Viewpoint

Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX pubs.acs.org/IC

Copper-Targeting Approaches in Alzheimer’s Disease: How To

Improve the Fallouts Obtained from in Vitro Studies
Charleǹ e Esmieu,†,‡ Djamila Guettas,†,‡ Amandine Conte-Daban,†,§ Laurent Sabater,† Peter Faller,†,⊥
and Christelle Hureau*,†
†
LCC−CNRS, Université de Toulouse, CNRS, Toulouse, France

ABSTRACT: According to the amyloid cascade hypothesis, metal

ions, mainly Cu and Zn ions, bound to the amyloid-β (Aβ) peptides
are implicated in Alzheimer’s disease (AD), a widespread neuro-
degenerative disease. They indeed impact the aggregation pathways of
Aβ and are involved in the catalytic generation of reactive oxygen
species (ROS) that participate in oxidative stress, while Aβ
aggregation and oxidative stress are regarded as two key events in
AD etiology. Cu ions due to their redox ability have been considered
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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 Aβ−
Cu interaction. Among them, small synthetic ligands and small
peptide scaffolds have been designed and studied for their ability to
remove Cu from Aβ. Some of those ligands are able to prevent Cu(Aβ)-induced ROS production and can modify the
aggregation pathways of Aβ 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(Aβ) 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.

1. GENERAL CONSIDERATIONS of various lengths are obtained, Aβ1−40 and Aβ1−42 are still
1.1. Alzheimer’s Disease (AD): A Brief Introduction. regarded as the most abundant and their binding to metal ions
the most investigated.5,9,10,15,16 The mechanism of formation
Dementias are frequent and serious neurological disorders that
of the amyloid plaques and its role in the pathology of AD are
represent the main mental pathologies of the elderly. The
controversial topics. However, the identification of Aβ in senile
dementia syndrome is characterized by a gradual deterioration
plaques and mutations in the APP, presenilin 1 and presenilin
of cognitive functions, especially memory, with a significant
2 genes, leading to the accumulation of Aβ and early onset
impact on the personality, activities of daily life, social
familial dementia,11,17,18 strengthened the proposition of the
functioning, and patient autonomy. AD is the most common
“amyloid cascade hypothesis” first formulated in 1992.19
form of dementia with a prevalence of around 47.5 million According to this hypothesis, the deposition of Aβ aggregates
patients worldwide.1 This number is expected to triple within is the initial pathological trigger in the disease, which
the next 35 years, and therefore it represents a major public subsequently leads to the formation of neurofibrillary tau
health issue.1,2 Two morphological hallmarks are observed in tangles, neuronal cell death, and later on, dementia.11,17,19,20
AD.3−6 Both are abnormal accumulations of fibrils in different Several Aβ aggregates have been identified that include dimers,
locations: (i) intracellular deposition of paired helical filaments oligomers, protofibrils, fibrils, and amorphous aggregates.3,21
of the hyperphosphorylated tau protein in neurofibrillary Their size varies from dimer to larger architectures of about 10
tangles, which also occurs in other neurodegenerative diseases nm in diameter, while oligomers (i.e., small soluble assemblies
such as Parkinson’s disease; (ii) extracellular amyloid plaques made of a few peptides) are considered more toxic than high-
(also known as senile plaques) made of amyloid-β (Aβ) molecular-weight aggregates.22,23 Note that here, for simplicity,
peptides under aggregated structures. Hence, targeting Aβ and high-molecular-weight assemblies (amorphous and/or fibrillar)
tau accumulation is crucial for the diagnosis, treatment, and are grouped under the term aggregates. The neurotoxicity of
prevention of AD.7,8 In this Viewpoint, we will focus on Aβ
because of its interaction with metal ions, which has been
Special Issue: Metals in Biology: From Metallomics to Trafficking
intensively studied in the last decades.5,9,10 The Aβ peptides
are produced from the amyloid precursor protein (APP) by β- Received: April 5, 2019
and γ-secretases (Figure 1).11−14 Although several Aβ isoforms

© XXXX American Chemical Society A DOI: 10.1021/acs.inorgchem.9b00995

Inorg. Chem. XXXX, XXX, XXX−XXX
Inorganic Chemistry Viewpoint

Figure 1. Simplified view of the formation of amyloid plaques. A change in color from yellow to blue corresponds to a change in the folding of the
peptide upon metal-ion binding. Cofactors = Cu and Zn. β and γ represent the β- and γ-secretases.

Aβ aggregates is linked to various interconnected mechanisms synaptic cleft can reach micromolar levels37−39 making Cu and
such as (i) oxidative stress,24,25 (ii) hyperphosphorylation of Zn ions available for coordination to Aβ.
the tau protein, leading to its inability to bind to microtubules, 1.2.a. Coordination of Metal Ions to Aβ. In vitro, CuI/II,
to the formation of neurofibrillary tau tangles and further to ZnII, and FeII showed direct coordination to Aβ,9,16,40−44 while
enhancement of the production of the Aβ peptide,26,27 (iii) FeII bound to Aβ does oxidize rapidly and finally converts into
synaptic dysfunction due to the accumulation of soluble forms insoluble FeIII(OH)3, making the in vivo interaction between
of the Aβ peptides within the neurons,18,25 and (iv) the Fe and Aβ unlikely.45,46 The high-affinity metal-ion binding
insertion of the Aβ peptides into cell membranes with the site of Aβ lies within residues 1−16, a sequence regarded as the
formation of ion channels inducing the dyshomeostasis of appropriate model to investigate the Aβ coordination ability in
Ca2+.14,22 Besides these AD-specific features, considerable vitro.16 CuII binding sites to Aβ peptides have been extensively
evidence indicates the presence of other hallmarks that are studied and reviewed (see refs 40 and 47). At neutral pH, Aβ
common for multiple neurodegenerative diseases; these coordinates Cu in its +I and +II redox forms with very distinct
include prominent activation of inflammatory and innate environments,16 with a conditional affinity constant at pH 7.4
immune responses as well as excessive iron deposition and of 1010 M−1 for CuII and from 107 to 1010 M−1 for CuI.43 The
mitochondrial damage.28 ZnII ion is tetrahedrally bound to Aβ,9 with a conditional
The interconnection between the various dysfunctions or affinity constant of about 105 −106 M.
malfunctions is an obstacle to an unquestionable attribution of 1.2.b. Metal Ions−Aβ and Oxidative Stress. It is now
the original cause(s) of AD. Today, the accumulation of Aβ accepted that reactive oxygen species (ROS) such as O2•−,
peptide is no longer regarded as the only direct cause of the H2O2, and HO•, overgenerated under oxidative stress
disease but rather as a key event, which is linked to a complex conditions, play a key role in neurodegenerative diseases,24,48
and branched cascade of several imbalances that favor the while oxidative stress is one of the pathological hallmarks of
neuronal death. While the APP is expressed ubiquitously, the AD, although it remains uncertain whether this is a cause or a
amyloid deposits formed in AD are localized in very specific consequence of pathogenic processes occurring in the brain.49
areas.4 It could thus be hypothesized that, in addition to the A relationship between ROS and Aβ toxicity exists, mediated
accumulation of Aβ (related to genetic causes or age), other by redox-active metal ions bound to Aβ that produce
factors may be involved in the formation of amyloid deposits. ROS.24,48,50 Indeed, in the presence of biological reductants
1.2. Metal Ions in AD. Among all of the biological factors such as ascorbate or glutathione, ROS can be generated by the
linked to the etiology of AD, metal ions held an essential place redox cycling of CuI/II, which then contributes to neuronal
because they are ill-regulated in AD brains.10,29−32 The total oxidative damage. The involvement of Fe(Aβ) in ROS
metal amount (ZnII, CuI/II, and FeII ions) detected in frozen production is still unclear; thus, ROS production has been
brain tissues (lobes, amygdala, hippocampus, etc.) in AD predominantly studied with Cu(Aβ). The in vitro techniques
patients varies from healthy controls, indicating of a global used to investigate ROS production rely on UV−vis
misregulation of metal ions in the AD patient. Depending on monitoring of the consumption of ascorbate and on indirect
the nature of the tissue, the levels are either up-regulated or methods for detection of the ROS produced.51 They mainly
down-regulated.33 It has also been reported that the senile include the reduction of ferricytochrome C to ferrocytochrome
plaques are enriched in metal ions (about millimolar) in AD by O2•− monitored by UV−vis, the oxidation of Amplex Red
patients,34,35 and several in vitro experiments documented that by H2O2, leading to a fluorescent species, thiobarbituric acid
metal ions can modulate Aβ aggregation (vide infra). The reactive substances assay relying on the reaction of 2-
importance of those results with respect to the proposed deoxyribose with HO• and detection by UV−vis of the
impact of metal ions in the amyloid cascade would, however, resulting highly colored dye, and hydroxylation of coumarin-3-
deserve further investigations by other methods.36 In addition, carboxylic acid (CCA) by HO•, leading to a fluorescent
concentrations of the labile pool of Cu and Zn ions in the derivative (Figure 2a).51
B DOI: 10.1021/acs.inorgchem.9b00995
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Inorganic Chemistry Viewpoint

Figure 2. Schematic representation of the most common techniques used to study Cu(Aβ) ROS production, Aβ aggregation, and their cytotoxicity:
(a) evaluation of the catalytic production of ROS by Cu(Aβ) in the presence of ascorbate and O2 and examples of detection techniques; (b)
detection of Aβ aggregation and examples of detection techniques; (c) evaluation of the Cu(Aβ) toxicity on cell cultures and evaluation of the
neuroprotective effect of drug candidates (ligands 1 and 2) using MTT assay.

1.2.c. Aβ Aggregation. The impact of Cu and Zn ions on culture protocols are well documented in the literature.60
the aggregation pathways has been widely studied in vitro Cellular models composed of primary neuronal cultures
under different conditions including pH, temperature, (cortical neuronal cells) or 2D neuron-like cell lines (e.g.,
concentration, nature of the metal ion, and metal ion over PC12, SH-SY5Y, and M17) are commonly used to study
peptide ratio, and important effects were reported.10,42,52−54 neuron cell death (induced by external stimuli) and to test the
Metal ions can modify the thermodynamics and kinetics of neuroprotective properties of specific compounds.61−63
aggregation as well as the morphology of the final aggregates Recently, new cellular platforms have emerged via the
formed. The influence of CuII on the Aβ aggregation seems to development of induced pluripotent stem cells (iPSCs).64
be mainly dependent on its ratio and might lead to the iPSCs offer the possibility of generating neuronal cultures from
production of highly cytotoxic oligomers.42 Regarding ZnII, blood and fibroblasts. The cell viability has been measured
once coordinated to Aβ, it has been mainly reported to using colorimetric tests, with the gold standard being the
promote fast aggregation of species different from apofibrils.52 classical 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
Peptide aggregation can be monitored by different techniques. bromide (MTT) assay relying on reduction of the MTT to
A widely used method to detect fibrils is fluorescence deep-purple formazan dye (Figure 2c).
enhancement of the Thioflavin-T (ThT) dye upon binding 1.3. Therapeutic Approaches to Fighting AD. Two
to the β sheets present in amyloid structures.55,56 Exper- main therapeutic approaches are currently considered in the
imentally, ThT fluorescence reports on the fibrillation kinetic
AD context depending on whether the symptoms or causes of
that corresponds to a sigmoidal-like curve divided into three
the disease are targeted.48,65−67 Disease-modifying approaches
parts: the first one is a lag phase, during which the nuclei are
that aim at affecting the pathways responsible for neuro-
formed. Then, a second phase corresponds to elongation of the
nucleus into protofibrils and fibrils. Finally, the plateau of the degeneration (i.e., the causes of the disease) include (i)
sigmoid reflects a thermodynamic equilibrium between fibrils reduction of the production of Aβ by inhibition of the β- and
and soluble species (Figure 2b). Other techniques have been γ-secretases, (ii) the increase of Aβ elimination, including by
used to monitor the formation of Aβ aggregates22 as active or passive immunotherapy, with the basis for these two
turbidimetry (a very simple but weakly insightful method first approaches being to counteract the deleterious accumu-
relying on the increase of absorbance near 400 nm), dynamic lation of the Aβ peptide, (iii) the targeting of metal ions in
light scattering, and gel electrophoresis/Western blotting57,58 order to affect both aggregation and oxidative stress; this is the
or to identify their morphology by transmission electronic topic of the present Viewpoint. The detection of a high level of
microscopy (TEM) and atomic force microscopy (AFM) metal ions in the brain areas affected by AD and the
(Figure 2b).58,59 observation of the pathogenic consequences of the metal
1.2.d. Toxicity Studies on Cells. In addition to the ions−Aβ interactions (ROS, aggregation, etc.) were at the
aforementioned methods that measure ROS production and origin of therapeutic strategies aimed at preventing metal ions
monitor Aβ aggregation, the toxicity associated with these two from binding to Aβ. Here, Cu is under focus. Indeed, because
events can be evaluated in cells. Samples of Aβ and Cu(Aβ) of its redox ability, it participates in the production of ROS24
have been measured toward their neurotoxicity, while cell and it modulates aggregation of the Aβ peptide, possibly
C DOI: 10.1021/acs.inorgchem.9b00995
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Inorganic Chemistry Viewpoint

Figure 3. Schematic representation of the action of a chelator in the synaptic cleft (a) versus the action of a metallophore (b).

stabilizing oligomers,42 regarded as the most toxic species of ions. The selectivity is the ability of the ligand to bind the
the aggregation process.22,68 targeted metal ion without strong interference from other
metal ions. More precisely, the selectivity of a ligand for one
2. METAL TARGETING THERAPY metal ion versus another one is defined as the ratio of the
affinity values of the ligand for the two metal ions. With regard
2.1. Definitions: Chelator and Metallophore. Metal- to the targeted Cu ions under focus here, the ligand has to
targeting therapy refers to a medical treatment that reduces the compete with Aβ (if we assume that the misplaced Cu is
toxic effects of metal ions by their removal or redistribution: mainly bound to the Aβ peptide). Thus, the affinity of the
(i) chelators have the intent of disrupting the deleterious ligand for Cu ions has to be higher that of Aβ (about 1010 M−1
interaction of metal ions with biomolecules, thus inactivating at pH 7.4 for Aβ1−40/42 in the case of CuII).75 In addition, the
them or helping their excretion (Figure 3a), and (ii) ligand has to possess a Cu over Zn selectivity higher that of Aβ
metallophores that have the additional ability to transfer (which equals about 104 in the case of CuII), and because Zn is
(across a membrane) the removed metal ions to the biological more concentrated than Cu in the synaptic cleft, the intended
compartment, where they become beneficial.69,70 In AD, it selectivity should be largely higher than 104.52
consists of redistributing the metal ions to regulate their In addition to the conditions described above, for neuro-
homeostasis, reordering the metal-ion traffic between extrac- degenerative diseases, the ability of the ligand to cross the
ellular and intracellular compartments (Figure 3b).30,43,71−73 blood−brain barrier (BBB) is a main factor to take into
Here, for a matter of simplicity, the term “ligands” is used for account.76−79 Apart from respecting adapted Lipinski’s rules
molecules capable of binding metal ions, with no precision on for passive transport through the BBB,80,81 other approaches
their releasing ability [i.e., a ligand can be either a chelator are currently developed, mainly relying on active transport.82,83
(binding only) or a metallophore (binding and redistrib- 2.3. Ligands of Cu Ions. Among the ligands developed in
ution)]. the AD chelatotherapy context (for a very recent review, see ref
2.2. Chemical and Biological Principles for in Vivo 48), some are monofunctional, aimed at targeting metal ions
Metal Targeting. The essential properties of a ligand based and associated deleterious processes, and others are multi-
on chemical and biomedical considerations have been functional, impacting several pathological features. In addition
progressively defined along with advances of research. The to the binding cavity, multifunctional ligands incorporate
metabolic stability of the ligands and resulting complexes is a moieties to scavenge radicals, to recognize Aβ aggregates, and
key parameter to fulfill: it has to be high enough to withstand to help BBB penetration. Proligands are ligands whose binding
the variety of physiological conditions, mainly the pH. Other ability is obtained with an appropriate stimulus.
required properties may be combined as an appropriate In the following, monofunctional ligands are under focus.
absorption, distribution, metabolism, and excretion profile.74 They have been grouped into three categories: (i) synthetic
Other thermodynamic properties have to be considered. The ligands, which regroup hydroxy/aminoquinolines (Figure 4
affinity of a ligand is the strength with which it binds the metal and Table 1), tetraazamacrocyles, aminophenol- and amino-
D DOI: 10.1021/acs.inorgchem.9b00995
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Inorganic Chemistry Viewpoint

Figure 4. Structures of hydroxy- and aminoquinoline ligands and derivatives.

Table 1. Hydroxy- and Aminoquinolines Reported from the Literature as Well as Their Impact on the ROS Production and
Scavenging, Prevention of Aggregation, Aggregates Disassembly, and Associated Recovery of Cells Survival
ROS Aggregation
pCua arrest of production scavenging prevention disaggregation cell toxicity refs
Cu(Aβ) 7.3−7.8b 75, 115
L1 6.6 √ × √ × × 85, 88, 89, 92, 93, 116, 117
L2 n.r. n.r. n.r. √ √ √ 108, 118
L3 7.89b √ × n.r. n.r. n.r. 104, 105
L4 10.45c √ n.r. √ n.r. n.r. 107
L5 and L6 n.r. n.r. √ √ n.r n.r. 108
L7 n.r. n.r. √ n.r. n.r. n.r. 108
L8 10.75c √ n.r. n.r. n.r. n.r. 109, 112
L9 10.75b √ n.r. n.r. n.r. n.r. 113, 114
a
pCu = −log[Cu]unbound; [Cu] = [L] = 10 μM. bFrom the conditional affinity value. cFrom the apparent affinity value, the pH is 7.4.

pyridine-based ligands, phosphine, and bis- 2.3.a. Synthetic Ligands. 2.3.a.1. Hydroxy- and Amino-
(thiosemicarbazonato) (Figure 5 and Table 2); (ii) peptidic quinoline Ligands. The quinoline motif is widely used in
ligands (Figure 6 and Table 3); (iii) ligands possessing an Aβ- therapeutic chemistry. Quinoline derivatives substituted at the
targeting moiety in which the binding unit is embedded 8 position with a heteroatom [such as the 8-hydroxyquinoline
(Figure 7 and Table 4). Their structures are shown in Figures (8-HQ) or 8-aminoquinoline derivatives] can be used as
4−7, while their characteristics and the studies carried out in ligands.85 The hydroxyquinoline ligands and their amino-
the AD context are described in the following paragraphs and counterparts developed on target metal ions in AD are
their main properties gathered in Tables 1−4. Note that, in described in this first paragraph (Figure 4 and Table 1).
Tables 1−4, the affinity of the ligand is reported as the pCu 2.3.a.1.1. Hydroxyquinoline. Among all of the hydrox-
parameter, which corresponds to the concentration of yquinoline derivatives, 8-HQ is the most interesting one
unbound Cu at a given pH (7.1 or 7.4) using the HySS because of its bioactivities and therapeutic potentials. It has
program84 when appropriate, and the results of the experi- been shown that substitution with a chlorine group at the C5
ments carried out in the literature are reported as follows: √ position or both the C5 and C7 positions of 8-HQ increases
the lipid solubility and the donor effect on the phenolic group,
means a “beneficial effect”, ∼ means “intermediate beneficial
which improves metal-ion binding. L1 [iodochlorhydroxyquin,
effect”, × means “no effect”, and “n.r.” means “not reported”,
clioquinol (CQ), also named PBT1 for Prana BioTechnology
with several processes reviewed: (i) arrest of Cu-induced ROS 1) and L2 (5,7-dichloro-2-[(dimethylamino)methyl]quinolin-
production, (ii) ROS scavenging by the ligand itself, (iii) 8-ol, PBT2) exert antineurodegenerative effects in transgenic
inhibition of Cu-induced aggregation, (iv) dissassembly of model mice (Tg2576 and APP/PS1).85−88 CuII/Zn binding to
Cu(Aβ) aggregates, and (v) recovery of cell survival for cells L1 was studied in solution89,90 and in the solid state.91 The
confronted with toxicity associated with Cu(Aβ) aggregates. effects of L1 on metal-induced Aβ aggregation92−94 and on its
Note that, in the following text and tables, unless otherwise ability to cross the BBB95 were reported. L1 can partially
specified, ROS production and aggregation under focus are dissolve amyloid plaques from human post-mortem brains96
CuII-induced. and induces a significant inhibition by almost 50% of Aβ
In the present Viewpoint, beyond describing the reported accumulation in APP Tg2576 transgenic mice.95 A pilot phase
results on CuII targeting strategies, our main aim is to gain II study of orally dosed L1 reported that it improved the
deeper insight into the interaction between Cu, Aβ, and ligands cognition and behavior of AD patients,97 but side effects such
to guide the design and investigations of the next generations as mutagenicity and neurotoxicity were observed,98 which led
of ligands, and that is why we examine and discuss several key to the arrest of L1 clinical development. Therefore, a new
parameters and criteria. dichloro-8-hydroxyquinoline derivative, the tridentate L2
E DOI: 10.1021/acs.inorgchem.9b00995
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Inorganic Chemistry Viewpoint

Figure 5. Structures of selected ligands: tetraazamacrocycles (a), aminophenol-based ligands (b), aminopyridine-based ligands (c), phosphine (d)
and bis(thiosemicarbazonato) ligands (e).

(PBT2) ligand, was developed. It does not show severe side 8-HQ scaffold, leading to L5−L7, respectively.108 Their
effects and has successfully completed phase IIa trials in antioxidant capacity (i.e., the ability to scavenge ROS) are
patients with early AD,99,100 but L2 did not reach statistically enhanced compared to the parent L1 and L2 ligands, a feature
significant reduction in amyloid plaque levels in the brains of that is linked to the increased stabilization of the resulting
patients with AD.85 The proposed mechanism of action relies phenoxyl radical due to the absence of halogenated substituent
on a metallophore activity, meaning that the compounds are on the quinoline ring and the extended conjugation provided
able to lessen the level of CuII bound to Aβ, in combination by substitution with the Schiff base. Regarding aggregation, L5
with the weakening of ROS production, resolubilization of and L6 are described as inhibitors of the formation of CuII-
amyloid plaques, and promotion of metal-ion uptake by induced oligomers, while L6 has a greater effect compared to
cells.72,101,102 While CuII coordination to L2 has been L5, which may be attributed to its higher hydrophilicity and/or
described,103 the efficiency of CuII removal from Aβ has increased hydrogen-bonding interaction of the carboxamide
been mostly investigated in vitro for the L3 analogue.104,105 It function with the β-strand of the Aβ peptides.108
has been shown that L3 can extract CuII from Aβ but also 2.3.a.1.3. Aminoquinoline Derivatives. In order to improve
forms ternary species L3[CuII]Aβ, where Aβ binds Cu via one the CuII affinity and the CuII over ZnII selectivity, bis-
of its His residues. This results in a decreased level of ROS (aminoquinoline) 71,109−112 and aminoquinoline deriva-
production. However, one may consider that in vivo the Aβ tives113,114 affording a 4N tetradentate binding unit have
His will be replaced by imidazole-containing molecules and recently been developed. In particular, L8 extracts CuII from
that L3[CuII](Im) does produce ROS to a significant Aβ peptide and can redistribute it to physiological ligands
extent.104 under reductive conditions. In addition, L8 does inhibit ROS
2.3.a.1.2. Hydroxyquinoline Derivatives. Other structural production.109−111 Noticeably, after 3 weeks of treatment by
modifications have been made to improve L1−L3 efficiency. an oral route with a close analogue of L8, a memory deficit
They include the connection of two hydroxyquinoline ring mouse model induced by a single intracerebroventricular
moieties (L4) with various one-atom linkers, leading to a injection of Aβ peptide showed full recovery of the deficit of
tetradentate binding motif and thus forming a 1:1 (ligand/ episodic memory.112 Later on, in order to improve the BBB
metal) complex (compared to the possible formation of a 2:1 permeability, tetradentate monoquinoline ligands based on a 8-
complex for L1−L3). As expected, the bis(hydroxyquinoline) HQ moiety substituted at C2 by a more modular and low-
derivatives have a higher affinity for CuII and ZnII than the 8- weight bis-chelating side chain were designed.113 L9 (obtained
HQ parent compounds, but a moderate improvement in the with n = 2 and m = 2, shown in Figure 4) is the best ligand of
ability to prevent Aβ aggregation.106,107 Ligand L4 is shown as the series because it has the appropriate CuII affinity, CuII over
an illustration of the wide series of bis(hydroxyquinoline) ZnII selectivity, and the ability to fully inhibit in vitro ROS
ligands studied because it recapitulates all of the expected production.114
effects and the crystallographic structures of CuII and ZnII 2.3.a.2. Tetraazamacrocycles. Complexes of macrocyclic
complexes have been reported as well.107 Hydrazone and semi- ligands are widely used in medicinal chemistry for imaging
and thiosemicarbazone moieties have been incorporated into a (MRI contrast agents, radiopharmaceuticals, and luminescent
F DOI: 10.1021/acs.inorgchem.9b00995
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Inorganic Chemistry Viewpoint

Table 2. Other Ligands Reported from the Literature as Well as Their Impact on ROS Production and Scavenging, Prevention
of Aggregation, Aggregates Disassembly, and Associated Recovery of Cells Survival
ROS aggregation
a
pCu arrest of production scavenging prevention disaggregation cell toxicity refs
Cu(Aβ) 7.3−7.8c 75, 115
L10 11.5b,c × n.r. √ ∼ √ 61, 121, 122
L11 11.8b,c √ n.r. √ n.r. n.r. 121, 122
L12 11.2b,c √ n.r. √ n.r. n.r. 121, 122
L13 12.7b,c × n.r. √ ∼ √ 61, 121, 122
L14 n.r. ∼ n.r. √ √ (2 equiv) √ 62
L15 n.r. ∼ n.r. √ √ √ 62
L16 11.5b,c √ n.r. √ n.r. n.r. 121, 122
L17 10.0b,c √ n.r. √ n.r. n.r. 121, 122
L18 10.8c n.r. √ √ n.r. n.r. 124
L19 11.4c n.r. √ √ n.r. n.r. 124
L20 9.6c √ n.r. ∼ n.r. n.r. 125, 126
L21 6.6c n.r. × × × n.r. 127
L22 10.8c n.r. n.r. √ × n.r. 127
L23 5.4c,d √ n.r. √ n.r. n.r. 128
a
pCu = −log[Cu]unbound; [Cu] = [L] = 10 μM. bAt pH 7.1 (if not specified, then pH 7.4). cFrom the conditional affinity value. dRelative to CuI.

probes). Indeed, tetraazamacrocycle properties such as their aggregates imaged by TEM, showing that, in the presence of
high affinity for metal ions associated with kinetic inertness, the ligands, apo-type fibrils are recovered.
their high selectivity for CuII over ZnII, their modularity 2.3.a.3. Aminophenol Derivatives. L18 and L19 (H2GL1
(possible N- or C-functionalizations via the incorporation of and H2GL2) are considered to be multifunctional agents
pendant arms), and their low molecular weight prompted their because of the phenolic moieties that can act as ROS
use in medicine. 119,120 In the AD context, the two scavengers, while the attached carbohydrate moieties enhance
tetraazamacrocycles cyclen (L10) and cyclam (L13) have solubility, minimize toxicity, and improve BBB penetration
served as scaffolds for the construction of several substituted (Figure 5 and Table 2).123 At neutral pH, L18 and L19 possess
ligands (L11, L12, and L14−L17, described later on; see a moderate-to-high affinity for CuII (1015 M−1), while the
Figure 5 and Table 2). L10 and L13 are able to modulate affinity for ZnII ions is weak (106 M−1), leading to an
aggregation, thus improving neuronal cell survival but not fully appropriate CuII over ZnII selectivity. In vitro studies have
arresting ROS production.61 Two cyclam derivatives displaying shown an efficiency to scavenge free radicals and to decrease
lipophilic pyridine groups in order to enhance their antioxidant the formation of Aβ aggregates as measured by turbidim-
activity and help them to cross the BBB (L14 and L15) were etry.124 L20 is an analogue to L18 and L19, in which the
reported.62 They could modulate aggregation and lower H2O2 phenolato groups are sulfonated to improve the solubility and
production. In addition, L14 and L15 are able to attenuate ease in vitro studies.125 L20 showed significant potential to
CuII(Aβ)-induced toxicity by decreasing the intracellular ROS arrest ROS production even in the presence of ZnII and to
on cell lines. Although performed in the absence of an external restore the apo-like (in the absence of ZnII) or ZnII-induced-
type aggregation of the Aβ40 peptide.125,126
physiological reductant such as ascorbate as a trigger of the
2.3.a.4. Aminopyridine Ligands. The characterizations of
ROS production reaction, this ability has been attributed to the
CuII and ZnII complex formation with the aminopyridine
interruption of the Cu(Aβ) redox cycle by L14 and L15. L15
ligand L21 show that it cannot compete with Aβ for any of the
is more efficient than L14, in line with the antioxidant expected
metal ions. In order to form more stable complexes, L22, a
effect of additional pyridine rings. Other tetraazamacrocycles, ligand that connects two L21 moieties, was used (Figure 5 and
L11, L12, L16, and L17, functionalized with one or two Table 2).127 It forms a stable 1:1 (ligand/metal) complex at
methylpicolinate arms have been reported later on.121,122 All of neutral pH with both CuII and ZnII through tetradentate
those ligands are able to prevent and stop ROS production and coordination, with affinity values (1015 M−1 for CuII and 1010
to restore the formation of apo-like fibrils by removing CuII. M−1 for ZnII) largely higher than those of Aβ. The ZnII
Importantly, the arrest of ROS was challenged during ROS aggregation studies displayed that L22 is able to remove ZnII
production [not only using preincubation of the ligand with from Aβ aggregates and thus to solubilize them as probed by
CuII(Aβ) prior to triggering of the ROS production reaction]. Tyr10 fluorescence titration of the resulting soluble fraction.
In other words, experiments were conducted in the presence of 2.3.a.5. Phosphine Derivatives. The ligands discussed
both CuI and CuII, which may best mirror the physiological above specifically target CuII. Knowing that Cu is engaged in
environment in the synaptic cleft. For the first time, it has been Aβ coordination in both redox forms, a CuI chelator has also
evidenced that the kinetics of CuII removal from Aβ by the been investigated in AD. L23 (triaza-7-phosphaadamantane;
ligand has to be considered because the unsubstituted L10 and Figure 5 and Table 2) is the first CuI ligand able to retrieve
L13 ligands were inefficient in stopping ROS production while both CuI and CuII from the Aβ peptide. Indeed, L23 can
L11, L12, L16, and L17 were, because of the pendant arm(s) reduce CuII bound to Aβ and then coordinates the resulting
that foster(s) CuII grasping. In addition, in contrast to the CuI in oxidation-resistant complexes.128 The same effect was
previous two studies, the aggregation was kinetically monitored also observed in the presence of ZnII, thus showing that the
by ThT fluorescence and the morphology of the apo and Cu PTA ligand has an appropriate CuI versus Zn selectivity.129
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Figure 6. Structures of selected peptidic ligands.

Table 3. Peptidic Ligands Reported from the Literature as Well as Their Impact on the ROS Production and Scavenging,
Prevention of Aggregation, Aggregates Disassembly, and Associated Recovery of Cells Survival
ROS aggregation
pCua arrest of production scavenging prevention disaggregation cell toxicity refs
Cu(Aβ) 7.3−7.8b 75, 115
L26 7.2b √ n.r. √ n.r. n.r. 139
L27 6.7b √ n.r. √ n.r. n.r. 139
L28 n.r. √ n.r. √ n.r. n.r. 139
L29 n.r. √ (4 equiv) n.r. × n.r. √ (4 equiv)c 63
L30 12.7b n.r. n.r. √ n.r. n.r. 140
L31 14.1b n.r. n.r. √ n.r. n.r. 140
L32 8.8b √ n.r. n.r. n.r. n.r. 141
a
pCu = −log[Cu]unbound; [Cu] = [L] = 10 μM. bFrom the conditional affinity value. cHere not due to aggregation but ROS-induced toxicity
(measurement performed in the presence of ascorbate).

2.3.a.6. Bis(thiosemicarbazonato) Derivatives. CuII and reagents, and apparatuses by trained researchers, and they
ZnII complexes of bis(thiosemicarbazonato) ligands (L24 and can be functionalized. In addition, a simple strategy like the use
L25; Figure 5) can be transported across the BBB and can of the D analogues, cyclization, substitution, or branched
cross neuron membranes and release metal ions. Thus, they peptides can be applied to lessen recognition and further
have been studied in neuron-like cells and AD animal models degradation of the peptide by peptidases in vivo.135 Last, they
in order to evaluate the effects of intracellular increase of have been extensively studied for their ability to bind CuII ions
metal-ion levels.130,131 Positive effects with regard to soluble and, to a lesser extent, ZnII ions.136 In the following, the use of
Aβ levels and restoration of the cognitive performance in APP/ peptidic ligands with the ATCUN-like motif, His-containing
PS1 transgenic AD model mice were reported for both metal peptides, and peptidomimetic ligands is described in the
ions, while a fine-tuned redox mechanism was at play for the context of Cu targeting in AD (Figure 6 and Table 3).
CuII complexes: the intracellularly reduced CuII(L24) species 2.3.b.1. ATCUN-like Motif. The ATCUN (amino-terminal
was efficient but not the CuII(L25) analogue, which is resistant Cu and Ni binding) motif is characterized by the H2N-Xxx-
to intracellular reduction and subsequent metal release.132 The Zzz-His sequence, with Xxx and Zzz being any amino acid
increased levels of intracellular bioavailable CuI and ZnII residues except proline. It has a high affinity for CuII, which is
initiated a signaling cascade involving the activation of higher by about 3 orders of magnitude that of Aβ. Inside such a
proteases that degraded Aβ. Although there is no report peptide sequence, CuII is coordinated by four N atoms: the N-
about such investigations, the additional ability of L24 to terminal amine, the first two amides, and the N atom of
extract CuII from Aβ peptides and aggregates is anticipated. imidazole.137 Some reports indicate that CuII in an ATCUN
Hence, metallophore effects similar to those reported for L2 motif is stable and cannot be reduced to CuI.137,138 Overall
could be foreseen.72,133,134 these beneficial features make ATCUN peptides good
2.3.b. Peptidic Ligands. The use of biocompatible peptides candidates as ligands in the AD context. The preparation of
and proteins as metal ligands to capture Cu in AD pathology tripeptides (His-Zzz-His, L26−L28) ligands was described.139
has been explored.48 Peptides have many advantages: they are Unexpectedly, the binding constants reported for these three
suitable for physiological conditions (appropriate water peptides are lower than those of other ATCUN motifs by 3−4
solubility, for instance), they can be easily and routinely orders of magnitude.137,138 Anyway, the removal of CuII from
synthesized with commercially available building blocks, Aβ was directly probed by the competition between L27 and
H DOI: 10.1021/acs.inorgchem.9b00995
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Inorganic Chemistry Viewpoint

Figure 7. Structures of selected ligands in which the binding unit is integrated in the Aβ recognition moiety: (A) stilbene-like moieties; (B) IMPY-
like moieties; (C) PiB-like moieties.

Aβ as well as indirectly because the addition of L26−L28 at chelate two metal ions. L31 shows a slightly better ability than
the beginning of the aggregation process restores the Aβ apo- L30 to bind CuII from Aβ, in line with its higher CuII affinity
aggregation behavior. L26−L28 were able to moderately value (Table 3), but neither L30 nor L31 could effectively
prevent ROS production, while on the basis of complete CuII compete with Aβ for ZnII in vitro, affording an appropriate CuII
removal from Aβ by L26−L28 and the anticipated redox over ZnII selectivity. Later on, L32 was designed based on the
inertness of the resulting CuII-ATCUN complexes, a total assumption that the main Cu redox state in the synaptic cleft is
preclusion of ROS production would be expected. Another not clearly defined. L32 was inspired by the amino acid side
tripeptide L29 (Gly-Gly-His) was reported to prevent ROS chains responsible for Cu binding in Aβ to be able to
production in the superstoichiometric ratio L29/CuII (4:1) coordinate both CuII and CuI ions. L32 displays His
and accordingly restore the viability of PC12 cells confronting coordination moieties that have been introduced on the
ROS production.63 Given the intrinsic affinity, formation of nitrilotriacetic acid platform, and it has moderate binding
stoichiometric CuII complexes, and redox properties of such a affinity constants for both CuI and CuII but slightly higher than
ATCUN motif, it is quite surprising that 4 equiv (not just 1 those of Aβ. As a proof of concept, L32 was able to target both
equiv) of L29 was required to observe the arrest of ROS redox states of Cu and prevent and stop ROS production.141
production. The beneficial effect observed was due to a more sluggish
2.3.b.2. His-Containing Peptides and Peptide-like Li- electron transfer between CuI and CuII for the Cu(L32)
gands. Upon the preparation and activity against Aβ complex compared to Cu(Aβ),142,143 due to a more con-
aggregation of two His-rich branched peptides with one lysine strained geometry of L32.
as a linking unit, L30 and L31 have been reported.140 The 2.3.c. “2-in-1” Integrated Bifunctional Ligands. Many
strategy based on branched peptides is interesting because of bifunctional and/or multifunctional molecules have been
resistance of these structures to proteolytic cleavage. CuII and extensively studied in the past decade and recently reviewed
ZnII coordinations of the branched peptides have been in several articles; the reader can refer to refs 48, 123, 144, and
investigated at neutral pH, and, notably, any peptide can 145. Hence, in this paragraph, we will only recapitulate the
I DOI: 10.1021/acs.inorgchem.9b00995
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Table 4. “2-in-1” Integrated Bifunctional Ligands Reported from the Literature as Well as Their Impact on the ROS
Production and Scavenging, Prevention of Aggregation, Aggregates Disassembly, and Associated Recovery of Cells Survival
ROS aggregation
a
pCu arrest of production scavenging prevention disaggregation cell toxicity refs
Cu(Aβ) 7.3−7.8b 75, 115
L33 n.r. √ n.r. √ √ √ 88
L34 7.4b √ n.r. √ √ √ 150, 152
L35 n.r. √ n.r. ∼ ∼ × 150
L36 n.r. ∼ √ √ √ √ 153
L37 n.r. ∼ ∼ × ∼ √ 153
L38 n.r. ∼ √ √ ∼ n.r. 153
L39 n.r. n.r. √ ∼ ∼ n.r. 153
L40 n.r. ∼ n.r. √ √ n.r. 164
L41 n.r. × n.r. √ √ n.r. 164
L42 n.r. ∼ n.r. √ √ n.r. 164
L43 n.r. × n.r. √ √ n.r. 164
L44 8.5b √ √ √ √ √ 154
L45 n.r. √ n.r. ∼ √ × 88
L46 n.r. n.r. n.r. ∼ ∼ n.r. 155
L47 n.r. n.r. n.r. ∼ ∼ n.r. 155
L48 n.r. ∼ n.r. √ √ √ 156
L49 n.r. ∼ n.r. √ √ √ 156
L50 n.r. ∼ n.r. √ √ √ 156
L51 7.9b n.r. n.r. ∼ n.r. n.r. 117
L52 6.9b n.r. n.r. ∼ n.r. n.r. 117
L53 5.9b n.r. n.r. ∼ n.r. n.r. 117
a
pCu = −log[Cu]unbound; [Cu] = [L] = 10 μM. bFrom the conditional affinity value.

main bifunctional ligands that possess an appropriate binding peroxidase (HRP)/Amplex Red assay, to regulate CuII- or ZnII-
moiety buried inside a scaffold aimed at targeting Aβ induced Aβ aggregation and disassemble preformed aggregates.
aggregates (Figure 7 and Table 4). For example, imaging In cellulo and in vivo treatment with L34 (on N2a cells and
agents of Aβ plaques55,146−148 such as (E)-4-iodo-4′- 5xFAD mice, respectively) showed both improvement in the
(dimethylamino)stilbene (p-I-stilbene), 2-[4′- cell viability (in the presence of exogenous added Cu and
(dimethylamino)phenyl]-6-iodoimidazo[1,2-a]pyridine Aβ1−40−42) and noticeable cognitive improvements compared
(IMPY), and Pittsburgh compound B, 2-(4′-[ 11 C]- to the control mice according to the Morris water maze test.152
methylaminophenyl)-6-hydroxybenzothiazole (PiB), have More detailed structure−activity-relationship investigations
been used as host scaffolds for the design of new compounds have shown that the dimethylamino group is crucial for the
of this family of ligands. Those molecules are of interest recognition of Aβ aggregates and could serve as an anchor.
because they could target and thus remove specifically the Cu Indeed, the analogue L35, lacking the dimethylamino group,
bound to Aβ and have a molecular weight low enough to fulfill shows a modest regulation of the Aβ aggregation and
the adapted Lipinsky’s rules. This paragraph will focus on disassembly compared to the parent L34.150 Furthermore,
integration of the binding moiety in Aβ-targeting scaffolds, changing the dimethylamino to amino (L36) or donating
thus keeping the size of the molecules appropriate for further methoxy (3,5-dimethoxy, for L37) functionalities on the
therapeutic/theranostic purposes. phenyl ring results in a partial (L36) or complete (L37) loss
2.3.c.1. Stilbene-like. In a pioneering work, two N atoms of the regulating effect on Aβ aggregation when [Aβ] = [CuII]
have been introduced to the p-I-stilbene to generate L33.88 = 25 μM.153 Modification of the pyridine moieties into a
L33 can bind CuII and ZnII ions (with modest selectivity for quinolone (L38) or a pyrrole (L39) moiety causes intrinsic
CuII over other metal ions in ethanol),149 and is able to reduce problems of the solubility (L39) and toxicity on M17 cells in
Aβ aggregation, disassemble preformed Aβ aggregates, and the presence of CuII ions (L38) and, thus, such modifications
lessen H2O2 production by 70%. Despite these interesting have not been further explored.153 The N-dimethylaniline
abilities, further biological applications have been hindered by moiety inspired from p-I-stilbene has also been conjugated to
its poor stability in water due to the imine moiety (t1/2 < 5 min hydroxyquinoline, a motif found in the PBT2 molecule (L2),
when metalated with CuII). Thus, it may be anticipated that by an imine (L40) or an amine bond (L41) and to the low-
the results observed upon aggregation and ROS production are binding methoxyquinoline counterparts (L42 and L43).118 In
linked to the activity of products of hydrolysis rather than to contrast to L33, the water stability of the imine-containing
the ligand itself. To solve this issue, the water-stable amine ligands has not been reported. These four molecules have a
derivative L34 has been prepared and studied.150,151 L34 can limited effect on H2O2 production, as evaluated on a HRP/
form a mixture of 1:1 and 1:2 metal-to-ligand complexes at Amplex Red assay, but exhibit effective inhibition of Aβ
neutral pH. The affinity constants of L34 for CuII and ZnII are aggregation and also show significant abilities to disassemble
weaker than those of Aβ (Table 4). Similar to the parent Aβ aggregates, as shown by ThT assay and TEM. Last, a
imine-containing compound, L34 is able to limit H2O2 stilbene derivative integrating a tetradentate [N2O2] binding
production (reduction by 84%), as determined by horseradish moiety has been reported (L44).154 It gathers structural
J DOI: 10.1021/acs.inorgchem.9b00995
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Inorganic Chemistry Viewpoint

elements for interaction with Aβ, improved metal coordina- ions are one of those;43,48 thus, metal-targeting approaches
tion, control of ROS generation, antioxidant activity, water have been developed with a special emphasis for Cu ions,
solubility, and BBB permeability. This ligand can form more which is redox-active and thus may contribute to the oxidative
stable 1:1 metal-to-ligand stoichiometry complexes (with the stress encountered in AD24 in addition to its involvement in
conditional affinity constants for L44 with CuII or ZnII being modulation of the Aβ aggregation.42 The field has seen an
about 1012 and 109 M−1, respectively, at pH 7.4) than the increase of interest for more than a decade, encouraged by the
bidentate ligands described above and has a higher CuII affinity clinical outcomes achieved by L1 and L2 in AD animal models.
than Aβ (Table 4). Moreover, the geometry of the scaffold is This, in turn, has driven medicinal inorganic scientists to
not suitable for CuI coordination, which prefers a tetrahedral produce a growing range of new ligands. This review has
geometry, and thus L44 keeps the Cu ions under the +II state, described such ligands. Depending on their structures, they
thus preventing redox cycling and ROS production. L44 is a have been separated into four groups (Figures 4−7), while
key example of designed molecules able to control multiple bifunctional molecules only include those where the binding
parameters involved in the AD pathway, including metal- cavity is embedded in an Aβ-recognition motif. An attempt to
induced Aβ aggregation, toxicity induced by Aβ and metal-Aβ sort them in terms of efficiency against some of the key
on N2aAPPswe neuroblastoma cells, ROS generation, and deleterious events linked to the etiology of AD (i.e., Cu-
free-radical reactions. The excellent results obtained with L44 induced ROS production and Cu-altered Aβ aggregation) is
were attributed to its ability to directly interact with shown in Tables 1−4.
monomeric and oligomeric Aβ, as confirmed by the detection 3.1.b. Variability of the in Vitro Assays. The main
of L44 in the ternary complex (Aβ)[CuII]L44 (and (Aβ)[ZnII] observation that clearly appears when trying to review the
L44) by ion-mobility mass spectrometry and to its ability to ligands designed against metal-related toxicity in AD is the
remove metal ions from metal ions−Aβ complexes, generating wide diversity of the in vitro methods used to test them against
apo-Aβ species. Cu-induced ROS production and Cu-altered Aβ aggregation
2.3.c.2. IMPY-like. Upon the incorporation of chelating (see sections 1.2.b and 1.2.c). Regarding the aggregation
atoms (N and O) into an IMPY scaffold, an Aβ aggregates process, its stochastic nature makes it very difficult to
imaging probe led to the synthesis of L45, L46, and L47, reproduce from one group to another one. This mainly
which are able to control the inhibition of CuII- and ZnII- depends on the quality of the starting monomeric Aβ peptides
triggered Aβ aggregation and disassemble preformed aggre- and of the monomerisation protocol. Hence, the aggregation
gates but to a lesser extent than bifunctional p-I-stilbene-based itself and the formed aggregates do differ even without the
molecules. On the basis of control experiments on apo-Aβ addition of external molecules.42 For instance, it was reported
aggregation, it seems that L46 and L47 have a preferential that most of Aβ40 remains soluble after incubation at 37 °C for
reactivity toward metal induced over metal-free Aβ aggregation 2 days61 or does aggregate with very similar experimental
pathways. More detailed investigations on L45 indicated its conditions.121,122 Hence, the study of the effect of the ligands
ability to reduce H2O2 production (70%) but pointed out its is dependent on the aggregation experiment itself, making a
intrinsic toxicity for SK-NBE(2)-M17 cells.88,155 comparison between the different studies/groups difficult or
2.3.c.3. PiB-like. Inspired by PiB and its water-soluble even irrelevant.
counterpart ThT, new bifunctional molecules that contain When performed, the in cellulo experiments are using
both amyloid-binding and metal-chelating properties were different cell lines with different culture media. In addition,
developed. L48−L50 can extract CuII from Aβ and form dimer except for a few reports,63 CuII and Aβ are introduced in the
(1:2 copper-to-ligand) complexes at a working concentration cellular media but neither with control on the aggregation state
of 20 μM.156 L48−L50 showed reduction of the amount of nor in conditions of ROS production (i.e., without the addition
aggregated Aβ when added at the beginning of the aggregation of an external reductant).
and disaggregation of preformed Aβ aggregates. In addition, 3.1.c. Variability in the Determination of Ligand
L48−L50 could moderately reduce H2O2 produced by Thermodynamic Characterizations. The CuII (and ZnII)
Cu(Aβ) in vitro and in PC12 cells (25%, 27%, and 41%, affinity values of the ligands are not always reported, although
respectively) and restore the cell viability. On the same it is a primordial criterion for a metal-targeting approach
principle, L51−L53 are suitable ligands for at least partially because the ligand has to compete with Aβ. Those values,
sequestering CuII and ZnII metal ions present in Aβ peptide when reported, are mainly determined using potentiometric
and thus are able to prevent Aβ aggregation, as shown by titrations, competitions with a ligand of very well-known
turbidity assays (Table 4).117 affinity (such as ethylenediaminetetraacetic acid) monitored by
2.3.c.4. Other Bifunctional Ligands. While the interactions an appropriate spectroscopy [UV−vis and electron para-
of stilbene, IMPY, and PiB derivatives with metal ions have magnetic resonance (EPR)], or direct titrations monitored
been thoroughly characterized, other bifunctional molecules spectroscopically. In the former case, the affinity can be
incorporating metal-binding moieties into resveratrol-, chal- recalculated at any pH values, while in the second and third
cone-, and diphenylpropynone-based structures have also been ones, the affinity value is determined at the pH where the
studied in the context of AD (although to a lesser extent) and competition or titration experiments are performed. Potentio-
have been recently reviewed in ref 48. metric titrations lead to determination of the conditional
affinity (i.e., the affinity at a given pH with no competing
3. DISCUSSION molecules), while titrations lead to the apparent affinity (i.e.,
3.1. How To Improve Fallouts from in Vitro Experi- the affinity at a given pH in the presence of buffer). Here, the
ments. 3.1.a. Introducing Remarks. The fundamental causes difficulty in comparing the data reported arises from
of AD are still not yet fully understood. Nevertheless, the inappropriate terminology because often the apparent affinity
current ever-increasing knowledge of the disease has led to the is used instead of the conditional affinity.157 Also several kinds
identification of several promising therapeutic targets. Metal of values related to the affinity are given, including the
K DOI: 10.1021/acs.inorgchem.9b00995
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formation constants (βmlh; see eq 1), the affinity itself, and pCu 3.2.a. General Criteria. In spite of the variability of the
= −log [Cu]free, as used here. investigations reported in the literature, the designed ligands
generally fulfill some basic criteria that are briefly reminded
mCu + l L + hH ↔ [Cu mLlHh] ; below. The designed molecules have to be soluble and stable in
[Cu mLlHh] a biological medium (buffered water at neutral pH). The
βmlh = toxicity of the ligands on cell lines should be tested and weak.
[Cu]m [L]l [H]h (1)
The ligand should keep its binding ability including in the
A convenient way to compare the affinity is the use of the presence of oxidative stress (i.e., should not be rapidly
pCu value, which corresponds to the amount of unbound degraded by oxidative damages). AD occurs in the brain
metal ion at given pH, metal-ion concentration, and metal-to- separated from blood circulation by the BBB, which the ligand
ligand ratio. pCu has the main advantage of being calculated must cross. In addition, in vitro, the ligands should be able to
for complexes of all metal-ion-to-ligand stoichiometries, while retrieve Cu from Aβ, prevent and stop ROS production,
the affinity values correspond only to the formation of 1:1 prevent the formation of Aβ toxic aggregates, and further
species. Hence, for instance, pCu makes possible a comparison restore the viability of cells confronting to Cu(Aβ)-induced
between the ligands forming either 1:1 only or 1:1 and 1:2 toxicity.
(metal/ligand) complexes. It is worth noting that for the later 3.2.b. Thermodynamic Parameters. The affinity of the
types of ligands when the concentration decreases, 1:1 species intended ligands should be high enough to compete with Aβ
are favored compared to 1:2 (metal/ligand) complexes. for CuII binding but not too high to withdraw Cu from
3.1.d. Variability of the Experimental Conditions. essential metalloproteins.30,43,44,48,71,144 One rapid way to
Furthermore, the ways to perform the experiments are also probe the ability to remove Cu from Cu(Aβ) is to add the
different from one report to another. Some primordial ligand to Cu(Aβ) and monitor the exchange by appropriate
experimental conditions like the pH, temperature, buffer, spectroscopy (EPR, UV−vis, etc.). This method is straightfor-
reaction time, and solvent differ, and the results obtained are ward for tetradentate ligands making 1:1 species only but could
therefore barely comparable. For instance, the effect of the be much more complicated for ligands with lower denticity,
ligand on the aggregation can be monitored in minutes or days. leading to 1:2 (metal/ligand) species as well. If the ability to
Another illustration lies in the ROS production assay, where remove CuII from Aβ is kept in the presence of ZnII, this will
the ratio between the Cu and evaluated ligand can vary from 1 also allow determination of whether the selectivity of the
to several equivalents. It is also important to maintain reaction ligand is appropriate. Indeed, the importance of taking ZnII
conditions as close as the ones in the biological environment, into account when designing CuII-targeting ligands has been
in particular regarding the aqueous medium. However, because demonstrated.52,71,113,114,126 The ligand has to display a high
the water solubility is a major issue for the intended ligands, CuII over ZnII selectivity, i.e., higher than those of Aβ, which
this condition is not always fulfilled. equals about 104 at neutral pH. This value is quite high (as a
3.1.e. Guidelines for Improvement. To be able to draw matter of comparison, the selectivity of ethylenediaminetetra-
valuable comparisons between the ligands, there is thus an acetic acid is 102), and thus these criteria are difficult to fulfill.
urgent need of going through more standardized procedures, The lack of CuII selectivity over ZnII may be an explanation for
experimental conditions, and even (analytical) language; this the difference of efficiency observed in vitro, in cellulo (most of
would ideally include purification and monomerization the studies were performed with CuII or ZnII but not a mixture
processes for the Aβ peptide samples for further aggregation of the two metal ions), and in vivo, in the case of L1 and L2
experiments. It is worth mentioning that in vitro investigations ligands. In the synaptic cleft, ligands with insufficient selectivity
may also be complemented by measurements on 2D neuron- will bind the most abundant ZnII and be saturated by them,
like cells (e.g., obtained from PC12, SH-SY5Y, and M17) precluding any activity against CuII-related events. The ability
regarding the toxicity of the ligands and Cu(Aβ) (via the of a ligand to remove CuII from Aβ in the presence of ZnII or
formation of aggregates and/or ROS production) and viability the evaluation of its selectivity has been reported only in a few
recovery in the additional presence of ligands. For the cases.113,114,126
inorganic chemist community, studies on differentiated 2D 3.2.c. “Chelators versus Metallophores”. The fate of the Cu
neuron cells would be a first and accessible way to challenge removed from Aβ is a controversial issue. Among the large
straightforwardly the ligand effect in an environment number of ligands that have been proposed as therapeutics for
mimicking the biological medium, thus more appropriate AD, a handful of them have been clinically trialed. On the basis
than the test tube. However, a step forward would be the use of of in vivo results, metallophores such as L1 (CQ) and L2
more accurate models as 3D human neural cells because they (PBT2) have shown the most satisfying results.72,158 In
are more physiologically relevant and have successfully addition, alterations of metal-ion levels in AD patients involve
recapitulated the AD pathogenic cascades (neurons generating both a dyshomeostasis and anomalous interactions with
the extracellular aggregation of Aβ and tauopathy driven by Aβ proteins rather than a large overload of loosely bound metal
accumulation).64 While, ultimately, better would be transgenic ions.30,31,159 Thus, the current development of metal-targeting
animal models because they allow behavioral tests, 2D cell strategies in AD is directed toward favoring brain metal
lines are well-known, more easily accessible, less difficult to redistribution rather than decreasing metal-ion levels.31,32,72,160
handle, and cheaper than the use of living animals. It should be noted that the metallophore ability has only been
3.2. Toward Better Ligands? The important properties of tested for a few of the reported ligands, namely, L1, L2, L24,
ligands aimed at targeting CuII in the context of AD are shown and L25.30,72
in the graphical abstract and can be summarized as follows: CuI 3.2.d. Bi- and Tridentate Ligands versus Tetradentate
and CuII chelation, redox inertness, BBB permeability, good Ones. The ligands described here can be gathered into three
affinity, selectivity, kinetic and stability properties, and low categories depending on their denticity. Bidentate ligands such
toxicity. as CQ or bifunctional ligands integrating an Aβ recognition
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moiety (L33−L53) can form a 1:2 (metal/ligand) complex, the first ligands tested in clinical trials could be linked to their
but this will depend on the metal concentration and metal-to- inability to fulfill all required criteria, such as those emphasized
ligand ratio, with the biologically relevant value of this latter in the previous paragraphs.
parameter being hard to predict. The in vitro study of such 3.2.h. Further Improvements. New insights into, for
ligands is made difficult by the possibility of forming different instance, the interactions within Aβ−metal ion−L involved
complexes. The affinity of such ligands is quite weak, and it when bi- or tridentate ligands are used104 or the kinetic aspects
may be anticipated that, rather than removing the CuII ion of CuII removal,121,122,164 may be valuable. Future improve-
from Aβ, they form Aβ[CuII]L ternary species in physiological ments also include improving the mathematical model of the
conditions. Such an ability has been shown for the tridentate synaptic cleft165 and taking into account the other isoforms of
ligand L3, which leaves free one binding position in the CuII Aβ.104,158 One crucial point is to perform the investigations in
equatorial plane. This position can be occupied by the better mimicking media (no organic solvent), more realistic
imidazole group of His from the Aβ sequence, for cellular models, such as 3D ones and/or primary neurons in
instance.104,105 This led to another kind of metal-targeting cultures.64
approach, where the metal could stay bound to Aβ, but its
properties are modulated by the presence of the ligand such as, 4. CONCLUDING REMARKS
for instance, L44.154 With respect to tetradentate ligands, their The present Viewpoint has gathered, described, and discussed
in vitro study is eased because they make 1:1 species only. In most of the ligands targeting CuII reported in the last years in
addition, when rigid enough, they prevent redox cycling to CuI, the context of AD and of the amyloid and metal hypotheses. A
thus being able to redox-silence CuII extracted from Aβ.43 key point for inorganic chemists working in the field will be to
3.2.e. CuII versus CuI. Among all of the ligands, only a few deliver directly comparable data obtained with standardized
have been designed to bind CuI,128,141 with investigations methodologies on their ligands’ properties that can be useful to
being mainly focused on CuII-targeting ligands. However, the discriminate between the ligands prior to in vivo assays.

■
redox state of Cu in the synaptic cleft is not clearly identified
because micromolar levels of the reductant Ascorbate are AUTHOR INFORMATION
present.161,162 Thus, the question of the most appropriate
Corresponding Author
redox state of Cu to target is still open. One pragmatic way to
solve this issue is to design ligands able to bind both redox *E-mail: [email protected].
states.128 However, then, particular attention has to be paid to ORCID
the redox activity of the resulting Cu complexes, so that the Cu Peter Faller: 0000-0001-8013-0806
complexes do not produce ROS on their own. Christelle Hureau: 0000-0003-3339-0239
3.2.f. Ligands with an Aβ Recognition Moiety. Multifunc- Present Addresses
tional ligands aimed at retrieving CuII and targeting Aβ §
A.C.-D.: Laboratory for Nanobiology, Department of
aggregates have been the subject of intense investigations in Chemistry, KU Leuven, 3000 Leuven, Belgium.
the last years.48,123,144,145 However, one has also to take into ⊥
P.F.: Institut de Chimie, UMR 7177, CNRS, Université de
account that disassembling Aβ aggregates or preventing Strasbourg, 4 rue Blaise Pascal, 67000 Strasbourg, France.
aggregation could lead to the formation of lower molecular
weight and more toxic species than fibrils. For instance, the Author Contributions
‡
ability of such ThT-based ligands to inhibit Aβ fibril formation These authors contributed equally to this work.
and promote fibril disaggregation has been shown to increase Notes
the cellular toxicity. This was likely due to the formation of The authors declare no competing financial interest.
neurotoxic soluble Aβ42 oligomers.163 Hence, the strategy Biographies
consisting of generating molecules able to interact, inhibit, and
disassemble Aβ fibrils may not be the optimal one until a
complete understanding of the neurotoxic role of the various
Aβ aggregates and of the mechanisms underlying Aβ
aggregation is reached.
3.2.g. Concluding Remarks. The lack of strong positive
results obtained with the metal-targeting approach in general
and the failure of the clinical trials of L1 and L2 may cast some
doubts about the relevance of using ligands, especially Cu ones,
against AD and even, more essentially, on the role of metal
ions in AD. Prior to this reasonable question, one may wonder
whether medicinal inorganic chemists learn as much as they
can from in vitro studies. A more systematic approach, leading
to comparable results and studies from one group to another,
could help in learning more from in vitro researches. Also,
going deeper into the mechanisms at play between the various Charlène Esmieu received her Ph.D. degree in 2014 from the
actors (Aβ, ligand, Cu, Zn, and other competing biomolecules) University of Grenoble Alpes (under the supervision of Dr. S.
is required to improve fallouts from in vitro data. In such a Ménage) for the development of bioinspired dinuclear Cu complexes
context, testing ligands in a biological medium on neuron-like for N2O reduction. She then joined the group of Dr. G. Berggren for a
cells appears as a good balance between the insights that can first postdoctoral stay in Uppsala University (Sweden), where her
be obtained and the complexity of the methodology. We may work mainly focused on the synthesis and characterization of new
also wonder whether the absence of strong beneficial effects of bioinspired diiron models for proton reduction. In May 2018, she

M DOI: 10.1021/acs.inorgchem.9b00995
Inorg. Chem. XXXX, XXX, XXX−XXX
Inorganic Chemistry Viewpoint

joined the team of Dr. C. Hureau and her aim to develop new Cu Laurent Sabater worked on models of the Oxygen Evolving Center
ligands in the context of chelation therapy in AD. under the supervision Prof. A. Aukauloo and received his Ph.D. in
Inorganic Chemistry in 2005 from the University Paris-Sud Orsay. He
performed his first postdoctoral project with Dr. P. Battioni in Paris
Descartes University on the demetalation of crude oils. Then he did 2
years of postdoctoral research with Dr. G. Pratviel at the Laboratoire
de Chimie de Coordination (LCC) on the interaction of designed
molecules with DNA quadruplex related to cancer treatments. He is
currently doing research about interactions of metal ions and amyloid
peptides in the group of C. Hureau at the LCC.

Djamila Guettas was born in Tigzirt (Algeria) in 1992. She obtained

her B.Sc. degree in organic chemistry at University of Science and
Technology Alger (Algeria) and her M.Sc. in Chemistry at University
Pierre and Marie Curie, University Reims Champagne-Ardenne
(France). After obtaining her Ph.D. at University of Lyon 1 under the
supervision of Dr. G. Pilet, she joined C. Hureau’s team in Toulouse,
France, for her postdoctoral stay.
Peter Faller is Professor in Chemistry at the University of Strasbourg
(France) and Group Leader at the Institute of Chemistry (University
of Strasbourg/CNRS). He studied at the University of Zürich
(Switzerland), earning a Ph.D. in Biochemistry on metallothioneins
with M. Vasak. He did his postdoctoral studies on photosystem II at
the CEA near Paris (France) and at the University of Freiburg
(Germany). He was Professor and group leader for a decade at the
Laboratoire de Chimie de Coordination in Toulouse, France, before
moving to Strasbourg. Ongoing research projects of his group are in
bioinorganic and biological chemistry, mainly on the metal binding of
(amyloidogenic) peptides/proteins and metallodrugs with essential
first-row d-block metals.

Amandine Conte-Daban was born in Tarasteix (France) in 1991. She

obtained her B.Sc. degree in Physical Chemistry at University Paris-
Sud, and her M.Sc. degree in Medicinal Chemistry from the
University Paul Sabatier in Toulouse, France, in 2014. After obtaining
her Ph.D. diploma at the University Paul Sabatier in Toulouse,
France, in 2017 under the supervision of Dr. C. Hureau and Prof. P.
Faller, she joined Prof. P. Dedecker’s group at Katholieke Universiteit
Leuven in Belgium for a postdoctoral stay.

Christelle Hureau has a background in physical inorganic chemistry

with a focus on EPR and electrochemistry. She is interested in
chemistry linked with the biological world. In 2015, she became group
leader of the “Alzheimer and amyloids” team hosted by the LCC in
Toulouse, France, where she investigates structural, dynamic, and
kinetic aspects of metal-ion coordination to the Aβ peptide and new
chelating concepts to fight against AD.

■ ACKNOWLEDGMENTS
C.H. warmly acknowledges Béatrice Mestre-Voegtlé for her
relevant and constructive comments on the manuscript. C.H.
N DOI: 10.1021/acs.inorgchem.9b00995
Inorg. Chem. XXXX, XXX, XXX−XXX
Inorganic Chemistry Viewpoint

also acknowledges the European Research Council (Grant (21) Schnabel, J. Amyloid: Little proteins, big clues. Nature 2011,
StG-638712, “aLzINK”) for funding of researches closely 475, S12−S14.
associated with the topic of this Viewpoint. C.E. thanks (22) Lee, S. J. C.; Nam, E.; Lee, H. J.; Savelieff, M. G.; Lim, M. H.
Prestige Program (Grant: PCOFUND-GA-2013-609102) for Towards an understanding of amyloid-β oligomers: characterization,
funding of researches. toxicity mechanisms, and inhibitors. Chem. Soc. Rev. 2017, 46 (2),

■
310−323.
(23) Walsh, D. M.; Selkoe, D. J. A beta oligomers - a decade of
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