Rationally Designed Cu(I) Ligand to Prevent CuAß-Generated ROS Production in the Alzheimer's Disease Context.

In the context of Alzheimer's disease, copper (Cu) can be loosely bound to the amyloid-ß (Aß) peptide, leading to the formation of CuAß, which can catalytically generate reactive oxygen species that contribute to oxidative stress. To fight against this phenomenon, the chelation therapy approach has been developed and consists of using a ligand able to remove Cu from Aß and to redox-silence it, thus stopping the reactive oxygen species (ROS) production. A large number of Cu(II) chelators has been studied, allowing us to define and refine the properties required to design a "good" ligand, but without strong therapeutic outcomes to date. Those chelators targeted the Cu(II) redox state. Herein, we explore a parallel and relevant alternative pathway by designing a chelator able to target the Cu(I) redox state. To that end, we designed LH2 ([1N3S] binding set) and demonstrated that (i) it is perfectly able to extract Cu(I) from Cu(I)Aß even in the presence of an excess of Zn(II) and (ii) it redox-silences the Cu, preventing the formation of ROS. We showed that LH2 that is sensitive to oxidation can efficiently replace the [Zn(II)L] complex without losing its excellent ability to stop the ROS production while increasing its resistance to oxidation.

Redox processes in Cu-binding proteins: the "in-between" states in intrinsically disordered peptides.

We report on a concept that some of us first described a decade ago for pure electron transfer [V. Balland, C. Hureau and J.-M. Savéant, Proc. Natl. Acad. Sci. U. S. A., 2010, 107, 17113]. In the present viewpoint, based on more recent results, we refine and extend this "in-between state" concept to explain the formation of reactive oxygen species by copper ions bound to the amyloid-ß (Aß) peptide involved in Alzheimer's disease. In such intrinsically disordered peptides, the Cu coordination is versatile due to the lack of stable folding and the presence of multiple possible binding anchors. Hence, the Cu(I) and Cu(II) ions do impose their favoured sites, with Cu(I) bound in a linear fashion between two His residues and Cu(II) in a square-based pyramid bound to Asp1 amine and carbonyl groups and two His residues in the equatorial plane. Hence a direct electron transfer is prevented and alternatively an in-between state (IBS) mechanism applies, whose description and analysis with respect to other electron transfer processes is the topic of the present viewpoint.

Ability of Azathiacyclen Ligands To Stop Cu(Aß)-Induced Production of Reactive Oxygen Species: [3N1S] Is the Right Donor Set.

Alzheimer's disease (AD) is an incurable neurodegenerative disease that leads to the progressive and irreversible loss of mental functions. The amyloid beta (Aß) peptide involved in the disease is responsible for the production of damaging reactive oxygen species (ROS) when bound to Cu ions. A therapeutic approach that consists of removing Cu ions from Aß to alter this deleterious interaction is currently being developed. In this context, we report the ability of five different 12-membered thiaazacyclen ligands to capture Cu from Aß and to redox silence it. We propose that the presence of a sole sulfur atom in the ligand increases the rate of Cu capture and removal from Aß, while the kinetic aspect of the chelation was an issue encountered with the 4N parent ligand. The best ligand for removing Cu from Aß and inhibiting the associated ROS production is the 1-thia-4,7,10-triazacyclododecane [3N1S]. Indeed the replacement of more N by S atoms makes the corresponding Cu complexes easier to reduce and thus able to produce ROS on their own. In addition, the ligand with three sulfur atoms has a weaker affinity for CuII than Aß, and is thus unable to remove Cu from CuAß.

Sequence-Activity Relationship of ATCUN Peptides in the Context of Alzheimer's Disease.

Amino-terminal CuII and NiII (ATCUN) binding sequences are widespread in the biological world. Here, we report on the study of eight ATCUN peptides aimed at targeting copper ions and stopping the associated formation of reactive oxygen species (ROS). This study was actually more focused on Cu(Aß)-induced ROS production in which the Aß peptide is the "villain" linked to Alzheimer's disease. The full characterization of CuII binding to the ATCUN peptides, the CuII extraction from CuII(Aß), and the ability of the peptides to prevent and/or stop ROS formation are described in the relevant biological conditions. We highlighted in this research that all the ATCUN motifs studied formed the same thermodynamic complex but that the addition of a second histidine in position 1 or 2 allowed for an improvement in the CuII uptake kinetics. This kinetic rate was directly related to the ability of the peptide to stop the CuII(Aß)-induced production of ROS, with the most efficient motifs being HWHG and HGHW.

Impact of N-Truncated Aß Peptides on Cu- and Cu(Aß)-Generated ROS: CuI Matters!

In vitro Cu(Aß1-x )-induced ROS production has been extensively studied. Conversely, the ability of N-truncated isoforms of Aß to alter the Cu-induced ROS production has been overlooked, even though they are main constituents of amyloid plaques found in the human brain. N-Truncated peptides at the positions 4 and 11 (Aß4-x and Aß11-x ) contain an amino-terminal copper and nickel (ATCUN) binding motif (H2 N-Xxx-Zzz-His) that confer them different coordination sites and higher affinities for CuII compared to the Aß1-x peptide. It has further been proposed that the role of Aß4-x peptide is to quench CuII toxicity in the brain. However, the role of CuI coordination has not been investigated to date. In contrast to CuII , CuI coordination is expected to be the same for N-truncated and N-intact peptides. Herein, we report in-depth characterizations and ROS production studies of Cu (CuI and CuII ) complexes of the Aß4-16 and Aß11-16 N-truncated peptides. Our findings show that the N-truncated peptides do produce ROS when CuI is present in the medium, albeit to a lesser extent than the unmodified counterpart. In addition, when used as competitor ligands (i.e., in the presence of Aß1-16 ), the N-truncated peptides are not able to fully preclude Cu(Aß1-16 )-induced ROS production.

The Aggregation Pattern of Aß1-40 is Altered by the Presence of N-Truncated Aß4-40 and/or CuII in a Similar Way through Ionic Interactions.

Alzheimer's disease (AD) is one of the most common of the multifactorial diseases and is characterized by a range of abnormal molecular processes, such as the accumulation of extracellular plaques containing the amyloid-ß (Aß) peptides and dyshomeostasis of copper in the brain. In this study, we have investigated the effect of CuII on the aggregation of Aß1-40 and Aß4-40 , representing the two most prevalent families of Aß peptides, that is, the full length and N-truncated peptides. Both families are similarly abundant in healthy and AD brains. For either of the studied peptides, substoichiometric CuII concentrations accelerated aggregation, whereas superstoichiometric CuII inhibited fibril formation, likely by stabilizing the oligomers. The addition of either Aß4-40 or substoichiometric CuII affected the aggregation profile of Aß1-40 , by yielding shorter and thicker fibrils; amorphous aggregates were formed in the presence of a molar excess of CuII . The similarity of these two effects can be attributed to the increase in the positive charge on the Aß N terminus, caused both by CuII complexation and N truncation at position 4. Our findings provide a better understanding of the biological Aß aggregation process as these two Aß species and CuII coexist and interact under physiological conditions.

Concentration-Dependent Interactions of Amphiphilic PiB Derivative Metal Complexes with Amyloid Peptides Aß and Amylin*.

Invited for the cover of this issue are Jean-François Morfin and Éva Tóth at the CNRS in Orléans, and their collaborators from University of Debrecen, University of Coimbra and Université de Toulouse. The image depicts that when an amphiphilic compound is intravenously injected, monomer, pre-micellar and micellar forms can co-exist in the blood and have different affinities for amyloid peptides. Read the full text of the article at 10.1002/chem.202004000.

Concentration-Dependent Interactions of Amphiphilic PiB Derivative Metal Complexes with Amyloid Peptides Aß and Amylin*.

Metal chelates targeted to amyloid peptides are widely explored as diagnostic tools or therapeutic agents. The attachment of a metal complex to amyloid recognition units typically leads to a decrease in peptide affinity. We show here that by separating a macrocyclic GdL chelate and a PiB targeting unit with a long hydrophobic C10 linker, it is possible to attain nanomolar affinities for both Aß1-40 (Kd =4.4 nm) and amylin (Kd =4.5 nm), implicated, respectively in Alzheimer's disease and diabetes. The Scatchard analysis of surface plasmon resonance data obtained for a series of amphiphilic, PiB derivative GdL complexes indicate that their Aß1-40 or amylin binding affinity varies with their concentration, thus micellar aggregation state. The GdL chelates also affect peptide aggregation kinetics, as probed by thioflavin-T fluorescence assays. A 2D NMR study allowed identifying that the hydrophilic region of Aß1-40 is involved in the interaction between the monomer peptide and the Gd3+ complex. Finally, ex vivo biodistribution experiments were conducted in healthy mice by using 111 In labeled analogues. Their pancreatic uptake, ∼3 %ID g-1 , is promising to envisage amylin imaging in diabetic animals.

Unexpected Trends in Copper Removal from Aß Peptide: When Less Ligand Is Better and Zn Helps.

Cu, Zn, and amyloid-ß (Aß) peptides play an important role in the etiology of Alzheimer's disease (AD). Their interaction indeed modifies the self-assembly propensity of the peptide that is at the origin of the deposition of insoluble peptide aggregates in the amyloid plaque, a hallmark found in AD brains. Another even more important fallout of the Cu binding to Aß peptide is the formation of reactive oxygen species (ROS) that contributes to the overall oxidative stress detected in the disease and is due to the redox ability of the Cu ions. Many therapeutic approaches are currently developed to aid fighting against AD, one of them targeting the redox-active Cu ions. Along this research line, we report in the present article the use of a phenanthroline-based peptide-like ligand (L), which is able to withdraw Cu from Aß and redox-silence it in a very stable 4N Cu(II) binding site even in the presence of Zn(II). In addition and in contrast to what is usually observed, the presence of excess of L lessens the searched effect of ROS production prevention, but it is counterbalanced by the co-presence of Zn(II). To explain such unprecedented trends, we proposed a mechanism that involves the redox reaction between Cu(II)L and Cu(I)L2. We thus illustrated (i) how speciation and redox chemistry can weaken the effect of a ligand that would have appeared perfectly suitable if only tested in a 1:1 ratio and on CuAß and (ii) how Zn overcomes the undesired lessening of ROS arrest due to excess of ligand. In brief, we have shown how working in biologically relevant conditions is important for the understanding of all of the reactions at play and this must be taken into consideration for the further rational design of ligands aiming to become drug candidates.

Measurement of Interpeptidic CuII Exchange Rate Constants of CuII-Amyloid-ß Complexes to Small Peptide Motifs by Tryptophan Fluorescence Quenching.

The interpeptidic CuII exchange rate constants were measured for two Cu amyloid-ß complexes, Cu(Aß1-16) and Cu(Aß1-28), to fluorescent peptides GHW and DAHW using a quantitative tryptophan fluorescence quenching methodology. The second-order rate constants were determined at three pH values (6.8, 7.4, and 8.7) important to the two Cu(Aß) coordination complexes, components Cu(Aß)I and Cu(Aß)II. The interpeptidic CuII exchange rate constant is approximately 104 M-1 s-1 but varies in magnitude depending on many variables. These include pH, length of the Aß peptide, location of the anchoring histidine ligand in the fluorescent peptide, number of amide deprotonations required in the tryptophan peptide to coordinate CuII, and interconversion between Cu(Aß)I and Cu(Aß)II. We also present EPR data probing the CuII exchange between peptides and the formation of ternary species between Cu(Aß) and GHW. As the nonfluorescent GHK and DAHK peptides are important motifs found in the blood and serum, their ability to sequester CuII ions from Cu(Aß) complexes may be relevant for the metal homeostasis and its implication in Alzheimer's disease. Thus, their kinetic CuII interpeptidic exchange rate constants are important chemical rate constants that can help elucidate the complex CuII trafficking puzzle in the synaptic cleft.

Hybrid Bis-Histidine Phenanthroline-Based Ligands to Lessen Aß-Bound Cu ROS Production: An Illustration of Cu(I) Significance.

We here report the synthesis of three new hybrid ligands built around the phenanthroline scaffold and encompassing two histidine-like moieties: phenHH, phenHGH and H'phenH', where H correspond to histidine and H' to histamine. These ligands were designed to capture Cu(I/II) from the amyloid-ß peptide and to prevent the formation of reactive oxygen species produced by amyloid-ß bound copper in presence of physiological reductant (e.g., ascorbate) and dioxygen. The amyloid-ß peptide is a well-known key player in Alzheimer's disease, a debilitating and devasting neurological disorder the mankind has to fight against. The Cu-Aß complex does participate in the oxidative stress observed in the disease, due to the redox ability of the Cu(I/II) ions. The complete characterization of the copper complexes made with phenHH, phenHGH and H'phenH' is reported, along with the ability of ligands to remove Cu from Aß, and to prevent the formation of reactive oxygen species catalyzed by Cu and Cu-Aß, including in presence of zinc, the second metal ions important in the etiology of Alzheimer's disease. The importance of the reduced state of copper, Cu(I), in the prevention and arrest of ROS is mechanistically described with the help of cyclic voltammetry experiments.

A Water-Soluble Peptoid Chelator that Can Remove Cu2+ from Amyloid-ß Peptides and Stop the Formation of Reactive Oxygen Species Associated with Alzheimer's Disease.

Cu bound to amyloid-ß (Aß) peptides can act as a catalyst for the formation of reactive oxygen species (ROS), leading to neuropathologic degradation associated with Alzheimer's disease (AD). An excellent therapeutic approach is to use a chelator that can selectively remove Cu from Cu-Aß. This chelator should compete with Zn2+ ions (Zn) that are present in the synaptic cleft while forming a nontoxic Cu complex. Herein we describe P3, a water-soluble peptidomimetic chelator that selectively removes Cu2+ from Cu-Aß in the presence of Zn and prevent the formation of ROS even in a reductive environment. We demonstrate, based on extensive spectroscopic analysis, that although P3 extracts Zn from Cu,Zn-Aß faster than it removes Cu, the formed Zn complexes are kinetic products that further dissociate, while CuP3 is formed as an exclusive stable thermodynamic product. Our unique findings, combined with the bioavailability of peptoids, make P3 an excellent drug candidate in the context of AD.

The aroylhydrazone INHHQ prevents memory impairment induced by Alzheimer's-linked amyloid-ß oligomers in mice.

Converging evidence indicates that neurotoxicity and memory impairment in Alzheimer's disease is induced by brain accumulation of soluble amyloid-ß oligomers (AßOs). Physiological metals are poorly distributed and concentrated in the senile plaques typical of Alzheimer's disease, where they may be coordinated to the amyloid-ß peptide (Aß). Indeed, zinc and copper increase Aß oligomerization and toxicity. Metal-protein attenuating compounds represent a class of agents proposed for Alzheimer's disease treatment, as they reduce abnormal interactions of metal ions with Aß, inhibit Aß oligomerization and prevent deleterious redox reactions in the brain. The present work investigates the protective action of an isoniazid-derived aroylhydrazone, INHHQ, on AßO-induced memory impairment. Systemic administration of a single dose of INHHQ (1 mg/kg) prevented both short-term and long-term memory impairment caused by AßOs in mice. In-vitro studies showed that INHHQ prevents Cu(Aß)-catalyzed production of reactive oxygen species. Although the mechanism of protection by INHHQ is not yet fully understood at a molecular level, the results reported herein certainly point to the value of aroylhydrazones as promising neuroprotective agents in Alzheimer's disease and related disorders.

Copper-Targeting Approaches in Alzheimer's Disease: How To Improve the Fallouts Obtained from in Vitro Studies.

According to the amyloid cascade hypothesis, metal ions, mainly Cu and Zn ions, bound to the amyloid-ß (Aß) peptides are implicated in Alzheimer's disease (AD), a widespread neurodegenerative disease. They indeed impact the aggregation pathways of Aß and are involved in the catalytic generation of reactive oxygen species (ROS) that participate in oxidative stress, while Aß aggregation and oxidative stress are regarded as two key events in AD etiology. Cu ions due to their redox ability have been considered to be the main potential therapeutic targets in AD. A considerable number of ligands have been developed in order to modulate the toxicity associated with Cu in this context, via disruption of the Aß-Cu interaction. Among them, small synthetic ligands and small peptide scaffolds have been designed and studied for their ability to remove Cu from Aß. Some of those ligands are able to prevent Cu(Aß)-induced ROS production and can modify the aggregation pathways of Aß in vitro and in cellulo. Examples of such ligands are gathered in this Viewpoint, as a function of their structures and discussed with respect to their properties against Cu(Aß) deleterious fallouts. Nevertheless, the beneficial activities of the most promising ligands detected in vitro and in cellulo have not been transposed to human yet. Some parameters that might explain this apparent contradiction and key concepts to consider for the design of "more" efficient ligands are thus reported and discussed. En passant, this Viewpoint sheds light on the difficulties in comparing the results from one study to another that hamper significant advances in the field.

Cu and Zn coordination to amyloid peptides: From fascinating chemistry to debated pathological relevance.

Several diseases share misfolding of different peptides and proteins as a key feature for their development. This is the case of important neurodegenerative diseases such as Alzheimer's and Parkinson's diseases and type II diabetes mellitus. Even more, metal ions such as copper and zinc might play an important role upon interaction with amyloidogenic peptides and proteins, which could impact their aggregation and toxicity abilities. In this review, the different coordination modes proposed for copper and zinc with amyloid-ß, α-synuclein and IAPP will be reviewed as well as their impact on the aggregation, and ROS production in the case of copper. In addition, a special focus will be given to the mutations that affect metal binding and lead to familial cases of the diseases. Different modifications of the peptides that have been observed in vivo and could be relevant for the coordination of metal ions are also described.

Ascorbate Oxidation by Cu(Amyloid-ß) Complexes: Determination of the Intrinsic Rate as a Function of Alterations in the Peptide Sequence Revealing Key Residues for Reactive Oxygen Species Production.

Along with aggregation of the amyloid-ß (Aß) peptide and subsequent deposit of amyloid plaques, oxidative stress is an important feature in Alzheimer's disease. Cu bound to Aß is able to produce reactive oxygen species (ROS) by the successive reductions of molecular dioxygen, and the ROS produced contribute to oxidative stress. In vitro, ascorbate consumption parallels ROS production, where ascorbate is the reductant that fuels the reactions. Because the affinity of Cu for Aß is moderate compared to other biomolecules, the rate of ascorbate consumption is a combination of two contributions. The first one is due to peptide-unbound Cu and the second one to peptide-bound Cu complexes. In the present Article, we aim to determine the amounts of the second contribution in the global ascorbate consumption process. It is defined as the intrinsic rate of ascorbate oxidation, which mathematically corresponds to the rate at an infinite peptide to Cu ratio, i.e., without any contribution from peptide-unbound Cu. We show that, for the wild-type Cu(Aß) complex, this value equals 10% of the value obtained for peptide-unbound Cu and that this value is strongly dependent on peptide alterations. By examination of the dependence of the intrinsic rate of ascorbate oxidation, followed by UV-vis spectroscopy, for several altered peptides, we determine some of the key residues that influence ROS production.

Kinetics Are Crucial When Targeting Copper Ions to Fight Alzheimer's Disease: An Illustration with Azamacrocyclic Ligands.

Targeting copper ions to either remove or redistribute them is currently viewed as a possible therapeutic strategy in the context of Alzheimer's disease (AD). Thermodynamic parameters, as for instance the copper(II) affinity of the drug candidate or the copper(II) over zinc(II) selectivity, are considered in the design of the drug candidate. In contrast, kinetic factors have been overlooked despite their probable high importance. In the present article, we use a series of azamacrocyclic ligands to demonstrate that kinetic issues must be taken into account when designing copper-targeting drug candidates in the context of AD.

N4 -Tetradentate Chelators Efficiently Regulate Copper Homeostasis and Prevent ROS Production Induced by Copper-Amyloid-ß1-16.

The disruption of copper homeostasis and the oxidative stress induced by Cu-amyloids are crucial features of Alzheimer's disease pathology. The copper specific N4 -tetradendate ligands TDMQ20 and 1 are able to fully inhibit in vitro the aerobic oxidation of ascorbate induced by Cu-Aß1-16 , even in the presence of 100 molar equivalents of ZnII with respect to CuII , whereas other ligands with N2 O2 or N3 O2 coordination spheres failed to do so. This essential result indicates that, in addition to metal selectivity, the coordination sphere of copper chelators should exhibit a N4 -tetradendate motif to be able to reduce an oxidative stress in the zinc-rich physiological environment of brain. The N4 -scaffolds of these two aminoquinoline-based ligands, TDMQ20 or 1, suitable for a square-planar coordination of copper(II), allowed them to enhance both the selectivity for copper and the ability to reduce the oxidative stress induced by copper-amyloid in a zinc-rich environment.

A Metallo Pro-Drug to Target CuII in the Context of Alzheimer's Disease.

Alzheimer's disease and oxidative stress are connected. In the present communication, we report the use of a MnII -based superoxide dismutase (SOD) mimic ([MnII (L)]+ , 1+ ) as a pro-drug candidate to target CuII -associated events, namely, CuII -induced formation of reactive oxygen species (ROS) and modulation of the amyloid-ß (Aß) peptide aggregation. Complex 1+ is able to remove CuII from Aß, stop ROS and prevent alteration of Aß aggregation as would do the corresponding free ligand LH. Using 1+ instead of LH in further biological applications would have the double advantage to avoid the cell toxicity of LH and to benefit from its proved SOD-like activity.

N-Terminal Cu-Binding Motifs (Xxx-Zzz-His, Xxx-His) and Their Derivatives: Chemistry, Biology and Medicinal Applications.

Peptides and proteins with N-terminal amino acid sequences NH2 -Xxx-His (XH) and NH2 -Xxx-Zzz-His (XZH) form well-established high-affinity CuII -complexes. Key examples are Asp-Ala-His (in serum albumin) and Gly-His-Lys, the wound healing factor. This opens a straightforward way to add a high-affinity CuII -binding site to almost any peptide or protein, by chemical or recombinant approaches. Thus, these motifs, NH2 -Xxx-Zzz-His in particular, have been used to equip peptides and proteins with a multitude of functions based on the redox activity of Cu, including nuclease, protease, glycosidase, or oxygen activation properties, useful in anticancer or antimicrobial drugs. More recent research suggests novel biological functions, mainly based on the redox inertness of CuII in XZH, like PET imaging (with 64 Cu), chelation therapies (for instance in Alzheimer's disease and other types of neurodegeneration), antioxidant units, Cu transporters and activation of biological functions by strong CuII binding. This Review gives an overview of the chemical properties of Cu-XH and -XZH motifs and discusses the pros and cons of the vastly different biological applications, and how they could be improved depending on the application.