Tuning Structures and Properties for Developing Novel Chemical Tools toward Distinct Pathogenic Elements in Alzheimer's Disease

Article abstract of DOI:10.1021/acschemneuro.7b00454

Multiple pathogenic factors [e.g., amyloid-β (Aβ), metal ions, metal-bound Aβ (metal-Aβ), reactive oxygen species (ROS)] are found in the brain of patients with Alzheimer's disease (AD). In order to elucidate the roles of pathological elements in AD, chemical tools able to regulate their activities would be valuable. Due to the complicated link among multiple pathological factors, however, it has been challenging to invent such chemical tools. Herein, we report novel small molecules as chemical tools toward modulation of single or multiple target(s), designed via a rational structure-property-directed strategy. The chemical properties (e.g., oxidation potentials) of our molecules and their coverage of reactivities toward the pathological targets were successfully differentiated through a minor structural variation [i.e., replacement of one nitrogen (N) or sulfur (S) donor atom in the framework]. Among our compounds (1-3), 1 with the lowest oxidation potential is able to noticeably modify the aggregation of both metal-free Aβ and metal-Aβ, as well as scavenge free radicals. Compound 2 with the moderate oxidation potential significantly alters the aggregation of Cu(II)-Aβ₄₂. The hardly oxidizable compound, 3, relative to 1 and 2, indicates no noticeable interactions with all pathogenic factors, including metal-free Aβ, metal-Aβ, and free radicals. Overall, our studies demonstrate that the design of small molecules as chemical tools able to control distinct pathological components could be achieved via fine-tuning of structures and properties.

Full text of DOI:10.1021/acschemneuro.7b00454

Research Article
Cite This: ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
pubs.acs.org/chemneuro
Tuning Structures and Properties for Developing Novel Chemical
Tools toward Distinct Pathogenic Elements in Alzheimer’s Disease
†
‡
§
†
†
∥
Jiyeon Han, Hyuck Jin Lee, Kyu Yeon Kim, Shin Jung C. Lee, Jong-Min Suh, Jaeheung Cho,
,§
,†
Junghyun Chae,* and Mi Hee Lim*
†
Department of Chemistry, Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Republic of Korea
School of Life Sciences, Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Republic of Korea
Department of Chemistry, Sungshin Women’s University, Seoul 02844, Republic of Korea
‡
§
∥
Department of Emerging Materials Science, Daegu Gyeongbuk Institute of Science and Technology (DGIST), Daegu 42988,
Republic of Korea
*
S Supporting Information
ABSTRACT: Multiple pathogenic factors [e.g., amyloid-β (Aβ),
metal ions, metal-bound Aβ (metal−Aβ), reactive oxygen species
(
(
ROS)] are found in the brain of patients with Alzheimer’s disease
AD). In order to elucidate the roles of pathological elements in
AD, chemical tools able to regulate their activities would be
valuable. Due to the complicated link among multiple pathological
factors, however, it has been challenging to invent such chemical
tools. Herein, we report novel small molecules as chemical tools
toward modulation of single or multiple target(s), designed via a
rational structure-property-directed strategy. The chemical
properties (e.g., oxidation potentials) of our molecules and their
coverage of reactivities toward the pathological targets were successfully differentiated through a minor structural variation [i.e.,
replacement of one nitrogen (N) or sulfur (S) donor atom in the framework]. Among our compounds (1−3), 1 with the lowest
oxidation potential is able to noticeably modify the aggregation of both metal-free Aβ and metal−Aβ, as well as scavenge free
radicals. Compound 2 with the moderate oxidation potential significantly alters the aggregation of Cu(II)−Aβ . The hardly
4
2
oxidizable compound, 3, relative to 1 and 2, indicates no noticeable interactions with all pathogenic factors, including metal-free
Aβ, metal−Aβ, and free radicals. Overall, our studies demonstrate that the design of small molecules as chemical tools able to
control distinct pathological components could be achieved via fine-tuning of structures and properties.
KEYWORDS: Alzheimer’s disease, chemical tools, redox properties, metal-free amyloid-β, metal-bound amyloid-β, free radicals
INTRODUCTION
In order to gain a better understanding of the pathogenesis
of AD, chemical tools capable of targeting and modulating
■
Various pathological factors [e.g., amyloid-β (Aβ), metal ions,
metal-bound Aβ (metal−Aβ), reactive oxygen species (ROS)]
are reported to be involved in the pathogenesis of Alzheimer’s
20−39
pathogenic factors have been devised.
A variety of anti-
amyloidogenic compounds that interact with metal-free Aβ
species and mediate peptide aggregation have been con-
1
−10
disease (AD).
Aβ peptides, produced via the proteolytic
20−22
structed.
Small molecules, shown to specifically modify
cleavage of amyloid precursor protein (APP), tend to aggregate
into oligomers, protofibrils, and fibrils.
Aβ oligomers are suggested to be major toxic species that cause
neuronal atrophy and death.
the aggregation of metal−Aβ over metal-free Aβ, have also
1
,2,8−10
Recently, soluble
23−25
been invented.
In addition, several antioxidants have been
shown as chemical tools toward ROS-induced oxidative
2
,10−13
26,27
Additionally, highly
stress.
Moreover, given that the individual elements have
concentrated metals (e.g., copper, zinc, iron) found in senile
plaques are observed to directly interact with Aβ generating
metal−Aβ complexes, which can facilitate Aβ aggregation and
been detected to be intertwined with each other in the AD-
affected brain, the design of small molecules as multifunctional
tools for regulating the interconnections among AD patho-
8
,13−15
28−39
stabilize toxic Aβ oligomers.
Moreover, complexes of Aβ
genic components has currently received attention.
For
and redox-active metal ions, including Cu(I/II), are presented
to overproduce ROS via Fenton-like reactions leading to
damage of nucleic acids, lipids, and cellular organelles.
example, some small molecules, including (E)-5-(4-
hydroxystyryl)quinoline-8-ol (10c), N,N-dimethyl-p-phenyl-
35
2,16−18
Due to the complex link among multiple pathological elements
to AD pathology, however, a cure for the disease has still not
been discovered.
Received: November 16, 2017
Accepted: December 11, 2017
8
,19
©
XXXX American Chemical Society
A
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ACS Chemical Neuroscience
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37
39
tures [e.g., DMPD, 4 ], incorporating one N donor atom at
the para position of the N,N-dimethyl group, are known to
42,43
undergo one- or two-electron oxidation.
Such oxidation is
indicated to direct the capabilities of compounds against
3
7,39,44
regulation of metal-free Aβ, metal−Aβ, and free radicals.
Second, in order to interact with metal ions [i.e., Cu(II),
Zn(II)] as well as metal ions bound to Aβ, the bidentate
ligands containing N and S donor atoms were introduced into
45,46
the framework of 1−3 (Figure 1b).
Lastly, to tune the
extent of compounds’ oxidation, 1−3 were obtained through
the modification of only one N or S donor atom (Figure 1c).
The oxidation potentials of small molecules have been
previously demonstrated to be critical for interactions and
reactivities with pathological targets, including metal-free Aβ,
39,44
Figure 1. Rational design of the small molecules (1−3) and
previously reported chemical tools able to react with pathological
factors found in AD. (a,b) Structures of DMA and the bidentate
ligands used for construction of 1−3. (c) Structural variations of 1−3,
obtained via change of only one N or S donor atom in the framework,
and difference in their oxidation and targets. (d) Examples of
multifunctional chemical tools [10c, (E)-5-(4-hydroxystyryl)-
metal−Aβ, and free radicals.
As summarized in Figure 1c,
our new molecules (1−3), developed via a very slight
structural variation, are observed to have differences in their
oxidation and coverage of pathological targets for interactions
and reactivities (vide infra).
Synthesis and Characterization of 1−3. As described in
Scheme 1, new small molecules (1−3) were prepared.
Compound 1 was afforded by ring opening of ethylene sulfide
with DMPD. Compounds 2 and 3 were obtained through
copper-catalyzed C−S bond formation between 4-iodo-aniline
and the corresponding alkanethiols, followed by conversion of
terminal groups into thiol (for 2) or amino (for 3)
functionality. Synthesis of 1−3 was confirmed by spectroscopic
and spectrometric methods (Figures S1−S3). Moreover, our
molecules were verified to interact with Cu(II) and Zn(II)
35
37
quinoline-8-ol; DMPD, N,N-dimethyl-p-phenylenediamine; 4,
1
4
4
39
N -((1H-pyrrol-2-yl)methyl)-N ,N -dimethylbenzene-1,4-diamine ].
3
7
1
enediamine (DMPD), and N -((1H-pyrrol-2-yl)methyl)-
4
4
39
N ,N -dimethylbenzene-1,4-diamine (4), were shown to
alter the aggregation of metal-free Aβ and metal−Aβ, as well
as quench free radicals (Figure 1). Taken together, efforts on
engineering chemical tools able to target single or multiple
pathogenic component(s) and control their activities have
been made to provide molecular-level insights into the
pathology of AD. The development of such tools, however,
has been challenging.
(
Figure S4) as we designed. Furthermore, 1−3 are suggested
to be blood-brain barrier (BBB) permeable based on the
calculated and experimentally obtained values (logBB > −1.0
and −logP < 5.4; Table S1).
e
Herein, we report new small molecules (1−3; Figure 1) that
distinguishably interact and react with the pathological targets
found in AD (i.e., metal-free Aβ, metal−Aβ, and free radicals).
Our compounds, 1−3, were designed via a rational structure-
property-directed strategy. As depicted in Figure 1, through a
minor structural variation [i.e., change of one nitrogen (N) or
sulfur (S) donor atom in the backbone], we are able to tune
the properties of compounds (e.g., oxidation potentials and
interactions with metal-free and metal-bound Aβ) and
subsequently afford reactivities against disparate pathological
features. Overall, our studies demonstrate that small molecules
as chemical tools for modulating distinct pathological
components of AD can be rationally constructed through a
structure-property-based design strategy. Such design tactics
would be further applied for devising chemical reagents utilized
for other neurodegenerative disorders.
Redox Properties of 1−3. To determine whether a minor
structural variation of compounds could lead to distinguishable
oxidation potentials of the compounds we designed (Figure 1),
their redox properties were investigated by cyclic voltammetry
(
CV) and UV−visible spectroscopy (UV−Vis). The oxidation
potentials of each compound were measured by CV (Figures
a,b and S5). The lower value of the anodic peak potential
E ) indicates that the compound is relatively easy to be
2
(
pa
47
oxidized. The E value of 1 containing a moiety of DMPD is
pa
ca. 0.22 V [versus Ag/Ag(I)], significantly lower than those of
2
and 3 (Epa = ca. 0.54/0.80 and 0.75 V, respectively)
composed of a 4-(dimethylamino)-benzenethiol (BT) group
Figure 2a and Table S2). Although the E values of 2 and BT
(
pa
could not be measured in H O due to their limited solubility, 1
2
has a much lower Epa value than 3 in H O, similar to the
2
observation in DMSO (Figure S5 and Table S3). Based on the
Epa values of DMPD and BT (ca. 0.23 and 0.93 V,
respectively), the structural portion of DMPD would be
mainly responsible for the lower Epa value of 1.
RESULTS AND DISCUSSION
■
Structure−Property-Directed Principle for Designing
Chemical Tools Targeting Distinct Pathological Com-
ponents of AD. To design the backbone of the new
compounds (1−3), we rationally selected two structural
moieties, that is, DMA and bidentate ligands composed of N
and S donor atoms (Figure 1a,b). The conjugation of two
structural groups could achieve different redox properties of
small molecules and their distinguishable interactions with
metal ions, metal-free Aβ, metal−Aβ, and free radicals. First,
DMA (Figure 1a) is a structural portion employed for
previously reported chemical tools targeting metal-free Aβ or
In addition to the moieties of DMPD and BT, the oxidation
of the thiol groups in 1 and 2 was monitored by the DTNB
assay [DTNB = 5,5′-dithio-bis-(2-nitrobenzoic acid)] with L-
cysteine as a positive control (Figures 2c and S6a). A new
absorption band at ca. 412 nm was exhibited upon reaction of
the thiol groups in 1 and 2 with the disulfide bond in DTNB,
indicative of the formation of 2-nitro-5-thiobenzoic acid
2−
dianion (TNB ) (Figure 2c). The different intensity of the
2−
optical bands of TNB , resulting from the interactions of
3
4,37,40,41
metal−Aβ.
In addition, the DMA-containing struc-
DTNB with compounds [Figure 2c, blue (for 1) and red (for
B
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2
)], presents that the thiol group in 1 is less oxidizable than
that in 2.
Based on the redox properties of 1−3, analyzed through CV
and the DTNB assay, their oxidation was additionally traced
for 24 h by UV−Vis (Figures 2d and S6b). The absorption
band of 1 decreased at ca. 254 nm and increased at ca. 300 nm
upon incubation, indicative of oxidation producing the
37,39
delocalized electron(s) on the phenyl ring.
Different
from 1, the intensity of the optical band of 2 was slightly
reduced at ca. 280 nm, whereas the peak intensity was
enhanced in the range of ca. 290 to 400 nm. The spectral
changes of 3 were not detected even for 24 h (Figure S6b),
suggesting that the compound was not easily oxidized under
our experimental conditions. Therefore, the moiety of DMPD
in 1 is suggested to be more associated with ligand oxidation
than the BT portion found in 2 and 3, which is consistent with
the results of CV (Figure 2a). More importantly, the different
degree of oxidation of the compounds is demonstrated to be
achieved via a minor structural difference (i.e., replacement of
only one donor atom in the backbone; Figure 1c). These
distinct redox properties of compounds are observed to direct
their distinguishable interactions as well as modulating
reactivities toward metal-free Aβ, metal−Aβ, and free radicals
(
vide infra).
Interactions of 1−3 with Metal-free Aβ. To verify how
the distinct redox properties of 1−3 could influence their
reactivities toward Aβ aggregation, the interactions of 1−3
with metal-free Aβ species were identified at the molecular
level employing electrospray ionization mass spectrometry
2
(
ESI−MS) and tandem MS (ESI−MS ) (Figures 3a and S7).
Upon incubation of metal-free Aβ with or without the
4
0
Figure 2. Redox properties of 1−3. The oxidation of 1−3 and their
structural portions was monitored by (a, b) CV (in DMSO), (c) the
3+
compounds, the +3-charged Aβ40 monomer ([Aβ40 + 3H] )
was detected at 1444 m/z in the ESI−MS spectra (red peaks;
Figure 3a). Among our small molecules, 1 was observed to
oxidize metal-free Aβ40 showing the peak at 1449 m/z
DTNB assay, and (d) UV−Vis. The E values at 250 mV/s are
pa
summarized in (b). The quantitative E values measured in DMSO
pa
and H O are summarized in Tables S2 and S3. Conditions for CV
2
3
+
studies: [compound] = 1 mM; 0.1 M tetra-N-butylammonium
perchlorate (in DMSO); various scan rates [25 (red), 50, 100, 150,
corresponding to [Aβ40 + O + 3H] (red asterisk; Figure
3a), while such peptide modification was not monitored in the
case of metal-free Aβ40 treated with both 2 and 3.
2
00, and 250 mV/s (blue)]; room temperature; three electrodes
composed of the glassy carbon working electrode, platinum counter
electrode, and Ag/Ag(I) reference electrode. Conditions for the
DTNB assay: [DTNB] = 50 μM; [compound] = 50 μM; pH 8.0;
room temperature; incubation for 10 min. Conditions for UV−Vis
investigations: [compound] = 50 μM; pH 7.4; room temperature;
incubation for 24 h.
Furthermore, we determined which amino acid residues of
metal-free Aβ40 were plausibly oxidized upon treatment of 1
2
through ESI−MS (Figure S7). Note that we incubated 1 with
metal-free Aβ for 3 h to obtain the relatively high intensity of
4
0
the peak at 1449 m/z corresponding to the oxidized Aβ for
40
2
2
better ESI−MS analyses (Figure S7a). The ESI−MS
spectrum of Aβ with 1 added displayed that the methionine
Scheme 1. Synthetic Routes to 1−3
40
3
5 (M35) residue in Aβ was oxidized (e.g., methionine
40
4
8,49
sulfoxide; Figure S7b).
In the previous studies, the
oxidation of M35 in Aβ has been suggested to modify Aβ
50,51
aggregation pathways.
Together, among our molecules
(
1−3), 1 with the lowest E value is capable of oxidizing
pa
metal-free Aβ , implying that the interactions of compounds
4
0
with metal-free Aβ40 could be linked to their redox properties.
Interactions of 1−3 with Cu(II)−Aβ. In addition to
metal-free Aβ , the interactions of 1−3 with Cu(II)-treated
4
0
Aβ species were investigated (Figure 3b). Note that
4
0
compound interactions with Zn(II)−Aβ could not be studied
since Zn(II)-bound Aβ species were not observed under our
MS conditions. Upon incubation of Aβ40 in the presence of
3
+
Cu(II), Cu(II)-added Aβ40 (i.e., [Aβ40 + Cu(II) + H] ) was
revealed at 1465 m/z, along with metal-free Aβ40 at 1444 m/z
(
red and light blue peaks; Figure 3b). Compound 1 with the
lowest oxidation potential was shown to degrade and oxidize
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2
Figure 3. Interactions of 1−3 with metal-free and Cu(II)-treated Aβ , analyzed by ESI−MS and ESI−MS . The +3-charged Aβ monomers in the
4
0
40
samples incubated with 1−3 in the (a) absence and (b) presence of Cu(II) were detected in the ESI−MS spectra. Metal-free Aβ and Cu(II)−
Aβ are denoted as red and blue peaks, respectively. The oxidized ions are indicated with the red asterisks. The number of the asterisks represents
4
0
4
0
the number of the oxygen atoms incorporated into Aβ . The degraded Aβ with 89 Da loss (orange peaks) was presented from the 1-treated
4
0
40
2
sample in the presence of Cu(II). (c) The oxidized amino acid residues of Aβ40 incubated with 1 and Cu(II) were identified through ESI−MS .
Conditions: [Aβ ] = 100 μM; [CuCl ] = 100 μM; [compound] = 500 μM; incubation for 3 h (for metal-free samples) or 1 h [for Cu(II)-added
40
2
samples]; 20 mM ammonium acetate, pH 7.2; 37 °C; no agitation. All samples were diluted with ddH O by 10-fold before injection to the mass
2
spectrometer.
Cu(II)-treated Aβ40 (orange peaks and red asterisks,
respectively; Figure 3b).
oxidation of H13, H14, and M35 in Aβ is indicated to alter Aβ
aggregation pathways.
Compound 2, which possesses a higher oxidation potential
than 1 (Figure 2a), indicated different interactions with
50,51
The degraded Aβ by 89 Da could be induced by oxidative
40
cleavage of the aspartate 1 (D1) residue forming isocya-
5
2−54
Cu(II)-treated Aβ , compared with 1. In detail, 2 significantly
nate.
The D1 residue was reported to be preferentially
40
52−54
reduced the intensity of peaks at 1465 m/z corresponding to
oxidized through alkoxyl radical pathways,
which could
3
+
[
Aβ40 + Cu(II) + H] , suggesting that the coordination of
impact the structural rearrangement of Cu(II) coordination in
Aβ. In addition to the degraded Aβ40 (at 1415 m/z), the peak
at 1409 m/z could be assigned as the degraded Aβ40 with
Cu(II) to Aβ was modified by incubation with 2 (Figure 3b).
4
0
Distinct from 1, 2 was not able to degrade and oxidize Aβ even
in the presence of Cu(II). In order to further analyze the
interaction of 2 with Cu(II)−Aβ, we additionally employed
further loss of one water (H O) molecule. In a similar manner,
2
the peaks at 1421 m/z and 1427 m/z (peptide species bound
12
Cu(II)-treated Aβ , another isoform of Aβ (Figure 4).
4
2
to one and two H O, respectively) might indicate the
2
When 2 was incubated with Cu(II) and Aβ , the peak of
4
2
degradation of Aβ .
4
0
Cu(II)−Aβ disappeared, supporting that this molecule might
4
2
The oxidation sites in the amino acid sequence of Cu(II)-
also disrupt Cu(II) binding to Aβ (blue peaks; Figure 4a).
2
42
added Aβ40 induced by 1 were determined by ESI−MS
Moreover, compound 2 was observed to be transformed to
(
Figure 3c). Under Cu(II)-present conditions, histidine 13
58
BT (153 Da) or the oxidized BT (BT ; 304 Da) under
ox
and 14 (H13 and H14) were shown to be oxidized, along with
M35, previously reported as the plausibly oxidizable residues in
Cu(II)-present conditions. These transformed compounds
from 2, BT and BT , were shown to subsequently form the
ox
4
8,50,51,55,56
Aβ.
H14 in Aβ,
Since Cu(II) is known to be bound to H13 and
their oxidation (e.g., 2-oxo histidine) upon
noncovalent adduct with the Aβ42 dimer at 1868 m/z (green
peaks; Figure 4a). Moving forward, we applied ion mobility
mass spectrometry (IM−MS) to monitor the effects of 2 on
3
,5,45
57
incubation of Aβ with 1 might explain the low abundance of
Cu(II)−Aβ40 in the spectra (Figure 3b). Additionally, the
conformation of the Aβ dimer (Figure 4b). The arrival time
42
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molecules, could not interact with both metal-free Aβ and
Cu(II)−Aβ. Taken together, our MS studies support that the
interactions between our compounds and metal-free or Cu(II)-
treated Aβ could be differentiated depending on their redox
properties, which were rationally tuned via a very minor
structural variation (Figure 1c).
Modulating Abilities of 1−3 toward Aggregation of
Metal-free Aβ and Metal−Aβ. To confirm the compounds’
regulatory activities against metal-free Aβ and metal−Aβ, the
capability of 1−3 to control peptide aggregation with and
without metal ions was evaluated. The resultant Aβ species
upon treatment with compounds were analyzed by gel
electrophoresis/Western blotting (gel/Western blot; for size
distribution) with an anti-Aβ antibody (6E10) and trans-
mission electron microscopy (TEM; for morphology). In the
inhibition experiments (Figure 5), freshly prepared Aβ was
introduced with our compounds in the absence and presence
of metal ions [i.e., Cu(II) and Zn(II)] for 24 h. In the
disaggregation experiments (Figure S9), the compounds were
added to preformed metal-free or metal-added Aβ aggregates,
generated by 24 h incubation, and the resultant samples were
additionally incubated for 24 h (Figure S9a). The larger Aβ
aggregates (e.g., fibrils), generated from both experiments, are
known to limit their penetration through the gel matrix; thus,
Figure 4. Interactions of 2 with Cu(II)-treated Aβ , monitored by
4
2
ESI−MS and IM−MS. (a) The +5-charged Cu(II)-treated Aβ dimer
4
2
in the samples incubated without (left) or with 2 (right). The
noncovalent complex formation of BT (153 m/z) or BTox (304 m/z)
with Aβ42 was detected in the ESI−MS spectra (green peak, 1868
m/z). (b) The altered arrival time distributions (ATDs) of Cu(II)−
Aβ upon incubation with 2 indicate the conformational change of
5
9,60
they are in general visualized by TEM.
When Aβ
aggregation is varied with treatment of compounds producing
4
2
small-sized Aβ species, smearing bands in the gel/Western blot
the peptide. Ions selected for the IM−MS analysis are marked with
59,60
are presented.
gray and orange circles in the ESI−MS spectra. Conditions: [Aβ ] =
4
2
When metal-free Aβ /Aβ and metal−Aβ /Aβ were
4
0
42
40
42
1
00 μM; [CuCl ] = 100 μM; [2] = 500 μM; incubation for 1 h; 20
2
incubated with 1, noticeable smearing bands of the resultant
Aβ40 and Aβ42 species with lower molecular weights were
observed in the gel/Western blots (Figure 5b). These results
suggest that 1 is able to significantly modulate Aβ aggregation
pathways in the absence and presence of metal ions. In
addition, 1 is shown to disaggregate preformed metal-free
Aβ40/Aβ42 and metal−Aβ40/Aβ42 aggregates or affect their
further aggregation (Figure S9b). TEM images of the Aβ
aggregates formed upon treatment of 1 in both inhibition and
disaggregation studies were amorphous and smaller sized
instead of larger and fibrillar aggregates observed from
compound-free Aβ samples (Figures 5c and S9c).
mM ammonium acetate, pH 7.2; 37 °C; no agitation. All samples
were diluted with ddH O by 10-fold before injection to the mass
2
spectrometer.
distribution (ATD) of the Aβ42 dimer, monitored after
addition of 2 into the sample of Cu(II)-treated Aβ , was
4
2
enhanced at 11.57 ms, implying that 2 could trigger the
conformational compaction of the dimeric form. Thus, 2 is
able to noticeably interact with Cu(II)-treated Aβ over metal-
free Aβ, especially showing the noncovalent complexation
between the compound and the Aβ42 dimer with conforma-
tional changes when Cu(II) is present. These interactions of 2
with Cu(II)−Aβ are exhibited to be related to its modulating
Unlike 1, in the case of 2, this molecule could not
significantly regulate metal-free Aβ40 and metal−Aβ40
aggregation in both inhibition and disaggregation experiments
(Figures 5b and S9b). Different from Aβ40, the smearing bands
of Cu(II)−Aβ42 aggregates, larger than 140 kDa, were
observed upon incubation with 2, which could indicate its
specific reactivity toward the aggregation of Cu(II)−Aβ42 over
metal-free Aβ42 and Zn(II)−Aβ42 (Figures 5b and S9b). In
accordance with the results of gel/Western blot, shorter-sized
Aβ aggregates were shown only in the samples containing 2
and Cu(II)-treated Aβ42 in both inhibition and disaggregation
experiments (Figures 5c and S9c). Lastly, 3 could not modify
the aggregation of metal-free Aβ /Aβ and metal−Aβ /Aβ
4
2
activity toward the aggregation of Cu(II)−Aβ (vide infra).
42
Lastly, the hardly oxidizable compound 3, relative to 1 and
, displayed no noticeable interactions with Cu(II)-treated
2
Aβ . For example, 3 was not able to degrade and oxidize Aβ as
4
0
well as generate noncovalent complexes with peptides (Figure
b). We further investigated the effect of 3 on the
conformational change of Cu(II)−Aβ through IM−MS
3
40
(Figure S8). When the ions (at 1465 m/z) corresponding to
Cu(II)−Aβ treated with and without 3 were analyzed, the
4
0
ATD of Cu(II)−Aβ was not considerably differentiated
40
(Figure S8). These results indicate that 3 could not affect the
4
0
42
40
42
conformation of the peptide, although the peak intensity of
based on the results of gel/Western blot (Figures 5b and S9b).
No noticeable morphological changes of 3-added Aβ
aggregates, produced with and without metal ions, were
visualized (Figures 5c and S9c).
Cu(II)−Aβ upon incubation with the compound was slightly
4
0
enhanced as depicted in the ESI−MS spectra (Figure 3b). In
summary, the easily oxidizable compound, 1, is observed to
considerably interact with both metal-free and Cu(II)-treated
The reactivities of 1−3 against the aggregation of metal-free
Aβ. Compound 2 (with a higher E value than 1) significantly
Aβ and metal−Aβ are consistent with the trend of their E
pa
pa
affects Cu(II) binding to both Aβ40 and Aβ42 and produces a
noncovalent adduct with the Aβ42 dimer showing structural
compaction. The least oxidizable compound, 3, among our
values (Figure 2), as well as the observation for the interactions
with metal-free and metal-bound Aβ species (Figures 3 and 4).
Compound 1 with the lowest Epa value could trigger the
E
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Figure 5. Reactivities of 1−3 toward the aggregation of metal-free and metal-induced Aβ and Aβ . (a) Scheme of the inhibition experiments. (b)
4
0
42
Analysis of the size distributions of the resultant Aβ species from panel a by gel/Western blot using 6E10. Conditions: [Aβ or Aβ ] = 25 μM;
4
0
42
[
CuCl or ZnCl ] = 25 μM; [compound] = 50 μM; pH 6.6 [for Cu(II)-containing samples] or pH 7.4 [for metal-free and Zn(II)-containing
2 2
samples]; 37 °C; constant agitation. (c) TEM images of the samples from panel b. The inset image represents the minor species. Scale bar = 200
nm.
oxidation of metal-free Aβ (at M35) as well as the degradation
(
at D1) and oxidation (at H13, H14, and M35) of Cu(II)−Aβ.
Such interactions between 1 and Aβ could lead to modify
metal-free and metal-induced Aβ aggregation. Compound 2
with the higher E value than 1 could disrupt the coordination
pa
of Cu(II) to Aβ and generate a noncovalent complex with the
Aβ42 dimer showing structural compaction with Cu(II) being
present; thus, the molecule could noticeably control Cu(II)−
Aβ aggregation. In the case of 3 with the highest E value
4
2
pa
among our molecules, the compound was not able to interact
with metal-free Aβ and Cu(II)−Aβ, which could support no
reactivities of the molecule toward the aggregation of both
metal-free Aβ and metal−Aβ. Taken together, the ability of
compounds to modulate the aggregation of Aβ and Aβ with
40
42
or without metal ions is demonstrated to be highly relevant to
their characteristics, including redox properties and inter-
actions with the targets.
Figure 6. Scavenging capability of 1−3 against free organic radicals in
N2a cell lysates, determined by the TEAC assay. The TEAC values of
compounds are summarized in (b): *n.d. indicates not determined.
The TEAC values of ethanamine (EA) and ethanethiol (ET) were
not able to be obtained due to their limited activity.
Scavenging Capability of 1−3 against Free Radicals.
To determine if the redox properties of compounds can direct
their regulatory activity against free radicals, the ability of 1−3
to quench free radicals was evaluated by the Trolox equivalent
antioxidant capacity (TEAC) assay employing the lysates of
Neuro-2a (N2a) neuroblastoma cells (Figure 6a). The TEAC
assay verifies the capability of compounds to scavenge free
radicals, such as ABTS+ [ABTS = 2,2′-azino-bis-(3-ethyl-
benzothiazoline-6-sulfonic acid)], relative to that of an analog
of vitamin E, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-
against free radicals is well characterized; thus, small molecules
with the TEAC values higher than 1.0 are regarded as potent
•
61
antioxidants than Trolox. Compound 1 has a TEAC value of
1.6 ± 0.1, suggesting that this molecule is a better scavenger
against free radicals than Trolox (Figure 6). Compound 2 also
quenches free radicals [the TEAC value as 0.33 (± 0.04)] but
3
9,61−63
carboxylic acid).
The quenching ability of Trolox
F
DOI: 10.1021/acschemneuro.7b00454
ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience
Research Article
its activity is much lower than 1 and Trolox. As expected, 3
with the highest oxidation potential exhibits the lowest TEAC
value [0.03 (± 0.01)]. Collectively, the trend of the TEAC
values of 1−3 is observed to be well matched with the degree
of their oxidation (Figure 2a,b).
In order to identify which structural portions contribute to
the antioxidant capability of our compounds (especially, 1 and
METHODS
■
All reagents were purchased from commercial suppliers and used as
received unless otherwise noted. Cyclic voltammograms were
recorded under N (g) with a CHI620E model potentiostat (Qrins,
Seoul, Republic of Korea). A three-electrode setup is composed of a
Ag/Ag(I) reference electrode [RE-1B Reference electrode [Ag/Ag(I);
Qrins], a Pt wire auxiliary electrode (SPTE Platinum electrode;
Qrins), and a glassy carbon working electrode (Qrins). Aβ40 and Aβ42
2
2), we further investigated the ability of structural components
(
Aβ42 = DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVG-
of 1−3 [i.e., DMPD, BT, ethanamine (EA), ethanethiol (ET);
Figure 6] to scavenge free radicals. The TEAC value of DMPD
is 1.0 ± 0.03, much higher than that of BT [0.02 ± 0.03],
which could explain the better antioxidant capability of 1
containing DMPD, compared to 2 and 3 possessing BT. In the
case of EA and ET, their TEAC values could not be measured
due to their limited activity against free radicals. In addition to
the individual moieties, the entire structure of the compounds
might be shown to be important for their antioxidant capacity
GVVIA) were purchased from Anaspec (Fremont, CA, USA). Trace
metal ions were removed from the solutions used in the studies by
treating the solutions with Chelex overnight (Sigma-Aldrich, St. Louis,
MO, USA). Optical spectra were recorded using an Agilent 8453
UV−vis spectrophotometer. Absorbance values for biological assays,
including the parallel artificial membrane permeability assay adapted
for the blood−brain barrier (PAMPA−BBB), TEAC assay, and MTT
assay, were measured using a Molecular Devices SpectraMax M5e
microplate reader (Sunnyvale, CA, USA). TEM images were taken by
a JEOL JEM-2100 transmission electron microscope [UNIST Central
Research Facilities (UCRF), Ulsan, Republic of Korea]. ESI−MS,
(
1 versus DMPD; Figure 6b). Overall, the antioxidant
2
capability of compounds is confirmed to be associated with
their chemical structures that consequently differentiate their
oxidation potentials.
ESI−MS , and IM−MS analyses were performed using a Waters
Synapt G2-Si quadrupole time-of-flight (Q-Tof) ion mobility mass
spectrometer (Waters, Manchester, UK) equipped with an ESI source
[
DGIST Center for Core Research Facilities (CCRF), Daegu,
1
13
Republic of Korea]. H and C NMR spectra were recorded using
a 400 MHz Agilent NMR spectrometer (UCRF). The high-resolution
mass spectra of compounds were obtained through a Q exactive plus
orbitrap mass spectrometer (Thermo Fisher Scientific, Carlsbad, CA,
USA). The detailed experimental procedures are described in the
Supporting Information.
CONCLUSIONS
■
Novel small molecules, composed of DMA and bidentate
ligands, were newly constructed for regulating distinct factors
linked to AD pathology to different extents. The development
of such compounds was achieved via our rational structure-
property-directed design principle employing a very straight-
forward structural modification. The distinguishable character-
istics of the small molecules, including redox properties and
interactions with pathological components, were obtained
through our simple structural variation. The relatively easily
oxidizable compound, 1, is demonstrated to effectively modify
multiple pathogenic factors, metal-free and metal-bound Aβ as
well as free radicals. In addition, 2, which is less easily oxidized
than 1, presents noticeable reactivity toward the aggregation of
Cu(II)−Aβ , along with less scavenging ability against free
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acschemneur-
o.7b00454.
■
*
S
Experimental details, MW, clogP, HBA, HBD, PSA, log
BB, and −log P
DMSO and H O, H and C NMR spectra, metal
binding, and oxidation potentials in DMSO and H O for
values, quantitative data of CVs in
e
1
13
2
4
2
2
radicals than that of 1. The hardly oxidizable compound, 3,
could not influence the actions of all pathogenic components,
metal-free Aβ, metal−Aβ, and free radicals. Therefore, our
studies reveal that a minor structural change could successfully
tune their properties (especially, oxidation potentials) as well
as regulatory reactivities toward distinct pathogenic elements
found in AD.
In order to evaluate biological applications of our small
molecules, their cytotoxicity was examined employing human
neuroblastoma SH-SY5Y cells through the MTT assay [MTT
1−3, oxidation of L-Cys and 3, interactions of 1 with
metal-free Aβ40, analyses of Cu(II)−Aβ in the absence
and presence of 3, reactivities of 1−3 towards preformed
metal-free and metal-added Aβ and Aβ aggregates,
4
0
42
and toxicity of 1−3 in 5Y cells (PDF)
AUTHOR INFORMATION
Corresponding Authors
M.H.L. E-mail: mhlim@unist.ac.kr.
J.C. E-mail: jchae@sungshin.ac.kr.
■
*
*
=
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
ORCID
mide] (Figure S10). Compound 1 was observed to have no
cytotoxicity up to 10 μM (cell viability, ca. 100%; Figure S10).
In the case of 2 and 3, the compounds were less toxic up to 25
μM (cell viability, ca. 80−100%) (Figure S10). When the cells
incubated with Aβ and 1 (the compound with regulatory
reactivities toward multiple pathological factors) in the absence
and presence of metal ions, the cell survival was enhanced by
ca. 10−20% (Figure S10). To improve the utilization of such
molecules in biological systems, further structural optimization
would be necessary. To conclude, our structure-property-
directed design demonstrates the feasibility of building up
novel structural entities useful for devising chemical tools
toward modulation of single or multiple pathogenic factor(s)
found in AD.
Jaeheung Cho: 0000-0002-2712-4295
Mi Hee Lim: 0000-0003-3377-4996
Author Contributions
M.H.L., J.H., and H.J.L. designed research. J.H. and H.J.L.
conducted the electrochemical studies, analyses of gel
electrophoresis/Western blot and TEM, biochemical assays,
and cell studies. K.Y.K and J.C. synthesized 1−3. J.H., S.J.C.L.,
and J.C. carried out mass spectrometric studies with data
analysis. J.H., H.J.L., J.-M.S., J.C., and M.H.L. contributed to
manuscript writing. All authors have given approval to the final
version of the manuscript.
Notes
The authors declare no competing financial interest.
G
DOI: 10.1021/acschemneuro.7b00454
ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience
Research Article
(
20) Lorenzo, A., and Yankner, B. A. (1994) β-amyloid neurotoxicity
ACKNOWLEDGMENTS
■
requires fibril formation and is inhibited by congo red. Proc. Natl.
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This work was supported by the National Research
Foundation of Korea (NRF) grant funded by the Korean
government [NRF-2016R1A5A1009405 and NRF-
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