synaptic plasticity.
The implementation of multitarget therapies is known to be able to circumvent the complications associated with the administration of multiple single-target drugs, e.g. conflicting bioavailabilities, pharmacokinetics, and metabolism. In fact, multitarget treatments for other diseases like HIV and cancer have already demonstrated improved therapeutic properties over their single-target counterparts.
"CHELALZ proposal aimed to develop multitarget two-component conjugates and evaluate their therapeutic properties for AD. The main results are structured according to the three Specific Objectives (SOs):
SO1. Development of biocompatible and effective copper chelators (MPACs) to lessen copper-induced toxicity
Based on our previous research (Chem. Eur. J. 2016, 22, 7268), we have selected the peptidic sequence His-Xaa-His to obtain copper chelators which are able to displace Aβ-bound copper(II) ions and minimize copper-induced ROS generation. The choice of the chelating unit has been supported by the computational study carried out by Prof. Mariona Sodupe (Universitat Autònoma de Barcelona).
We have synthesized and included in the central residue of the tripeptides two fluorophores: 3CCA and 4DMN. 3CCA may work as ROS-probe for oxidative environments (amyloid diseased cells). 4DMN is an environment-sensitive fluorophore that allows monitoring its aggregation and distinguish between distinct amyloid morphologies.
In total, four peptide-based chelators have been obtained with high yields; two of which contain a terminal lysine residue to anchor them onto the surface of gold nanoparticles (SO2).
The peptidic chelators shows a higher copper-binding affinity than Aβ protein and they can effectively displace Aβ-bound Cu(II) ions.
The capability of these peptides to bind magnesium ions could not be, however, demonstrated.

SO2. Development of PEGylated gold nanoparticles (AuNPs) that bind Aβ aggregates.
Highly monodisperse AuNPs with two sizes have been synthesized and further functionalized with a brush-like conformation of polyethylene glycol (PEG) (D2.1 Table 1). Different terminal groups were used to yield negative, neutral and positive surface charges.
Under the co-supervision of the biophysicist Dr. Raimon Sabaté (UB), I carried out assays to follow the aggregation kinetics of the protein in the presence of the nanoparticles. The most active particles were those of bigger size, and they affected mainly the nucleation rate rather than the elongation rate (Fig. 2). Also, the non-charged particles exerted a markedly higher effect than the negatively-charged particles, which agrees with the lack of electrostatic repulsion between particles and protein in the former ones.
Afterwards, we found that the self-assembly appears to be controlled by a single activation energy in both nucleation and elongation steps. 15-nm particles changes dramatically the aggregation pathway of Abeta.
During the secondment at the IBEC, the particles resulted non-toxic against mouse brain cells.

SO3. Obtention of multitarget AuNP-chelator conjugates.
The chelating peptides have been successfully coupled onto PEGylated AuNPs via amide bonding. We have developed a new procedure to quantify the peptide loading of the particles in a faster way than the traditional analysis of aminoacids (which requires multiple steps and it time-consuming). The newly-developed method (D3.1) uses fluorescence spectroscopy, which is available in most of the labs.
We have confirmed the ability, although moderate, of the nanoconjugates to interact and displace copper ions from binding Aβ.

Please find enclosed Figures 1-3 and Table 1, where all the milestones and deliverables are listed.

Dissemination and exploitation of results
Scientific publications
• Journal Article: Chemistry: A European Journal 2018, 24(20), 5153. Inside Cover. Open Access.

Dissemination and communication activities
• Presentation in several national and international scientific conferences.
- Press-release in science-disseminating web-site: “Trapping copper with peptides to diminish metal-related toxicity in Alzheimer’s disease"" (INVITED CONTRIBUTION). .
• Participation in the science fair “Festa de la Ciència de la UB”
• Publication of the"
The exploration of effects resulting each added functionality on its own and from their combination in a single entity (leading to an expected synergism among other benefits) is a valuable contribution to the EU research on AD therapeutics, and a future transference of results into the pharmaceutical sector is not only planned but also likely to be successful. The main aspects that CHELALZ has brought out beyond the state of the art are:
1. Our studies on the interaction of copper ions with different histidine-containing tripeptide sequences have demonstrated that the albumin-like binding motif, namely H-Xaa-Xaa-His, not only is key for an efficient inhibition of the copper-mediated aggregation of Aβ and the associated oxidative damage but also that the inclusion of a second histidine as first residue enhances further the affinity and the antioxidant action, despite not coordinating the metal ion (D1.3: Mena et al. Chem. Eur. J. 2018). This observation is expected to impact significantly the scientific community because, among other implications, it shakes the widely-accepted grounds pointing to the ATCUN motif as the optimized sequence that Nature uses for metal-detoxification.
2. Besides the 3CCA probe, the bimodal photoluminescent probe 4DMN has been developed and integrated in a copper-chelating peptide for the first time. 4DMN fluorophore could be used to detect and monitor the Aβ aggregation while providing structural information about the aggregates. The results will contribute to the development of diagnosis tools for early detection of amyloid-related diseases such as Alzheimer’s or Parkinson’s disease.
3. This project has shown for the first time that PEGylated AuNPs of small sizes affect strongly the aggregation of Aβ with a marked dependence of the particle size and surface polarity. These results pave the way towards the control of the proteins folding through biocompatible nano-sized agents (artificial nano-chaperones).
4. The attachment of moderate Cu(II)-chelators -Ac-His-Xaa-His peptides- onto biocompatible gold nanoparticles enhances the ability to displace copper from Aβ. This avoids the need for using strong MPACs that may alter the homeostasis of the metal in other tissues and the function of metalloproteins.This is a big step forward less toxic MPAC-based therapies.