Multiple reactivities of flavonoids towards pathological elements in Alzheimer's disease: Structure-activity relationship

Article abstract of DOI:10.1039/d0sc02046j

Amyloid-β (Aβ) accumulation, metal ion dyshomeostasis, oxidative stress, and cholinergic deficit are four major characteristics of Alzheimer's disease (AD). Herein, we report the reactivities of 12 flavonoids against four pathogenic elements of AD: metal-

Full text of DOI:10.1039/d0sc02046j

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Multiple reactivities of flavonoids towards
pathological elements in Alzheimer's disease:
Cite this: DOI: 10.1039/d0sc02046j
structure–activity relationship†
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
bc
d
Geewoo Nam,‡a Mannkyu Hong,
‡
Juri Lee,b Hyuck Jin Lee,
Yonghwan Ji,a
bc
b
Juhye Kang,b Mu-Hyun Baik
and Mi Hee Lim
*
*
Amyloid-b (Ab) accumulation, metal ion dyshomeostasis, oxidative stress, and cholinergic deficit are four
major characteristics of Alzheimer's disease (AD). Herein, we report the reactivities of 12 flavonoids
against four pathogenic elements of AD: metal-free and metal-bound Ab, free radicals, and
acetylcholinesterase. A series of 12 flavonoids was selected based on the molecular structures that are
responsible for multiple reactivities including hydroxyl substitution and transfer of the B ring from C2 to
C3. Our experimental and computational studies reveal that the catechol moiety, the hydroxyl groups at
C3 and C7, and the position of the B ring are important for instilling multiple functions in flavonoids. We
establish a structure–activity relationship of flavonoids that should be useful for designing chemical
reagents with multiple reactivities against the pathological factors of AD.
Received 11th April 2020
Accepted 7th September 2020
DOI: 10.1039/d0sc02046j
rsc.li/chemical-science
Ab),9–12 and reactive oxygen species (ROS)13 corroborates the
complex nature of the disease. Ab, the major component of
Introduction
senile plaques, is an aggregation-prone peptide composed of
36–43 amino acid residues.7 Ab aggregation is a subject of
intensive research and Ab oligomers were recently identied as
potential toxic species capable of disrupting neuronal homeo-
stasis.14 Metal ions present two neurochemical implications: (i)
they function as essential cofactors and structural anchors in
enzymatic reactions and protein folding and (ii) they exhibit
neurotoxicity by catalyzing the generation of ROS.10 For these
reasons, metal ions are tightly regulated in biological systems.
Dyshomeostasis and miscompartmentalization of metal ions
such as Cu(II) and Zn(II) are observed in the brains of AD
patients and linked to neurodegeneration.10 Moreover, metal
ions can affect the aggregation and conformation of Ab by
directly binding to the peptide.9,10,15 Based on their physiolog-
ical roles and reactivity with Ab, metal ions are considered an
important part of AD pathogenesis.16 Research regarding the
pathological relationship between metal ions and Ab intro-
duced the concept of metal–Ab as a pathogenic factor based on
its potential toxicity and involvement in producing ROS.10,16,17
Lastly, oxidative stress, characterized by the imbalance between
the formation and removal of ROS, has been indicated in
a spectrum of diseases including AD for detriments such as
lipid peroxidation and peptide oxidation.18,19
Alzheimer's disease (AD) is a progressive neurodegenerative
disorder responsible for a majority of dementia cases.1,2 Despite
extensive research aimed at developing therapeutics, there is no
cure and our capability of controlling the development and
progression of AD is severely limited.2,3 Currently available
symptomatic treatments offer temporary relief using acetyl-
cholinesterase (AChE) inhibitors and N-methyl-D-aspartic acid
receptor antagonists.3 AChE terminates cholinergic trans-
mission at the synapse by catalyzing the hydrolysis of the
neurotransmitter, acetylcholine (ACh).4 The cholinergic
hypothesis identies the reduced level of ACh as a primary
culprit in the pathogenesis of AD.5 In recent years, however, the
inability of AChE inhibitors to effectively halt the progressive
neurodegeneration in AD has led to a re-evaluation of this
hypothesis.6 The prevailing perception of AD pathology reects
the multifaceted quality of the disease.2
Identication of additional pathological contributors of AD
such as amyloid-b (Ab),7 metal ions,8 metal-bound Ab (metal–
aDepartment of Chemistry, Ulsan National Institute of Science and Technology
(UNIST), Ulsan 44919, Republic of Korea
bDepartment of Chemistry, Korea Advanced Institute of Science and Technology
(KAIST), Daejeon 34141, Republic of Korea. E-mail: miheelim@kaist.ac.kr
cCenter for Catalytic Hydrocarbon Functionalizations, Institute for Basic Science (IBS),
Daejeon 34141, Republic of Korea. E-mail: mbaik2805@kaist.ac.kr
dDepartment of Chemistry Education, Kongju National University, Gongju 32588,
Republic of Korea
Attempts to exploit these pathological elements for the
development of single target-based therapeutics have been
made, but they were largely shown to be clinically ineffec-
tive.20,21 As a result, research efforts have shied towards
understanding the connections among the various pathogenic
pathways in AD. There is growing recognition that chemical
† Electronic supplementary information (ESI) available: Experimental section and
Fig. S1–S5. See DOI: 10.1039/d0sc02046j
‡ These authors contributed equally to this work.
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reagents capable of simultaneously targeting and modulating
multiple pathological features are necessary as potential ther-
apeutic candidates and chemical tools in elucidating the
pathology of AD at the molecular level. In this work, we evalu-
ated the reactivities of avonoids against the pathological
elements of AD such as metal-free and metal-bound Ab, free
radicals, and AChE and determined the structural features
responsible for their versatile reactivities, as depicted in Fig. 1.
Flavonoids are a family of phytochemicals exhibiting low
toxicity,22 anti-/pro-oxidant activity,23 and numerous utilitarian
biological activities (e.g., anti-cancer,24 anti-viral,25 and anti-
bacterial26). The direct administration of avonoids, however,
presents limitations in practical applications. For example, the
solubility and stability of avonoids oen present challenges.
Therefore, understanding the molecular structures of avonoids
and identifying their pharmacophores could be valuable in
designing small molecules with multiple reactivities for investi-
gating and treating AD. In this work, the modulative reactivities
of 12 avonoids enumerated in Fig. 1 towards metal-free Ab,
metal–Ab, free radicals, and AChE were assessed and analyzed
based on their molecular structures to identify the structural
moieties critical for their reactivities. Moreover, computational
studies were carried out to obtain a more in-depth perspective of
their activities against free radicals and AChE.
Results and discussion
Rational selection
Quercetin (Que), luteolin (Lut), and orobol (Oro) were included
in our study because they have been previously tested against
several pathogenic elements of AD. Both Que and Lut exhibited
a variety of biological activities23,24,26 including the ability to
modulate the aggregation of metal–Ab and inhibit AChE
activity.27,28 Oro was found to interact with metal ions, alter the
aggregation of both metal-free and metal-bound Ab, scavenge
free radicals, and inhibit the catalytic activity of AChE.29,30 The
12 avonoids enumerated in Fig. 1 include three different
classes, namely avonols (row 1), avones (row 2), and iso-
avones (row 3), that offer signicant structural variance. These
avonoids were chosen according to three structural criteria: (i)
inclusion of the chromone framework containing the 4-oxo
group and the double bond between C2 and C3 on the C ring;
(ii) variation in hydroxyl groups at C3 (row 1 vs. row 2), C30, C40,
and C7 (columns 1, 2, and 3, respectively); (iii) change in the
position of the B ring from C2 to C3 (row 2 vs. row 3). Previous
studies identied the 4-oxo group and the unsaturated bond
between C2 and C3 on the C ring to be important in various
chemical and biological properties such as their metal-binding
ability and antioxidant activity.28,31,32 Moreover, the unsaturated
Fig. 1 Rational selection of 12 flavonoids. The presented library of flavonoids was chosen based on structural variations including the number
and position of hydroxyl groups and the location of the B ring to identify the structural features responsible for reactivities against multiple
pathological factors found in AD [i.e., metal-free Ab, metal–Ab, free radicals, and AChE].
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C2–C3 bond may contribute to interactions of the compounds rings, as previously reported.38 The aforementioned band of
with the hydrophobic regions of Ab such as the self-recognition Que and kaempferol (Kae), however, was diminished at greater
site and the C-terminal region that are essential for peptide Cu(II) concentrations, indicative of their oxidation.39 Together,
aggregation.33–36 The 4-oxo functionality can form 5- and 6- these observations conrm that all of the selected avonoids
membered metal chelation sites with the 3-OH or 5-OH group, interact with Cu(II) in solution. Unfortunately, quantitative
respectively. The catechol moiety on the B ring can also chelate measurements for metal-binding affinities of the avonoids
metal ions. In addition, the number and location of electron- were not possible due to the limited stability of their corre-
donating hydroxyl groups can impact the avonoids' redox sponding complexes.
potentials that direct their capacity to quench free radicals.37
These 12 avonoids were obtained through commercial sources
Modulation on the aggregation of metal-free Ab42 and metal–
or prepared in our laboratory in the cases of Oro29 and 5-
Ab42
hydroxyisoavone (HIF). The synthetic procedure and charac-
terization for HIF are presented in Scheme 1 and the ESI To determine the effects of the avonoids on the aggregation of
(Fig. S1†).
metal-free and metal-treated Ab42, the molecular weight (MW)
distribution and morphology of the resultant Ab42 species upon
incubation with the compounds were analyzed by gel electro-
phoresis with Western blotting (gel/Western blot) and trans-
mission electron microscopy (TEM), respectively. The amount
of b-sheet-rich aggregates generated through metal-free and
metal-induced Ab42 aggregation with treatment of the avo-
noids could not be quantitatively determined due to the optical
interference from the compounds within the analysis window of
the assays (e.g., thioavin-T assay). Two types of Ab aggregation
experiments were performed: inhibition and disaggregation
experiments, as summarized by Fig. 3 and 4. For the inhibition
experiments, freshly prepared metal-free or metal-treated Ab42
was incubated with the avonoids for 24 h to assess whether the
compounds could inhibit the formation of peptide aggregates.
In the disaggregation experiments, the samples containing Ab42
with and without metal ions were pre-incubated for 24 h to
produce peptide aggregates and then treated with the avo-
noids for an additional 24 h. The disaggregation studies
determined whether the avonoids could disassemble pre-
formed peptide aggregates or modulate their aggregation.
Under our experimental conditions, Ab42 alone spontaneously
aggregated to yield a heterogeneous mixture of Ab ensembles
including large aggregates that were too big to penetrate the gel
matrix and be visualized by gel/Western blot. Such large
aggregates were probed by TEM.40,41 On the other hand, smaller
Ab42 species such as monomers and low MW oligomers indi-
cated visible bands in gel/Western blot, but they were not
detected by TEM.42,43 To further examine the gel/Western blot
data, the band intensities of the gel images were analyzed using
the ImageJ soware.
Interaction with Cu(II)
To verify that the avonoids interact with metal ions in solution,
UV-Vis spectra were monitored. Signicant spectral changes in
the UV-Vis range were observed upon adding Cu(II) to avo-
noids, as illustrated in Fig. 2. Two characteristic p–p* transition
bands were noted: band I associated with the B ring of avo-
noids (l ¼ ca. 300–400 nm) and band II associated with the A
ring (l ¼ ca. 240–280 nm).38 Incubation with Cu(II) showed
optical shis of either or both band I and II, e.g., bathochromic/
hypsochromic and hyper/hypochromic shis, indicative of
interactions between Cu(II) and the oxygen (O) donor atoms in
the A, B, and C rings of the avonoids such as 4-oxo/5-OH, 3-
OH/4-oxo, and 30-OH/40-OH. An absorption band was observed
at ca. 400–440 nm with the addition of Cu(II). This band is
associated with Cu(II) binding to avonoids with implications at
the extended resonance structure encompassing the B and C
In the inhibition experiments, as shown in Fig. 3b, the
introduction of Que, Lut, and Oro showed a minor change in
the MW distribution of metal-free Ab42. The signal intensities of
the bands corresponding to smaller oligomeric Ab42 species
(from 7 to 15 kDa) were slightly reduced upon treatment of Que,
Lut, and Oro. In the case of Cu(II)–Ab42, Que, Lut, Oro, and Kae
modied the aggregation of the peptide: Que increased the
smearing band intensity at ca. 7–270 kDa, Lut and Kae
increased the signal in the range of ca. 7–50 kDa, and Oro
signicantly decreased the band intensity of the entire range (7–
270 kDa). Moreover, Que, Lut, and Oro altered the aggregation
of Zn(II)–Ab42, resulting in diverse MW distributions from ca. 15
to 50 kDa. Kae did not signicantly inuence the aggregation of
Scheme 1 Synthetic routes to 5-hydroxyisoflavone (HIF).
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Fig. 2 Interaction of the flavonoids with Cu(II) monitored by UV-Vis spectroscopy. Conditions: [flavonoid] ¼ 25 mM; [CuCl2] ¼ 0, 12.5, 25, 50, and
125 mM; 20 mM HEPES, pH 6.6, 150 mM NaCl or EtOH (for Chr and HF). The grey spectra were obtained by addition of 0.5, 1, and 2 equiv of CuCl2.
Ab42 in the absence and presence of Zn(II). Apigenin (Api),
In the disaggregation experiments, as illustrated in Fig. 4b,
genistein (Gen), galangin (Gal), chrysin (Chr), 5,7-dihydrox- Que and Oro very mildly varied the MW distribution of pre-
yisoavone (DHIF), 3,5-dihydroxyavone (DHF), 5-hydroxy- formed metal-free Ab42 aggregates. In the case of Cu(II)–Ab42,
avone (HF), and HIF did not exhibit signicant reactivity Que, Lut, Oro, and Kae exhibited reactivity towards preformed
against metal-free or metal-induced Ab42 aggregation. As Ab42 aggregates: (i) Que increased the intensity of the bands
summarized in Fig. 3c, the quantication of the gel/Western larger than ca. 15 kDa; (ii) Lut increased the signal corre-
data further indicated the degree of the avonoids' reactivities sponding to Ab species in the range of ca. 15–100 kDa while
towards the formation of metal-free and metal-added Ab42 decreasing that of the species larger than 100 kDa; (iii) Oro
aggregates. The changes in the band intensity in the gel for the reduced the signal intensity of all MW species below ca. 50 kDa;
metal-free conditions were denitively smaller relative to those (iv) Kae increased smearing in the MW range from ca. 15 to 270
of the metal-treated conditions.
kDa. The size distribution of preformed Zn(II)–Ab42 aggregates
As depicted in Fig. 3d, TEM images corroborated the effect upon treatment with Que and Oro differed from that of the
of the avonoids on Cu(II)-induced Ab42 aggregation. The compound-free peptide sample. Que led to the enhanced
avonoids exhibiting noticeable modulative reactivity towards intensity of the bands in the range ca. 15–240 kDa, while Oro
the aggregation of Cu(II)–Ab42 in gel/Western blot led to resulted in a more signicant change in the smearing band of
notable morphological changes in the peptide aggregates: (i) the overall range (ca. 7–270 kDa). Api, Gen, Gal, Chr, DHIF,
Que gave signicantly shorter brillar aggregates; (ii) Lut DHF, HF, and HIF did not exhibit notable reactivity with pre-
decreased the degree of branching in the brils; (iii) Oro formed metal-free and metal-induced Ab42 aggregates.
afforded thinner and shorter aggregates; (iv) Kae generated Demonstrating modulative reactivity towards preformed Cu(II)–
amorphous assemblies distinct from native Ab42 brils. As Ab42 aggregates, Lut and Kae did not impact the disaggregation
expected, Gal and HF did not signicantly alter the morphol- samples of metal-free Ab42 and Zn(II)–Ab42. The gel quantica-
ogies of Ab42 aggregates regardless of the presence of Cu(II) or tion data further supported the signicant effects of the reactive
Zn(II). When Zn(II)–Ab42 was incubated with Que, Lut, or Oro avonoids on the aggregation of metal-free Ab42 and metal–
that modied Zn(II)–Ab42 aggregation in the gel/Western blot Ab42, as presented in Fig. 4c.
studies, thinner chopped brils were detected. In the presence
As illustrated in Fig. 4d, morphological changes in pre-
of Kae, Gal, and HF, brillar aggregates similar to those from formed metal-free and metal-treated Ab42 aggregates in the
the compound-free Zn(II)–Ab42 sample were produced. None of presence of the avonoids were monitored by TEM. As for
the avonoids studied via TEM exhibited the ability to preformed Cu(II)–Ab42 aggregates, Que, Lut, Oro, and Kae
particularly change the aggregate morphology of metal-free exhibiting modulative reactivity in the gel/Western blots altered
Ab42
.
the morphologies of the resultant aggregates with indication of
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Fig. 3 Influence of the flavonoids on the aggregation of metal-free and metal-treated Ab42. (a) Scheme of the inhibition experiments. (b) Analysis
of the MW distribution of the resultant Ab42 species by gel/Western blot with an anti-Ab antibody (6E10). The original gel images are presented in
Fig. S2a.† (c) Quantification of Ab42 species visualized in the gel by the ImageJ software. The intensity of the gel from the sample was normalized
to that of the corresponding control (ISample/IControl). (d) TEM images of the samples from (b). Conditions: [Ab42] ¼ 25 mM; [CuCl2 or ZnCl2] ¼ 25
mM; [flavonoid] ¼ 50 mM; pH 7.4 [for metal-free and Zn(II)-containing samples] or pH 6.6 [for Cu(II)-added samples]; 37 ꢀC; 24 h incubation;
constant agitation.
a mixture of amorphous aggregates and thinner chopped brils. with two potential metal chelation sites, i.e. 4-oxo/5-OH on the
Treatment of Que and Oro to preformed Zn(II)–Ab42 aggregates A/C rings and 30-/40-OH on the B ring, demonstrated notable
produced shorter brils. As expected from the gel/Western blot modulation on the aggregation of both metal-free and metal-
experiments, the avonoids lacking modulative reactivity in the added Ab42. The pertinence of the 3-OH group in the avo-
disaggregation experiments indicated no signicant variation noids' modulative reactivity towards Ab42 was denoted by the
in the morphology of preformed peptide aggregates.
distinctions between (i) Kae and Api and (ii) Que and Lut. Kae
In general, increasing the number of hydroxyl substituents displayed notable reactivity with Cu(II)–Ab42 in both inhibition
on the backbone promotes the ability of the avonoids to and disaggregation experiments, while Api did not show such
modify the aggregation pathways of metal–Ab42. Among the reactivity. Moreover, Que altered the MW distributions of Zn(II)–
avonoids, the isoavone Oro carrying four hydroxyl groups Ab42 in both inhibition and disaggregation experiments, but Lut
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Fig. 4 Impact of the flavonoids on the disassembly and aggregation of preformed metal-free and metal-added Ab42 aggregates. (a) Scheme of
the disaggregation experiments. (b) Analysis of the MW distribution of the resultant Ab42 species by gel/Western blot with an anti-Ab antibody
(6E10). The original gel images are presented in Fig. S2b.† (c) Quantification of Ab42 species visualized in the gel by the ImageJ software. The
intensity of the gel from the sample was normalized to that of the corresponding control (ISample/IControl). (d) TEM images of the samples from (b).
Conditions: [Ab42] ¼ 25 mM; [CuCl2 or ZnCl2] ¼ 25 mM; [flavonoid] ¼ 50 mM; pH 7.4 [for metal-free and Zn(II)-containing samples] or pH 6.6 [for
Cu(II)-added samples]; 37 ꢀC; 24 h incubation; constant agitation.
only notably inhibited the generation of Zn(II)–Ab42 aggregates. and Ab. Catechol-type avonoids were reported to undergo
Structurally comparing Kae, Gal, and Api, a connection between oxidation to form o-quinones that in turn covalently bind to the
the 3-OH and 40-OH groups on the C and B rings, respectively, lysine residues of Ab.44 Our results conrm the importance of
can be recognized regarding the avonoid's inuence on the the catechol moiety in the avonoids' modulative reactivity
aggregation of metal–Ab42. The presence of the 3-OH or 40-OH towards Ab in the absence and presence of metal ions and
functionality alone (Gal and Api, respectively) does not result in further support that such reactivity could be maintained and
the reactivity towards metal–Ab42 aggregation; however, the may be altered by changing the location of the B ring catechol
presence of both hydroxyl groups on the B and C rings (Kae) group, as observed with Oro. Taken together, the aggregation
leads to noticeable reactivity. The catechol moiety on the B ring and disaggregation studies reveal three major structural
(Que, Lut, and Oro) fostered their interactions with metal ions features of avonoids associated with their ability to modify the
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aggregation pathways of metal-free Ab and metal–Ab: (i) the 3- by the 4-oxo functionality and the double bond between C2 and
OH group on the C ring, (ii) the catechol moiety on the B ring, C3.46 Conjugation of the A, B, and C rings resulting in the
and (iii) the position of the B ring.
stabilization of the avonoid radical through resonance may be
critical for their scavenging activity against free radicals.46 Our
results employing Chr, DHIF, HF, and HIF, however, indicate
that the 4-oxo group and the unsaturated C2–C3 bond do not
guarantee a molecule's ability to quench free radicals effec-
tively. The difference in the scavenging capacity against free
radicals between avonols and avones, as shown in Fig. 1 (row
1 and 2), respectively, may be prompted by the presence of
electron-donating groups, especially at C3, that could lower
their redox potentials (vide infra).47 These observations suggest
that the 3-OH group on the C ring is an important structural
feature for the free radical scavenging capability of avonoids.
Que, Lut, and Oro containing a catechol moiety on the B ring
displayed the best inhibitory activity against free organic radi-
cals according to the TEAC assay. Catechol and “catechol-type
avonoids” are recognized for their notable antioxidant
activity and mechanistic studies further support their signi-
cance.48,49 Conversely, the avonoids possessing the 40-OH
group without 30-OH, as depicted in Fig. 1 (column 2), except
Kae, did not demonstrate noticeably great antioxidant activity,
further emphasizing the signicance of the catechol moiety and
the 3-OH group for the ability to scavenge free radicals.
Scavenging free organic radicals
To evaluate the capacity of the avonoids for quenching free
organic radicals relative to the vitamin E analog Trolox, the
Trolox equivalent antioxidant capacity (TEAC) assay was con-
ducted with the cationic radical form of 2,20-azino-bis(3-
ethylbenzthiazoline-6-sulphonic acid) as the organic radical
substrate.45 As presented in Fig. 5a, the avonols Que, Kae, Gal,
and DHF (Fig. 1, row 1) exhibited notable scavenging capacity
against free organic radicals, suggesting the importance of the
3-OH group on the C ring for this activity. As expected, Que, Lut,
and Oro carrying a catechol moiety on the B ring showed
signicant antioxidant activity. Among the selected avonoids,
Que embodying both the 3-OH functionality and the catechol
moiety was the most effective scavenger against free organic
radicals. In contrast, Api, Chr, DHIF, HF, and HIF presented
negligible radical scavenging activity, compared to Trolox. The
antioxidant properties of the avonoids are reportedly affected
Redox potentials
To verify the antioxidant activity of our avonoid series, the
redox potentials of all compounds were computed following
a previously reported method.50 Attempts to experimentally
determine the redox potentials were made, but their instability
and limited solubility in both aqueous media and organic
solvents hampered electrochemical measurements. As
summarized in Fig. 5a, the computed redox potentials (Eꢀ vs.
SHE) of the avonoids support that the incorporation of
electron-donating hydroxyl groups into the molecular frame-
work lowers the redox potentials. Furthermore, the redox
potentials depended not only on the number of electron-
donating hydroxyl groups but also on their positions affecting
the degree of p-conjugation between the core skeleton of the
molecule and the functional group. Directly comparing the
changes in the calculated Eꢀ suggests that the impact of
a hydroxyl group on the B or C ring is more prominent than that
on the A ring. The 40-OH (column 2 vs. 3 shown in Fig. 1), 30-OH
(column 1 vs. 2), and 3-OH (row 1 vs. 2) groups on the B and C
rings decreased the Eꢀ values by ca. 0.18, 0.09, and 0.27 V,
respectively, but the 7-OH group on the A ring (column 3 vs. 4)
gave a difference of only ca. 0.03 V in the computed Eꢀ.
Fig. 5 Scavenging activity of the flavonoids against free organic
radicals determined by the TEAC assay and their computed redox
potentials. (a) Summary of the TEAC values for the flavonoids and their
computed redox potentials (Eꢀ vs. SHE). Conditions: EtOH; 25 ꢀC; labs
Fig. 5b illustrates the calculated dihedral angles and the
highest occupied molecular orbital (HOMO) levels of the neutral
forms of Que, Lut, and Oro. Lut exhibited the lowest HOMO
a
b
¼ 734 nm. This value was obtained from ref. 30. n.d., not deter- energy (ꢁ6.13 eV) out of the three compounds of interest, which
is in accord with the more positive Eꢀ (1.32 V vs. SHE). The HOMO
mined. The TEAC values of Api, Chr, and HIF could not be obtained
due to limited solubility or marginal antioxidant activity levels unde-
tected under our experimental conditions. (b) Isosurface plots (iso-
level of Que that embodies an additional hydroxyl group at C3 on
the framework of Lut with a relatively planar structure was
density value ¼ 0.03 a.u.) of the HOMO energy for Que, Lut, and Oro
elevated to ꢁ5.75 eV. On the other hand, the HOMO level of Oro,
and their dihedral angles between two planes calculated from carbon
coordinates of the A/C rings and the B ring, respectively.
a regioisomer of Lut, was at ꢁ5.80 eV, which was higher than that
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of Lut. The better antioxidant ability of Oro can be rationalized by uorometric assay with slight modications employing Elec-
the distinctions in the conjugated p-system of the HOMO for Lut trophorus electricus AChE (eeAChE).51 As a reference, tacrine,
and Oro. Considering the intrinsic electronic property of the C a potent inhibitor against AChE,52 was tested under our exper-
ring enone, the a-carbon of the enone (C3) possesses imental conditions (IC50 ¼ 38.7 ꢂ 5.0 nM). Thereaer, the
a dꢁ partial charge while the b-carbon (C2) has a partial d+ charge. inhibitory activity of all avonoids towards AChE was deter-
In the case of Lut, the electron-rich catechol is attached to the d+ mined under the same conditions. As shown in Fig. 6a, Que,
charged b-carbon C2, preferring the p-conjugation throughout Lut, Oro carrying a catechol moiety displayed notable inhibition
the B and C rings. On the other hand, in the case of Oro, the against the activity of AChE, exhibiting IC50 values in the
catechol moiety is attached to the dꢁ charged a-carbon C3 of the nanomolar range. In the case of Lut and Oro, their inhibitory
enone; thus, the resonance between the B and C rings is weak- activity against AChE was comparable to that of tacrine. Kae and
ened. In the optimized structure of Oro, the dihedral angle Gal indicated high nanomolar IC50 values similar to that of Que,
between the A/C rings and the B ring was 36.7ꢀ, which is notably while those of Gen, DHIF, and DHF were within the micromolar
greater than that of Lut. The frontier orbitals showed that the range. Lastly, the activity of AChE was not inhibited by Api, Chr,
HOMO of Lut was composed of a conjugated p-system HF, and HIF under our experimental settings.
throughout the A/C rings and the B ring. In contrast, the orbital
Relating these experimental results to the molecular struc-
lobes in the HOMO of Oro displayed a biased localization tures, several features stand out. The catechol functionality is
towards the B ring resulting in a less electronically stable HOMO. important for the inhibitory activity against AChE (Que, Lut,
This distorted alignment of p-orbitals and the consequential and Oro). DHF exhibited an IC50 value in the low micromolar
disruption of p-conjugation between the two p-ring planes range and the incorporation of the 7-OH group led to an
elevated the HOMO levels of Oro, making its redox potential increase in the inhibitory activity as observed with Gal. The
more negative. Therefore, our experimental and computational distinct inhibitory activity between DHIF and HIF further
data support that three structural components including the 3- supports the involvement of the 7-OH group, whereas the
OH group, the catechol moiety, and the position of the B ring pertinence of the 3-OH functionality can be inferred from the
considerably inuence redox potentials of the avonoids and, general enhancement of the inhibitory capacity from Api, Chr,
subsequently, their antioxidant activity.
and HF to Kae, Gal, and DHF, respectively. Lastly, the enhanced
AChE inhibitory effects of Gen and DHIF, relative to Api and
Chr, implicate the potential inuence of the B ring position on
their ability to inhibit the catalytic activity of AChE.
Inhibition against AChE
The 12 avonoids were tested for their ability to inhibit the
catalytic activity of AChE following a previously reported
Fig. 6 Inhibitory activity of the flavonoids against AChE. (a) Summary of the IC50 values of the flavonoids against eeAChE determined by
a fluorometric assay. (b) Intermolecular interactions between the flavonoids and AChE (PDB 1C2O54) observed by aMD simulations. (c) Visual-
ization of the flavonoid–AChE interactions modeled through aMD simulations. N, O, and H (from hydroxyl groups) atoms in the flavonoids are
depicted in blue, red, and white, respectively. a n.d., not determined. Inhibitory activity of Api, Chr, HF, and HIF against AChE was too low to be
detected under our experimental conditions and, thus, an accurate IC50 value could not be determined.
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Based on the initial identication of these avonoid–AChE
interactions, a more detailed computational analysis was per-
formed on the representative binding modes of the three potent
inhibitors, Que, Lut, and Oro, against AChE. As shown in Fig. 6b
and c, a closer inspection of the binding congurations between
the avonoids and AChE revealed the hydrogen bonding
between the catechol moiety on the B ring and the carboxylate
group of E202. The presence of such hydrogen bonding inter-
actions in close proximity to S203 could limit the accessibility of
the substrate ACh to the catalytic triad responsible for cata-
lyzing the hydrolysis of the neurotransmitter. Such anchoring of
the avonoids near S203 through hydrogen bonding may
explain the relatively high inhibitory activity of Que, Lut, and
Oro towards AChE. These observations emphasize the pertinent
role of the catechol moiety on the B ring in controlling the
activity of AChE. Other binding modes, e.g. where the A ring is
arranged towards S203, were also sampled from the clustering
analysis, as illustrated in Fig. S3.† They represent a relatively
small population, suggesting that they play only a minor role in
the overall binding characteristic. Moreover, the p–p stacking
interactions between the A/C rings and the tryptophan, tyrosine,
and phenylalanine residues also provided additional stability
for binding of the avonoids to AChE. The chromone moieties
of Lut and Oro interact with W86 and F295, respectively, while
Que displayed a slightly longer p–p distance to Y341. The
additional 3-OH functionality on Que, distinguishing it from
Lut, did not show notable interactions with any amino acid
Computational studies for interactions with AChE
Based on their distinct structural characteristics and IC50
values against AChE, Que, Lut, Oro, HF, and DHF were chosen
for a detailed computational evaluation of their interactions
with AChE. Specically, the binding congurations and
interactions of the selected avonoids towards the AChE
dimer were analyzed by accelerated molecular dynamics
(aMD) simulations. Details of these simulations are given in
the ESI.† This method has been shown to improve the delity
of the simulated poses in studies of ligand–protein interac-
tions by enhancing the sampling efficiencies.53 The structural
clustering analysis of three potent avonoids, Que, Lut, and
Oro, against AChE identied three representative interactions
between the compounds and AChE (PDB 1C2O54), as depicted
in Fig. 6b and c. (i) The most dominant interaction observed by
the aMD simulations is the hydrogen bonding between the
catechol moiety on the B ring and neighboring amino acid
residues, where the E202 residue in AChE is the dominant
hydrogen bond accepting partner. (ii) The 7-OH group on the A
ring serves as a hydrogen bond donor to interact with
a carbonyl moiety from a backbone amide group of E81/G82,
E292/S293, or F338/L339 in the gorge region of the pocket.
(iii) A p–p stacking interaction between the chromone
framework of the avonoids on the A and C rings and the
amino acid residues containing a p-ring such as W86, Y341,
and F295 within the hydrophobic pocket of AChE was
detected.
Fig. 7 Evaluation of flavonoid-binding determinants against AChE (PDB 1C2O54). (a) Closest distance between the flavonoid and S203 of the
catalytic triad. (b) Box plot of the number of hydrogen bonds between the flavonoid and AChE. The box extends to the top 25% and bottom 75%
of the clustered data. The black line represents the mean value of each computed case. (c) SASA distribution of the hydrophobic residues in the
active site. (d) Average count of water molecules in the binding pocket for the apo and holo cases. Error bars represent the standard deviation.
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residues in the binding pocket of AChE, as presented in
Fig. S4a.† The hydrogen bond mediated by the 7-OH group
indicated longer distances with a large standard deviation than
those formed between the catechol moiety and E202.
The binding modes of DHF and HF, whose inhibitory
potency towards AChE was relatively weak (IC50 > 2.0 mM), were
also analyzed, as visualized in Fig. S5.† The two compounds do
not possess the catechol moiety on the B ring or the 7-OH group
on the A ring. The clustering analysis of the aMD trajectories
indicated distinct binding characteristics of DHF and HF
towards AChE compared to Que, Lut, and Oro. DHF did not
interact with E202, but it was still compact enough to position
itself within the hydrophobic pocket by preserving the hydrogen
bonding between the 3-OH group and the side chain of G122. In
the case of HF, we were not able to obtain an aMD cluster for the
compound populating more than 5% of the trajectories, in
accord with its low inhibitory activity against AChE. Neverthe-
less, we considered the most clustered conformation of HF for
further exploration and analysis.
Upon determining the binding congurations between the
selected avonoids and AChE, statistical analysis regarding four
specic physical parameters including the distance from S203,
the number of hydrogen bonds, the solvent accessible surface
area (SASA) of the hydrophobic residues lining the active site
pocket, and the number of water molecules in the active site
pocket were conducted to provide more in-depth details of the
avonoid–AChE interactions, as shown in Fig. 7 and S4b.† The
minimum distance between the avonoid and S203 was
measured. On average, aMD simulations with Que, Lut, Oro,
Fig. 8 Summary of the findings from the structure–activity relation-
ship studies regarding the flavonoids' modulative reactivities towards
multiple pathogenic elements found in AD.
cannot be considered a computational parameter in evaluating
the potency of compounds as Que and DHF showed comparable
SASA values (0.88 and 0.97 nm2, respectively), while their ability
to inhibit AChE activity varied signicantly. The HF-bound
structure indicated a mean SASA value of 1.23 nm2 with
signicantly large uctuations. This implies the unstable
organization of the hydrophobic residues upon binding of HF to
the active site.
Finally, the dynamics of the water molecules in the binding
pocket is another critical factor inuencing the binding proper-
ties of ligands.56 We analyzed the number of water molecules in
˚
the active site gorge within 8.0 A from the Ca atom of S203 for
simulated avonoid–AChE binding modes, relative to the number
of water molecules in the apo state, as illustrated in Fig. 7d. The
number of water molecules in holo states decreased by more than
5 in the presence of the avonoids, while 22 water molecules were
present in the apo state. In line with the computational ndings
described above, the HF-bound conformation contained the most
water molecules in the binding pocket (16 on average). In
contrast, Que and DHF disclosed lower numbers of water mole-
cules within the active site than that of HF. Lut and Oro exhibiting
stronger inhibitory activity against AChE led to the least amount
of water molecules in the hydrophobic pocket (on average, 12 and
7, respectively). Combining the experimental and computational
results, several structural features of the avonoids can be con-
nected to their inhibitory activity against AChE. Our structure–
activity relationship study regarding the inhibition towards AChE
underscores the importance of (i) the catechol moiety on the B
ring to facilitate hydrogen bonding with E202, (ii) the 7-OH group
for hydrogen bonding, and (iii) the chromone framework for p–p
stacking with the hydrophobic residues lining the active site
gorge. The aMD simulations also provide a detailed representa-
tion of the possible interactions between the avonoids and AChE
at the active site with multiple key parameters related to the
strength of their interactions.
˚
and DHF presented relatively short distances (below 2.5 A) to
S203, as illustrated in Fig. 7a, implying that they can remain in
the binding site interior. HF was positioned away (5.1 A) from
˚
the catalytic residue, which is consistent with its minimal
inhibitory potency. The distance to S203 alone is not sufficient
to discern more potent inhibitors, however, as DHF was also
found to maintain close contact with S203 despite its weak
inhibitory activity towards AChE. The number of hydrogen
bonds between the avonoids and the binding pocket residues
was calculated to assess the relative stabilities of the identied
binding poses, as presented in Fig. 7b. The lack of adequately
positioned hydrogen bond donors or acceptors on HF and DHF
led to poorly established hydrogen bonding with AChE, as
shown in Fig. S5.† Que, Lut, and Oro exhibited a signicantly
greater number of hydrogen bonds than HF and DHF, which
supports again the notion that the catechol moiety on the B ring
and the 7-OH group on the A ring are crucial structural features
for interacting with AChE.
The SASA values of the hydrophobic residues residing within
6.0 A from S203 were examined as a measure of the binding
˚
pocket stability in the presence of a ligand.55 As displayed in
Fig. 7c, the SASA values of the hydrophobic residues in the
presence of Lut and Oro were found to be 0.54 and 0.49 nm2,
respectively, suggesting that the hydrophobic residues effec-
tively minimized unfavorable contacts with the polar solvent
medium. In addition, the relatively low level of uctuations in
Conclusions
the computed SASAs indicated the increased stability of the The complexity of the multifaceted AD pathology has led to
binding pocket with Lut and Oro. The SASA alone, however, increased interest in the development of chemical reagents with
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multiple functions. Considering the versatile reactivity of the
three naturally occurring avonoids, a series of 12 avonoids
was selected and investigated with respect to their reactivities
towards four pathogenic elements that are implicated in AD
including metal-free Ab42, metal–Ab42, free radicals, and AChE.
Through our detailed investigations, several structural features
were identied to be connected to the reactivities towards the
aforementioned targets (Fig. 8). First, the catechol moiety on the
B ring of the avonoids notably promoted the molecules'
modulative reactivities against metal-free Ab, metal–Ab, free
organic radicals, and AChE. The two ortho-hydroxyl substituents
on the B ring of avonoids presented the ability to (i) chelate
metal ions in a bidentate manner forming a 5-membered ring,
(ii) undergo oxidation to produce an ortho-quinone that can
covalently bind to the lysine residues in Ab,44 (iii) effectively
scavenge free organic radicals via radical stabilization through
hydrogen bonding,57,58 and (iv) inhibit the catalytic activity of
AChE by sterically blocking off the catalytic active site by
interacting with the amino acid residues lining the active site
gorge. Second, the hydroxyl group at C3 contributed towards the
(i) chelation of metal ions in a bidentate manner manifesting
a 5-membered metal-binding site, (ii) modulation of metal-free
or metal-induced Ab aggregation when accompanied by
hydroxyl substituents on the B ring, (iii) scavenging free organic
radicals, and (iv) inhibition of AChE activity in conjunction with
the hydroxyl group at C7. Third, the isoavone variation
accompanied by the presence of the catechol moiety on the B
ring impacted the molecule's reactivities by altering the mole-
cule's thermodynamic properties such as HOMO energy. As for
the scavenging activity of the avonoids against free radicals,
the structural features lowering the computed redox potentials
were observed to increase the molecule's antioxidant capacity.
In addition to the catechol functionality on the B ring, the 7-OH
group on the A ring was associated with inhibition against AChE
through hydrogen bonding. The chromone framework of the
avonoids also demonstrated supplemental interactions with
the active site gorge of AChE through p–p stacking. Overall, our
structure–activity relationship study employing a series of 12
avonoids demonstrates that alterations in the number and
location of hydroxyl groups, the presence of a catechol moiety,
and the location of the B ring substantially contribute towards
the versatile reactivities of avonoids with multiple pathogenic
elements of AD.
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