Orally Administered Benzofuran Derivative Disaggregated Aβ Plaques and Oligomers in the Brain of 5XFAD Alzheimer Transgenic Mouse

Article abstract of DOI:10.1021/acschemneuro.0c00606

Amyloid-β (Aβ) aggregated forms are highly associated with the onset of Alzheimer's disease (AD). Aβ abnormally accumulates in the brain and induces neuronal damages and symptoms of AD such as cognitive impairment and memory loss. Since an antibody drug,

Full text of DOI:10.1021/acschemneuro.0c00606

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Research Article
Orally Administered Benzofuran Derivative Disaggregated Aβ
Plaques and Oligomers in the Brain of 5XFAD Alzheimer Transgenic
Mouse
DaWon Kim,# Gi Hun Bae,# Hye Yun Kim, Hanna Jeon, Kyeonghwan Kim, Jisu Shin, Sunhee Lee,
Seungpyo Hong, Ikyon Kim,* and YoungSoo Kim*
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ABSTRACT: Amyloid-β (Aβ) aggregated forms are highly associated with the onset of Alzheimer’s disease (AD). Aβ abnormally
accumulates in the brain and induces neuronal damages and symptoms of AD such as cognitive impairment and memory loss. Since
an antibody drug, aducanumab, reduces Aβ aggregates and delays clinical decline, clearance of accumulated Aβ in the brain is
accounted as a therapeutic approach to treat AD. In this study, we synthesized 17 benzofuran derivatives that may disaggregate Aβ
oligomers and plaques into inert monomers. By a series of Aβ aggregation inhibition and aggregates’ disaggregation assays utilizing
thioflavin T assays and gel electrophoresis, YB-9, 2-((5-methoxy-3-(4-methoxyphenyl)benzofuran-6-yl)oxy)acetic acid, was selected
as the final Aβ-disaggregator candidate. When it was orally administered to the 8-month-old male transgenic mouse model with five
familial AD mutations (5XFAD) via drinking water daily for two months, Aβ oligomers and plaques in hippocampus were reduced.
Consequently, decreased astrogliosis and rescued synaptic dysfunction were observed in the hippocampus of YB-9-treated 5XFAD
mice compared with the untreated transgenic control group.
KEYWORDS: Alzheimer’s disease, amyloid-β aggregates, benzofuran derivatives, hippocampus, synapse, 5XFAD mouse models
raised criticisms and concerns that Aβ might not be the
pathological culprit of AD, until the release of promising
clinical trial results of aducanumab, an antibody drug candidate
that directly clears Aβ oligomers and plaques from the
brain.7−10 Although the therapeutic regulation of cognitive
impairments was observed only in high dosage cases,
aducanumab successfully and significantly removed amyloid-
PET (positron emission tomography) positive plaques from
AD patient brains.11 Such clinical evidence supports two views
INTRODUCTION
■
Given that accumulation of amyloid-β (Aβ) in the brain is a
standard indication of Alzheimer’s disease (AD) onset,
reduction of the cerebral amyloid burden has been the major
therapeutic approach for drug discovery.1,2 Aβ is abnormally
released upon sequential cleavage of amyloid precursor protein
(APP) by β- and γ- secretases and then aggregates into plaques
and harmful oligomers, associated with atrophy, synaptic
dysfunction, and memory loss. Thus, small-molecule drug
candidates for AD have been actively developed to inhibit Aβ
production and aggregation and/or remove accumulated Aβ.3,4
Albeit Aβ is not an ideal protein target as it forms
heterogeneous and polymorphous aggregates with unknown
structures, well-characterized structures of APP secretases and
Aβ aggregation cofactors have enabled synthetic design of
chemical candidates.5,6 However, the absolute failure of APP-
and Aβ-modulating chemical drug candidates in clinical trials
Received: September 18, 2020
Accepted: December 8, 2020
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Figure 1. Chemical structures of YB compounds. Seventeen benzofuran derivatives were synthesized.
Figure 2. In vitro assays to evaluate inhibitory and disaggregating effects of YB compounds on Aβ aggregates. (a) Aβ aggregation inhibition test by
ThT assay. Fluorescence intensities of YB compounds (0.5, 5, 50 μM) incubated with Aβ42 (25 μM) are normalized to the control of 3-day Aβ
aggregates (3d, 100%). (b,c) Aβ aggregates disaggregation test by ThT assay. (b) Fluorescent intensities of selected YB compounds (0.5, 5, 50 μM)
incubated with preformed Aβ42 (25 μM) are normalized to the control of 3-day Aβ aggregates (3d, 100%), and (c) further selected compounds
were examine in the higher concentration of 500 μM. (d) SDS-PAGE with PICUP and silver staining for disaggregated Aβ with YB compound
incubation. Sizes of Aβ species according to the protein size markers (5 to 250 kDa) are monomers (5 kDa), dimers (10 kDa), oligomers (15 to 75
kDa), and high-molecular-weight aggregates including fibrils (above 250 kDa). The number above indicates the lane number. The whole gel image
is shown in the Supporting Information (Figure S1). Abbreviations: 3d = 3-day incubation of Aβ, 6d = 3-day preincubation of Aβ and additional 3-
day incubation of Aβ with/without compounds, Cpd = compound, FI = fluorescent intensity. Data represents the mean of triplicated experiments
SEMs, and one-way anova was applied followed by Bonferroni’s posthoc comparison test (*P < 0.033, **P < 0.002, ***P < 0.001).
B
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Figure 3. YB-9 reduces Aβ plaques and oligomers in the hippocampus of 5XFAD mice. YB-9, 0 (TG (−), male, n = 5) or 50 mg/kg/day (TG (+),
male, n = 5), was orally given to 8-month-old 5XFAD for 8 weeks, and their brains were compared to each other. (a) The representative images of
6E10-stained Aβ plaques (green) in whole brains (scale bars, 1 mm) of either control (TG (−)) or compound-treated (TG (+)) group. (b-c)
Quantitative measurements of plaque number (b) and area (c) performed manually in the range of whole brain (left, shown in Figure 3a),
hippocampal region (middle), and cortical region (right). (d) Histochemical analyses of Aβ deposition stained with 6E10 in either hippocampal
(up) or cortical (down) region. Hoechst was stained as a location indicator (blue). The third row of each group shows merged images of Aβ
plaques with Hoechst staining. Scale bars, 200 μm. (e) Sandwich ELISA of Aβ-insoluble fractions from hippocampal (left) and cortical (right)
sections. (f) Dot blot assays of the total Aβ (anti-Aβ: 6E10) and oligomers (antiamyloidogenic protein oligomer: A11). The data collected from
WT littermates and the whole blot image is shown in the Supporting Information (Figures S2 and S3). The error bars represent the mean of
quintuple experiments (n = 5 for each group) SEMs and one-way anova was applied followed by Bonferroni’s posthoc comparison test (*P <
0.033, **P < 0.002, ***P < 0.001). Abbreviation: conc.= concentration.
that Aβ aggregates are the pathological culprits and drug
targets of AD and that removal of accumulated Aβ is a better
fit as a therapeutic approach than inhibition of the event, which
is considered as a preventive method.12
mice were monomerized and removed from the brain by
multiple routes including an efflux to blood, and then, we
observed recovery of impaired cognitive functions and
upregulated neuroinflammation. Thus, the Aβ disaggregation
approach by chemicals would be a complementary version of
Aβ clearance immunotherapy.
Synthetic designs of chemical drug candidates mimicking the
Aβ aggregates clearance mechanism of immunotherapy seemed
impractical because of the unavailability of structural under-
standing of heterogeneous and polymorphous Aβ monomers
and oligomers. Thus, chemical candidates directly clearing Aβ
have not debuted in AD clinical trials yet. As chemical drugs
bear benefits for aged patients such as oral administration and
relatively lower cost, compared with antibody drugs, discovery
of chemical alternatives of Aβ-clearing immunotherapy would
be useful to treat AD. Previously, we reported a series of
studies demonstrating that small molecules could directly
target and disaggregate Aβ oligomers and plaques into
monomers in vitro and in vivo.13−15 By Aβ disaggregation,
oligomers and plaques in the brain of AD transgenic (TG)
RESULTS AND DISCUSSION
■
Syntheses of YB Benzofuran Derivatives. In this study,
we synthesized a chemical library of benzofuran derivatives and
examined their therapeutic efficacy to dissociate Aβ aggregates
in vitro and in vivo. Benzofuran-derived compounds were
previously reported to have high affinity and selectivity toward
Aβ deposits and to have oral availability with blood-brain
barrier permeability.16−18 As part of our continued research
interest in benzofurans, 17 YB compounds, 3-arylbenzofurans,
and their derivatives with a methoxyphenyl group at the C3
position (Figure 1 and Supporting Information) were
C
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Figure 4. YB-9 reduces Aβ-induced inflammation and synaptic protein loss in the hippocampus of 5XFAD mice. YB-9, 0 (TG (−), male, n = 5) or
50 mg/kg/day (TG (+), male, n = 5), was orally given to 8-month-old 5XFAD for 8 weeks, and their brains were compared to each other. (a)
Histochemical analyses of reactive astrocytes stained with GFAP (red) in either hippocampal (up) or cortical (down) region. Hoechst (blue) was
stained as a location indicator. The third row of each group shows merged images of activated astrocytes with Hoechst staining. Scale bars, 200 μm.
(b) Quantitative measurements of GFAP number in the hippocampus (up) and cortex (down) after the drug administration. (c) Western blotting
analyses of PSD-95 (85 kDa), synaptophysin (syn, 40 kDa), and β-actin (38 kDa) expression in hippocampal (up) and cortical (down) soluble
lysates. (d) Densitometry of PSD95 (left) and synaptophysin (right) in hippocampal (upper row) and cortical (down row) regions, represented in
ratio to β-actin. The data collected from WT littermates and the whole blot image is shown in the Supporting Information (Figures S4 and S5).
The error bars represent the mean of quintuple experiments (n = 5 for each group)
SEMs and one-way anova was applied followed by
Bonferroni’s posthoc comparison test (*P < 0.033, **P < 0.002, ***P < 0.001). Abbreviation: PSD95 = postsynaptic density protein 95, syn =
synaptophysin.
synthesized. Among them, 11 compounds were newly
synthesized, while the structures of YB-10, -11, -13, -14, -15,
and -17 were previously known.19,20
3 days (6d) to confirm the dissociating functions by ThT
assays (Figure 2b). At a compound concentration of 50 μM,
three YB compounds, YB-3, -9, and -10, appeared to contain
less-mature Aβ fibrils than the control (3d) by approximately
20%. They were selected for further evaluation at higher
concentration of 500 μM (Figure 2c), and all three of them
appeared to dissociate mature Aβ fibrils up to 70% compared
with the control (3d). On the basis of these observations, YB-
3, -9, and -10 among 17 benzofuran derivatives not only
blocked the formation of mature Aβ fibrils but also dissociated
the preformed β-sheet-rich Aβ aggregates efficiently.
However, the ThT assay solely cannot validate the
effectiveness of the compounds on the formation of Aβ
oligomers and protofibrils since it only binds to the mature β-
sheet structure like Aβ fibrils.22 In order to visualize alterations
of Aβ monomers, oligomers, and high-molecular-weight Aβ
including fibrils upon treatment of the selected compounds, we
further performed gel electrophoresis with photoinduced cross-
linking of unmodified proteins (PICUP), securing original
states of Aβ aggregates.23,24 Aβ(1−42) monomers (50 μM)
were aggregated alone for 3 days at 37 °C, and then, each
compound at concentrations of 250 μM was coincubated with
the preformed Aβ aggregates (3d) for three additional days
(6d) at 37 °C (Figure 2d). Prepared samples, Aβ monomers
(0d, lane 1), 3-day incubated Aβ (3d, lane 2), 6-day incubated
Screenings of YB Compounds via In Vitro Assays. The
compounds were tested for their Aβ aggregation inhibition as a
primary screening, and Aβ aggregates’ dissociation abilities
were tested using the selected compounds. We prepared
Aβ(1−42) monomers and quantified the level of Aβ fibril
formation by thioflavin T (ThT) fluorescence assay with or
without the addition of compounds.21 Each compound at
concentrations of 0.5, 5, and 50 μM was incubated with Aβ
monomers (25 μM) for 3 days at 37 °C, and fluorescence
intensity of ThT was measured. All data obtained from ThT
assays were normalized to the fluorescent intensity of the
control sample, 3-day incubated Aβ without the compound
(3d), and all 17 benzofuran derivatives showed significant
inhibiting effects on mature Aβ fibril formation (Figure 2a).
For the Aβ fibrils’ disaggregation test, we selected 11 YB
compounds, including YB-1, -3, -4, -5, -6, -7, -9, -10, -12, -14,
and -16, which inhibited mature Aβ fibril formation up to 50%
compared with the control. First, Aβ aggregates were obtained
by the preincubation of Aβ(1−42) monomers (25 μM) alone
without the compounds for 3 days (3d) at 37 °C, and then,
each compound (0.5, 5, and 50 μM) was added into the
preformed Aβ aggregates (3d) and coincubated for additional
D
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Aβ (6d, lane 3), and compound-treated Aβ (6d, lane 4−6)
were separated and visualized by silver staining. Compared
with the other two compounds (lane 4 and 6), YB-9 (lane 5)
displayed the dramatic reduction of high molecular weight Aβ
aggregates (over 250 kDa) to the extent of the Aβ monomer
solution (lane 1), indicating the effective disaggregation
function of YB-9. Taken together, based on the fact that YB-
9 reduced mature fibrils and high molecular weight aggregates
of Aβ in both ThT assay and electrophoresis, it was selected as
the final candidate for in vivo experiments using five familial
AD mutations (5XFAD) TG mouse model. Though no
benzofuran derivatives have been reported to dissociate Aβ so
far, the hydrophobic aromatic region and the two substituents
at the C5 and C6 positions of YB-9 make itself suitable for Aβ
dissociation as such characteristics help it to interact with and
fit to cross-β sheet structures throughout the amyloid fibrils
(Figure 1).
GFAP expression was observed both in the hippocampus and
cortex of YB-9-treated 5XFAD mice (Figure 4a), but the
statistical analyses of GFAP levels showed that reduction in
reactive astrocytes was only significant in the hippocampal area
while it remained negligible in cortical area (Figure 4b).
Since the reductions of Aβ plaques and oligomers and
reactive astrocytes by the administration of YB-9 might alter
the synaptic dysfunction in the mouse model, we analyzed the
levels of two synaptic proteins, presynaptic synaptophysin and
postsynaptic density protein 95 (PSD-95), using Western
blotting (Figure 4c,d).33 In hippocampal sections, both
synaptophysin and PSD-95 expression significantly increased
after the oral administration of YB-9, but those of in cortical
sections displayed similar or lower levels compared with the
nontreated group (Figure 4d).
CONCLUSIONS
■
In Vivo Efficacy of Selected YB Compound. To evaluate
the Aβ disaggregation efficacy of YB-9 in vivo, we employed 8-
month-old male 5XFAD TG mouse model which develops
extracellular Aβ deposition at the age of 2 months and exhibits
memory impairments at 4−6 months of age.25 In this
experiment, 8-month-old male 5XFAD mice (n = 5) were
treated with YB-9 at 50 mg/kg/day for eight full weeks by
daily oral administration and untreated littermate TG (n = 5)
and wild type (WT, n = 5) were prepared for comparison.
Effects of YB-9 on the levels of Aβ plaques in the brain were
observed by histochemical analyses with 6E10 staining (Figure
3a−d). Compared with the nontreated group, the YB-9
administered group significantly reduced the levels of 6E10-
positive Aβ plaques throughout the 5XFAD brain (Figure 3a).
Particularly, the number and area of 6E10-positive Aβ plaques
in the hippocampal region decreased more than 50% compared
with the nontreated group, while the plaques in the cortical
region were not reduced substantially (Figure 3b,c). To
confirm plaque reduction upon YB-9 administration, we
measured and compared Aβ plaque levels by sandwich
enzyme-linked immunosorbent assay (ELISA) (Figure 3e).
From the insoluble lysate fraction of hippocampus and cortex,
we lysed and extracted insoluble Aβ aggregates with a
guanidine lysis buffer (5 mM guanidine-HCl, 50 mM Tris-
HCl).26,27 Compared with hippocampal and cortical Aβ levels
of nontreated TG, we observed a significant reduction of Aβ, of
insoluble aggregates, in both brain regions of YB-9-treated TG
mice, consistent with the histochemistry results of 6E10
staining (Figure 3a−d).
Here, we discovered a novel benzofuran derivative YB-9 that
disaggregates Aβ oligomers and plaques in vitro and in vivo.
Among 17 benzofuran derivatives, YB-9 showed remarkable
inhibitory effects on Aβ aggregation and disaggregating effects
on preformed Aβ aggregation. The anti-Aβ functions of YB-9
affected Aβ-induced inflammation and the expression levels of
synapse plasticity-related proteins in 5XFAD TG mice
compared with the age-matched TG littermates. Notably, in
vivo data showed that YB-9 administration mainly affected the
hippocampal region compared with the cortical region of
5XFAD mouse brains. The distinctions between hippocampus
and cortex can be explained by the uneven and local
distributions of drug throughout the brain due to the complex
and multiple factors of brain tissues.34,35 Previous studies of
distinctive drug concentration in various brain areas after the
administration have suggested few possible mechanisms
regarding this issue, such as entry of drug into specific brain
areas both across capillaries and through the cerebrospinal
fluid or varying blood flow in different brain regions.27,36
Future studies will determine how exactly, at specific sites in
the brain regions, YB-9 circulates to and dissociates Aβ
aggregates in the AD brains. Our current data further suggests
animal behavior tests to encourage YB-9 as a promising Aβ-
disaggregating drug. Though we examined the reduction in Aβ
plaque burden after YB-9 treatment, the additional animal
behavior tests evaluating cognitive function impairments and
deficits would be required to corroborate the efficacy of YB-9.
Furthermore, to develop and use YB-9 for AD therapeutic to
humans, it is essential to determine the oral dosage and
evaluate the side effects at the dosage. While it is beyond the
scope of our study, the pharmacokinetics and pharmacody-
namics of YB-9 remain to be explored more in depth in the
future.
Considering that Aβ oligomers are the most toxic species,28
the level of oligomeric Aβ, presumably contained in the soluble
brain lysates, was confirmed by dot blot assays using anti-Aβ
6E10 and antioligomer A11 antibodies (Figure 3d). 6E10
detects all forms of Aβ while A11 is specific for oligomer
subspecies.29,30 The total concentration of Aβ soluble forms
including monomers and oligomers did not change in the YB-
9-treated group compared with the nontreated group.
However, we observed decrease in the level of A11-positive
Aβ oligomers in hippocampal regions of YB-9-administered
group. Given that Aβ aggregates including oligomers largely
contributes to the brain inflammation, we observed the level of
the local inflammatory response via measurements of reactive
astrocytes with glial fibrillary acidic protein (GFAP) staining
(Figure 4a,b). Reactive astrocytes are often located near dense
Aβ plaques and contribute to Aβ clearance in the AD
brain.31,32 As a result of histochemical analyses, the decrease in
METHODS
■
Chemical Syntheses of Benzofuran Derivatives. Syntheses of
YB-10, -11, -13, -14, -15, and -17 were previously reported.19,20 3-
Arylbenzofurans were synthesized utilizing BCl3-mediated cyclo-
dehydration of aryloxyketones, dihydrobenzofurans were prepared
by catalytic hydrogenation of the corresponding benzofurans in the
presence of Pd/C (see Supporting Information for details, Figure S6−
16). Only characterization data of the newly synthesized compounds
are listed as follows:
6-Methoxy-3-(4-methoxyphenyl)-2,3-dihydrobenzofuran-5-ol
(YB-1). Ivory solid, mp: 148.0−148.6 °C; 1H NMR (400 MHz,
CDCl3) δ 7.11 (d, J = 8.4 Hz, 2H), 6.85 (d, J = 8.4 Hz, 2H), 6.59 (s,
1H), 6.49 (s, 1H), 5.23 (s, 1H), 4.85−4.81 (m, 1H), 4.55 (s, 1H),
E
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4.32 (t, J = 6.8 Hz, 1H), 3.87 (s, 3H), 3.79 (s, 3H); 13C NMR (100
MHz, CDCl3) δ 158.6, 153.5, 146.5, 139.9, 134.9, 128.7, 121.7, 114.1,
110.7, 94.1, 79.8, 56.2, 55.3, 47.9; HRMS (ESI-QTOF) m/z [M +
H]+ calcd for C16H17O4273.1121, found 273.1134.
5-(Benzyloxy)-6-methoxy-3-(4-methoxyphenyl)benzofuran (YB-
2). White solid, mp: 120.2−120.6 °C; 1H NMR (400 MHz, CDCl3) δ
5-Methoxy-3-(4-methoxyphenyl)-2,3-dihydrobenzofuran-6-ol
1
(YB-6). Orange solid, mp: 113.4−114.1 °C; H NMR (400 MHz,
CDCl3) δ 7.13 (d, J = 7.6 Hz, 2H), 6.87 (d, J = 7.2 Hz, 2H), 6.53 (s,
2H), 5.71 (s, 1H), 4.84 (t, J = 8.0 Hz, 1H), 4.56 (t, J = 8.0 Hz, 1H),
4.31 (t, J = 7.2 Hz, 1H), 3.80 (s, 3H), 3.75 (s, 3H); 13C NMR (100
MHz, CDCl3) δ 158.6, 154.6, 146.1, 135.2, 128.8, 120.7, 120.7, 114.2,
108.0, 97.3, 79.9, 56.8, 55.3, 48.1; HRMS (ESI-QTOF) m/z [M +
H]+ calcd for C16H17O4 273.1121, found 273.1109.
7.62 (s, 1H), 7.49−7.34 (m, 8H), 7.11 (s, 1H), 7.01−6.99 (m, 2H),
5.17 (s, 2H), 3.96 (s, 3H), 3.87 (s, 3H); 13C NMR (100 MHz,
CDCl3) δ 158.9, 150.9, 149.1, 145.6, 139.7, 137.3, 128.5, 128.4, 127.9,
127.6, 124.6, 121.9, 118.6, 114.4, 105.6, 95.9, 72.3, 56.4, 55.4; HRMS
(ESI-QTOF) m/z [M + H]+ calcd for C23H21O4 361.1434, found
361.1433.
5-Methoxy-3-(4-methoxyphenyl)benzofuran-6-yl methanesulfo-
nate (YB-7). White solid, mp: 133.0−133.6 °C; 1H NMR (400 MHz,
6-Methoxy-3-(4-methoxyphenyl)benzofuran-5-ol (YB-3). Pale
1
red solid, mp: 88.3−89.1 °C; H NMR (400 MHz, CDCl3) δ 7.64
CDCl3) δ 7.74 (s, 1H), 7.55 (s, 1H), 7.50 (d, J = 8.0 Hz, 2H), 7.29 (s,
1H), 7.04 (d, J = 7.6 Hz, 2H), 3.94 (s, 3H), 3.88 (s, 3H), 3.22 (s,
3H); 13C NMR (100 MHz, CDCl3) δ 159.3, 148.9, 148.6, 142.6,
139.4, 128.7, 125.9, 123.7, 122.0, 114.6, 108.4, 102.8, 56.7, 55.4, 38.2;
HRMS (ESI-QTOF) m/z [M + H]+ calcd for C17H17O6 349.0740,
found 349.0759.
(s, 1H), 7.54 (d, J = 7.6 Hz, 2H), 7.32 (s, 1H), 7.06 (s, 1H), 7.00 (d, J
= 8.0 Hz, 2H), 5.59 (s, 1H), 3.96 (s, 3H), 3.86 (s, 3H); 13C NMR
(100 MHz, CDCl3) δ 158.9, 149.9, 145.4, 142.8, 139.9, 128.4, 124.7,
121.8, 119.3, 114.4, 104.1, 94.8, 56.3, 55.4; HRMS (ESI-QTOF) m/z
[M + H]+ calcd for C16H15O4 271.0965, found 271.0959.
5-Methoxy-3-(4-methoxyphenyl)benzofuran-6-yl acetate (YB-8).
1
White solid, mp: 110.2−110.9 °C; H NMR (400 MHz, CDCl3) δ
5-Methoxy-3-(4-methoxyphenyl)-2,3-dihydrobenzofuran-6-yl
acetate (YB-4). Yellow solid, mp: 85.1−85.9 °C; 1H NMR (400
7.69(s, 1H), 7.52 (d, J = 7.6 Hz, 2H), 7.26 (s, 2H), 7.03 (d, J = 7.6
Hz, 2H), 3.88 (s, 6H), 2.37 (s, 3H); 13C NMR (100 MHz, CDCl3) δ
169.4, 159.2, 149.2, 148.3, 141.6, 138.0, 128.7, 124.6, 124.2, 122.1,
114.5, 106.5, 102.3, 56.5, 55.3, 20.7; HRMS (ESI-QTOF) m/z [M +
H]+ calcd for C18H17O5 313.1071, found 313.1075.
MHz, CDCl3) δ 7.15 (d, J = 7.6 Hz, 2H), 6.87 (d, J = 7.6 Hz, 2H),
6.61 (d, 4J = 8.0 Hz, 2H), 4.87 (t, J = 8.6 Hz, 1H), 4.60 (t, J = 8.0 Hz,
1H), 4.34 (t, J = 8.0 Hz, 1H), 3.80 (s, 3H), 3.69 (s, 3H), 2.31 (s, 3H);
13C NMR (100 MHz, CDCl3) δ 169.1, 158.8, 153.9, 145.8, 139.7,
2-((5-Methoxy-3-(4-methoxyphenyl)benzofuran-6-yl)oxy)acetic
acid (YB-9). White solid, mp: 168.1−168.9 °C; 1H NMR (400 MHz,
134.2, 128.9, 128.2, 114.2, 109.7, 104.9, 80.0, 56.7, 55.3, 48.3, 20.7;
HRMS (ESI-QTOF) m/z [M + H]+ calcd for C18H19O5 315.1227,
found 315.1225.
6-(Benzyloxy)-5-methoxy-3-(4-methoxyphenyl)benzofuran (YB-
5). Orange solid, mp: 118.5−119.4 °C; 1H NMR (400 MHz, CDCl3)
δ 7.63 (s, 1H), 7.56−7.50 (m, 4H), 7.43−7.34 (m,3H), 7.24 (s, 1H),
7.12 (s, 1H), 7.04 (d, J = 8.0 Hz, 2H), 5.24 (s, 2H), 3.96 (s, 3H), 3.89
(s, 3H); 13C NMR (100 MHz, CDCl3) δ 159.0, 150.2, 147.4, 147.2,
139.9, 136.9, 128.6, 128.5, 127.9, 127.3, 124.7, 121.9, 119.2, 114.5,
102.3, 98.3, 71.5, 56.8, 55.4; HRMS (ESI-QTOF) m/z [M + H]+
calcd for C23H21O4 361.1434, found 361.1429.
Acetone-d6) δ 7.93 (s, 1H), 7.65 (d, J = 8.8 Hz, 2H), 7.38 (s, 1H),
7.25 (s, 1H), 7.06 (d, J = 8.8 Hz, 2H), 4.81 (s, 2H), 3.92 (s, 3H), 3.86
(s, 3H); 13C NMR (100 MHz, Acetone-d6) δ 169.3, 159.2, 150.2,
F
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147.7, 146.9, 140.6, 128.3, 124.4, 121.8, 119.6, 114.4, 103.0, 98.7,
66.1, 56.1, 54.7; HRMS (ESI-QTOF) m/z [M + H]+ calcd for
C18H16NaO6 351.0839, found 351.0858.
irradiated by visible light with three times of 1 s irradiation, and the
reaction was quenched by adding 3 μL of 5× sample buffer with β-
mercaptoethanol (Sigma-Aldrich, U.S.A.). The samples were further
heated for 5 min at 95 °C, and the peptides were separated by SDS-
PAGE electrophoresis on a 1.0 mm-thick 15% gradient polyacryla-
mide gel (Gradi-Gel II, ELPIS, South Korea) and then visualized by
silver staining according to the PlusOne Silver Staining Kit protocol
(GE Healthcare, USA).
5,6-Dimethoxy-3-(4-methoxyphenyl)benzofuran-2(3H)-one (YB-
12). Orange solid, mp: 120.6−120.9 °C; 1H NMR (400 MHz,
Animal Preparation. Eight-month-old male transgenic mice
(strain name: B6SJL-Tg(APPSwF1Lon, PSEN1*M146L*L286 V)
6799Vas/Mmjax) carrying five mutations associated with early onset
familial Alzheimer’s disease (5XFAD) were acquired from Jackson
Laboratory (Maine, U.S.A.) and have been conserved through mating
with C57BL/6 X SJL wild-type mice. All mice were bred in a
laboratory animal breeding room at Yonsei University (Seoul, South
Korea) under regulated conditions with 12h/12h light-dark phase.
Food and water were provided ad libitum. All animal experiments
were conducted in accordance with the National Institutes of Health
(NIH) Guide for the Care and Use of Laboratory Animals. The
research protocols were authorized by the Institutional Animal Care
and Use Committee of Yonsei University.
Oral Administration of YB-9. YB-9 in drinking water was freely
administered to 5XFAD (male) mice for 8 weeks with 50 mg/kg/day
dosages (n = 5). For control groups, TG mice (male, n = 5) and age-
matched WT mice (male, n = 5) were treated with water (5% DMSO
in drinking water). In order to regulate the exact dosage, all
administered compounds in drinking water were set according to the
average daily consumption in each cage and recalculated every other
day.
CDCl3) δ 7.17 (d, J = 6.4 Hz, 2H), 6.90 (s, 1H), 6.83 (s, 1H), 6.70
(d, J = 6.4 Hz, 2H), 5.83 (s, 1H), 3.93 (s, 3H), 3.84 (s, 3H), 3.66 (s,
3H); 13C NMR (100 MHz, CDCl3) δ 158.5, 148.5, 148.4, 147.1,
146.5, 129.8, 124.4, 120.9, 119.4, 113.4, 101.3, 94.9, 56.4, 56.2, 55.1;
HRMS (ESI-QTOF) m/z [M + H]+ calcd for C17H14O5 301.1071,
found 301.1054.
6-Methoxy-3-(4-methoxyphenyl)benzofuran-4-carboxylic acid
1
(YB-16). White solid, mp: 187.2−187.6 °C; H NMR (400 MHz,
Histochemistry of Mouse Brains. Brain tissues were fixed in 4%
paraformaldehyde (Biosesang, South Korea) overnight at 4 °C and
immersed in 30% sucrose for 48 h for cryoprotection. Brain sections
(35 μm) were cut with a cryostat (CM1860, Leica) and attached to
slides. The antigen retrieval on fixed brain sections was conducted
using 1% SDS (Biosesang, South Korea) in 1× PBS (Gibco, South
Korea) for 10 min, followed by blocking with 20% horse serum in 1×
PBS for an hour. Then, we incubated the slides at 4 °C overnight with
mouse monoclonal antibody 6E10 (1:200, BioLegend, U.S.A.) for Aβ
plaques or GFAP (1:300, MilliporeSigma, U.S.A.) for detection of
activated astrocytes. On the next day, the slides were incubated with
goat antimouse IgG conjugated with Alexa Fluor Plus 488 (1:200, Life
Technologies, U.S.A.) or goat antichicken IgG conjugated with Alexa
Fluor 568 (1:200, Life Technologies, U.S.A.) as a secondary antibody
for 6E10 or GFAP, respectively, for an hour at room temperature. The
amyloid plaques in the cortex and the hippocampus of the fixed brain
sections were visualized under a fluorescence microscope (DM500,
Leica), which is equipped with filter cubes containing excitation and
emission filters: A4 filter cube for Hoechst staining detection
(excitation filter: BP 360/40; dichromatic mirror: 400; emission
filter: BP 470/40), N2.1 filter cube for 6E10 staining detection
(excitation filter: BP 515−560; dichromatic mirror: 580; emission
filter: LP 590), and L5 filter cube for GFAP detection (excitation
filter: BP 480/40; dichromatic mirror: 505; emission filter: BP 527/
30). Numbers and area of plaques were obtained by Image-J software
(NIH). Analyses of plaque distributions were transcribed manually to
the computer-acceptable style while keeping the research colleagues
blind.
CDCl3) δ 7.58 (s, 1H), 7.44 (s, 1H), 7.26 (s, 1H), 7.25 (d, J = 7.2 Hz,
2H), 6.89 (d, J = 8.0 Hz, 2H), 3.92 (s, 3H), 3.79 (s, 3H); 13C NMR
(100 MHz, CDCl3) δ 170.1, 158.9, 157.1, 143.3, 130.8, 129.9, 125.3,
123.9, 122.5, 119.5, 113.8, 113.3, 100.9, 56.0, 55.2; HRMS (ESI-
QTOF) m/z [M + H]+ calcd for C17H15O5 299.0914, found
299.0936.
ThT Fluorescence Assay. The ThT fluorescence assay was
performed to confirm Aβ fibrilization and to quantify the β-sheet
complex of Aβ aggregates.37 In-house synthetic Aβ42 peptides were
dissolved in DMSO (1 mM), obtained from Sigma-Aldrich (Missouri,
U.S.A.), and distilled with deionized water to make Aβ solution (100
μM). During the inhibition assay, benzofuran derivatives dissolved in
DMSO (100 mM) and diluted with deionized water to three different
concentrations (0.5, 5, and 50 μM) were incubated with monomeric
Aβ42 (final concentration of 25 μM) at 37 °C for 3 days. During the
disaggregation assay, Aβ42 (100 μM) only was incubated at 37 °C for
3 days to initially prepare Aβ aggregates. Then, Aβ aggregates were
mixed with benzofuran derivatives (0.5, 5, 50, and 500 μM) and
reincubated for additional 3 days at 37 °C. After the incubation, 25 μL
of the samples and 75 μL of ThT solution (5 μM ThT in 50 mM
glycine buffer, pH 8.9) were loaded in a 96-well half area black plate.
ThT was purchased from Sigma-Aldrich (Missouri, U.S.A.), and a 96-
well half area black plate was purchased from Corning (New York,
U.S.A.). Fluorescence intensities were measured at 450 nm
(excitation) and 485 nm (emission) by using a microplate reader
(Infinite 200 PRO, Tecan).
Lysate Preparation. To prepare brain lysates, mice were
sacrificed, and hippocampal and cortical regions of mouse brains
were dissected separately. Each brain region was homogenized in ice-
cold RIPA buffer (20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.5% NP-
40, 4 mM EDTA, 0.1% SDS, 0.5% sodium deoxycholate) containing
1× protease inhibitor cocktail (Roche Diagnostics, Switzerland).
Homogenized brain tissues were incubated in ice for 20 min before
centrifugation at 14 000 rpm at 4 °C for 30 min. The supernatants
(soluble fraction) of brain lysates were collected. To gain Aβ-insoluble
fraction in brain lysates, the remaining pellet was rehomogenized
using guanidine hydrocholoride buffer (5 mM GdnHCl, 50 mM Tris-
HCl, pH 8.0) mixed with 1× proteinase inhibitor cocktail. The
mixtures were incubated at 37 °C for 3 h on a multi mixer in order to
SDS-PAGE with PICUP. In order to evaluate the levels of Aβ
oligomers, protofibrils, and fibrils, SDS-PAGE analysis along with
PICUP is usually performed.24 To confirm the disaggregating effects
of YB compounds, Aβ peptide (50 μM) incubated by itself for 3 days
at 37 °C was reincubated after the addition of benzofuran derivatives
(250 μM) for additional 3 days at the same temperature. For Aβ
peptides cross-linking, 10 mM tris(2,2′-bipyridyl)dichlororuthenium-
(II) hexahydrate (Ru(Bpy)) and 200 mM ammonium persulfate
(APS) were dissolved in sodium phosphate buffer (0.1 M, pH 7.4),
and they were diluted with the same buffer to make 1 mM and 20 mM
respectively. Both Ru(Bpy) and APS were purchased from Sigma-
Aldrich. Then, 1 μL of both 1 mM Ru(Bpy) and 20 mM APS was
added to 10 μL of each incubated sample. The mixed solutions were
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thoroughly dissolve Aβ-insoluble fractions and centrifuged at 14 000
rpm at 4 °C for 2 h. After the centrifugation, the supernatants,
supposedly insoluble brain extractions, were collected. The protein
concentrations in supernatants were quantified via Pierce BCA
protein assay kit (Thermo Fisher Scientific, U.S.A.).
Wisconsin Center for NanoBioSystems, University of
Wisconsin, Madison, Wisconsin 53705, United States
Gi Hun Bae − Department of Pharmacy and Yonsei Institute
of Pharmaceutical Science, Yonsei University, Incheon 21983,
Republic of Korea
Quantification Aβ-Insoluble Fraction of Brain Lysates with
ELISA and Aβ-Soluble Fraction with of Dotting Analyses.
Measurements of Aβ42 in insoluble fractions were performed by
Aβ42 sandwich ELISA kit (Invitrogen, U.S.A.). 0.5 μg of protein
samples was used according to the manufacturer’s instructions. To
confirm Aβ42 oligomer formation in both the hippocampal and
cortical regions of 5XFAD mice brains, dot blot analysis was
performed.29 Briefly, 17 μg (for 6E10) or 5 μg (for A11) of brain
lysates were loaded on a nitrocellulose membrane and dried for 30
min then blocked with a 5% skim milk (Becton, Dickinson and
Company, U.S.A.) solution for 1 h. The membrane was incubated
with 6E10 (1:2000, BioLegend, U.S.A.) or A11 (1:2000, Invitrogen,
U.S.A.) overnight at 4 °C. After the overnight incubation, membranes
were incubated wit HRP-conjugated goat antimouse or antirabbit
secondary antibody (1:10000, Bethyl Laboratories, U.S.A.) for 6E10
and A11, respectively. All washes were performed with TBS-T, three
times for 5 min, except for the last wash which took for 10 min.
Western Blots. Western blot analysis was conducted to detect
synaptophysin, a presynaptic marker, and PSD-95, a postsynaptic
marker.33 20 μg of brain lysates was separated by SDS-PAGE and
transferred to a nitrocellulose membrane. Then, the membrane was
blocked with a 5% skim milk solution and probed with primary
antibodies against either PSD-95 (1:2000, Invitrogen, U.S.A.) or
synaptophysin (1:1000, MilliporeSigma, U.S.A.). After being washed
with TBST, membranes were incubated with HRP-conjugated goat
antimouse secondary antibody (1:10000, Bethyl Laboratories,
U.S.A.). Then, membranes were washed three times with TBST
again, and the proteins were revealed using ECL kit (Thermo Fisher
Scientific, U.S.A.). The level of β-actin (a loading control) was
confirmed by a mouse anti-β-actin antibody (1:1000, MilliporeSigma,
U.S.A.) as a loading control.
Hye Yun Kim − Department of Pharmacy and Yonsei Institute
of Pharmaceutical Science, Yonsei University, Incheon 21983,
Republic of Korea; orcid.org/0000-0003-3757-5652
Hanna Jeon − Department of Pharmacy and Yonsei Institute
of Pharmaceutical Science, Yonsei University, Incheon 21983,
Republic of Korea
Kyeonghwan Kim − Department of Pharmacy and Yonsei
Institute of Pharmaceutical Science, Yonsei University,
Incheon 21983, Republic of Korea
Jisu Shin − Department of Pharmacy and Yonsei Institute of
Pharmaceutical Science, Yonsei University, Incheon 21983,
Republic of Korea
Sunhee Lee − Department of Pharmacy and Yonsei Institute of
Pharmaceutical Science, Yonsei University, Incheon 21983,
Republic of Korea
Seungpyo Hong − Department of Pharmacy, Yonsei Institute
of Pharmaceutical Science, and Yonsei Frontier Lab, College
of Pharmacy, Yonsei University, Incheon 21983, Republic of
Korea; Pharmaceutical Sciences Division and Wisconsin
Center for NanoBioSystems, University of Wisconsin,
Madison, Wisconsin 53705, United States; orcid.org/
0000-0001-9870-031X
Complete contact information is available at:
https://pubs.acs.org/10.1021/acschemneuro.0c00606
Author Contributions
#D.K. and G.H.B. contributed equally to this work. G.H.B. and
S.L. synthesized 17 benzofuran derivatives. D.K. performed all
experiments of amyloid aggregation assay, disaggregation
assays, and in vivo tests. H.J. performed preliminary in vitro
assessments of this study. K.K. synthesized Aβ peptides. J.S.
participated in the mouse model preparation. D.K., H.Y.K.,
I.K., and Y.K. analyzed all data of experiments and wrote the
paper. Y.K. and I.K. designed the research, performed the
literature review, and supervised the whole study.
Statistical Analysis. All graphs were obtained with GraphPad
Prism 8.0 software, and all statistical analyses were conducted with
one-way ANOVA followed by Bonferroni’s posthoc comparisons (*P
< 0.033, **P < 0.002, ***P < 0.001). The error bars represent the
standard error of the mean (SEM).
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acschemneuro.0c00606.
Funding
This work was supported by Korea Health Industry Develop-
ment Institute (KHIDI, HI18C0836), National Research
Foundation of Korea (NRF-2018R1A6A1A03023718, NRF-
2018R1D1A1B07048857, NRF-2018M3C7A1021858, and
NRF-2020R1A2C2005961), and POSCO TJ Foundation
(POSCO Science Fellowship).
Full images of the gel with silver staining, dot blot, and
Western blot analyses; histochemical analyses of WT
1
mice; H and 13C NMR data of synthetic compounds
(PDF)
Notes
AUTHOR INFORMATION
Corresponding Authors
■
The authors declare no competing financial interest.
Ikyon Kim − Department of Pharmacy and Yonsei Institute of
Pharmaceutical Science, Yonsei University, Incheon 21983,
Republic of Korea; orcid.org/0000-0002-0849-5517;
Email: ikyonkim@yonsei.ac.kr
YoungSoo Kim − Department of Pharmacy and Yonsei
Institute of Pharmaceutical Science, Yonsei University,
Incheon 21983, Republic of Korea; orcid.org/0000-0001-
5029-7082; Email: y.kim@yonsei.ac.kr
ACKNOWLEDGMENTS
■
All images are created by authors of this manuscript. All
experimental protocols including animal tests in the article
were approved by Yonsei University (IACUC-202003-1038-
01).
ABBREVIATIONS
■
AD, Alzheimer’s disease; Aβ, amyloid-β; APP, amyloid
precursor protein; 5XFAD, five familial AD mutations;
GFAP, glial fibrillary acidic protein; NIH, National Institutes
of Health; PICUP, photoinduced cross-linking of unmodified
proteins; TG, transgenic; ThT, thioflavin T; WT, wild type
Authors
DaWon Kim − Department of Pharmacy and Yonsei Institute
of Pharmaceutical Science, Yonsei University, Incheon 21983,
Republic of Korea; Pharmaceutical Sciences Division and
H
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Research Article
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