Orobol: An Isoflavone Exhibiting Regulatory Multifunctionality against Four Pathological Features of Alzheimer's Disease

Article abstract of DOI:10.1021/acschemneuro.9b00232

We report orobol as a multifunctional isoflavone with the ability to (i) modulate the aggregation pathways of both metal-free and metal-bound amyloid-β, (ii) interact with metal ions, (iii) scavenge free radicals, and (iv) inhibit the activity of acetylcholinesterase. Such a framework with multifunctionality could be useful for developing chemical reagents to advance our understanding of multifaceted pathologies of neurodegenerative disorders, including Alzheimer's disease.

Full text of DOI:10.1021/acschemneuro.9b00232

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Letter
Orobol: An Isoflavone with Regulatory Multifunctionality
against Four Pathological Factors of Alzheimer’s Disease
Geewoo Nam, Yonghwan Ji, Hyuck Jin Lee, Juhye Kang, Yelim
Yi, Mingeun Kim, Yuxi Lin, Young-Ho Lee, and Mi Hee Lim
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.9b00232 • Publication Date (Web): 14 Jun 2019
Downloaded from http://pubs.acs.org on June 15, 2019
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Orobol: An Isoflavone Exhibiting Regulatory Multifunctionality against
Four Pathological Features of Alzheimer’s Disease
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Geewoo Nam,†,‡,# Yonghwan Ji,‡,# Hyuck Jin Lee,†,§,# Juhye Kang,†,‡ Yelim Yi,† Mingeun Kim,† Yuxi
Lin,║ Young-Ho Lee,║,⊥ and Mi Hee Lim*,†
†Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic
of Korea
‡Department of Chemistry, Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Republic of
Korea
§Department of Chemistry Education, Kongju National University, Gongju 32588, Republic of Korea
║Protein Structure Research Group, Korea Basic Science Institute (KBSI), Chungbuk 28119, Republic of Korea
⊥Bio-Analytical Science, University of Science and Technology (UST), Daejeon 34113,
Republic of Korea
ABSTRACT: We report orobol as a multifunctional isoflavone with the ability to (i) modulate the aggregation pathways of
both metal-free and metal-bound amyloid-, (ii) interact with metal ions, (iii) scavenge free radicals, and (iv) inhibit the
activity of acetylcholinesterase. Such framework with multifunctionality could be useful for developing chemical reagents
to advance our understanding of multi-faceted pathologies of neurodegenerative disorders, including Alzheimer’s disease.
KEYWORDS: Multifunctionality, flavonoid, amyloid- metal-amyloid-, free radicals, acetylcholinesterase
Alzheimer’s
disease
(AD)
is
a
progressive
neurodegenerative disease responsible for the majority of
dementia cases.1,2 The lack of an effective cure and aging
world population depict an imminent epidemic and
pessimistic outlook. Although research efforts have been
dedicated towards understanding the pathology of AD, our
comprehension of the disease is fragmental and the
principal causative factors of the disease remain
unidentified.1 Attempts to pinpoint the primary culprit
behind AD have led to the proposal of several hypotheses
(e.g., amyloid cascade, metal ion, oxidative stress, and
cholinergic).3–6 Clinical failures of drugs formulated based
on the individual pathogenic targets [i.e., amyloid- (A),
metal ions, free radicals, and acetylcholinesterase (AChE)]
implicated in the aforementioned hypotheses have
prompted a shift in paradigm to elucidate their malignant
connections.1 For instance, the intimate association
between A and metal ions [e.g., Cu(II) and Zn(II)] have
led to the introduction of metal-bound A (metal–A) as a
hybrid pathogenic component in AD.7–9 Identification and
evaluation of such pathological relations in AD present
experimental challenges arising from the complexity
involving multiple intricate biological components under
physiologically relevant conditions. Thus, chemical tools
capable of interacting with multiple pathological elements
of AD could significantly expedite our efforts to elucidate
the relationships between them. As candidates of such
Figure 1. Rational identification of orobol as a multifunctional
small molecule for regulating four pathological factors implicated
in AD (i.e., metal-free A, metal–A, free radicals, and AChE).
chemicals, flavonoids, a naturally occurring family of
polyphenols, have reportedly exhibited a multitude of
chemical and biological activities (e.g., antioxidant,10
antimicrobial,11
antiviral,12
and
cytoprotective12).13
Numerous compounds have been tested as chemical tools
to understand AD pathology;14–24 however, examples of
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flavonoids capable of regulating four or more pathological
factors found in AD are very limited.
generated 4 in 19% yield. Lastly, the demethylation of 4
with boron tribromide (BBr3) afforded orobol (10% yield;
1
2
3
4
BF3·OEt2
+
75 °C, 4 h
5
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MeSO2Cl2, BF3·OEt2
8
DMF, 100 °C, 2 h
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1
8
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2
3
A
C
2’
B
5’
BBr3
1’
6’
3’
4’
5
4
CH2Cl2, 60 °C, 18 h
4
Orobol
Scheme 1. Synthetic routes to orobol.
Herein, we report, for the first time, a multifunctional
isoflavone (orobol, Figure 1) capable of modulating four
pathological targets (i.e., metal-free A, metal–A, free
radicals, and AChE), to the best our knowledge. Orobol is
characterized by an isoflavone framework with structural
variations from the basic flavone backbone (Figure 1 and
Scheme 1): (i) four hydroxyl substituents; (ii) catechol
functionality on the B-ring; (iii) translocation of the B-ring.
Orobol manifests two potential metal chelation sites (i.e.,
3’-OH/4’-OH and 4-oxo/5-OH), suggesting its ability to
interact with metal ions by forming a 5- or 6-membered
ring.17,18 The B-ring catechol moiety is a source of
antioxidant activity with radical stabilization capabilities
via intramolecular hydrogen bonding.25,26 Oxidation of
“catechol-type flavonoids” can produce an ortho-quinone
that covalently binds to primary amine-containing
residues of A (e.g., K16 and K28).27 Previous research
regarding synthetic aminoisoflavones demonstrated the
pertinence of both the catechol functionality and
isoflavone framework towards their ability to modify the
aggregation of A.17 Although orobol’s effect against AChE
has not been directly reported, structure-activity
relationship studies of flavonoids’ ability to suppress the
activity of AChE indicate the inhibitory potency of
isoflavones and the significance of the catechol functional
group.28 Despite the collective implications for the
versatility of orobol from previous studies, the
multifunctionality of the compound against four
pathological factors linked to AD pathology (i.e., metal-
free A, metal–A, free radicals, and AChE) has not been
directly evaluated until now.
Figure 2. Influence of orobol on the formation of metal-free
and metal-induced A42 aggregates. (a) Scheme of the
inhibition experiment. (b) Analysis of the MW distribution of
the resultant A42 species by gel/Western blot using an anti-A
antibody (6E10). The original gel images are presented in Figure
S4. (c) TEM images of the samples from (b). Conditions: [A42]
= 25 M; [CuCl2 or ZnCl2] = 25 M; [orobol] = 50 M; 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.
Figures S1 and S2).30 Orobol was predicted to exhibit
borderline blood-brain barrier (BBB) permeability with a –
logPe value of 5.35 (± 0.06) by the parallel artificial
membrane permeability assay adapted for the BBB (Table
S1).
Orobol’s effects on the aggregation pathways of metal-
free A42 and metal–A42 were first determined through gel
electrophoresis with Western blotting (gel/Western blot)
in both inhibition (inhibition of A42 aggregate formation;
Figure 2a) and disaggregation (disassembly and/or
aggregation of preformed A42 aggregates; Figure S3a)
experiments. In the inhibition experiments, orobol
noticeably altered the molecular weight (MW) distribution
of metal-free A42 species by increasing the intensity of the
smearing bands greater than ca. 20 kDa compared to
orobol-free A42 (Figure 2b; left). Orobol-treated Cu(II)–
A42 exhibited darker bands at ca. 20-240 kDa and less
intense bands at ca. 7-20 kDa, relative to Cu(II)–A42
without treatment of the compound. In the presence of
Zn(II), orobol enhanced the band intensity at ca. 7-100
kDa. In addition to the inhibition studies, the
disaggregation experiments illustrated the reactivity of
orobol towards preformed aggregates of metal-free A42
RESULTS AND DISCUSSION
Orobol was prepared following previously reported
procedures with minor modifications as shown in Scheme
1.29,30 An electrophilic aromatic substitution reaction
(Friedel-crafts acylation) of 1 with 2 produced 3 (17% yield)
using boron trifluoride ethyl etherate (BF3·OEt2) as both
the solvent and catalyst.29 The subsequent cyclization of 3
in the presence of methanesulfonyl chloride (MeSO2Cl2)
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Figure 3. Interaction of orobol with metal-free A42. (a)
Interaction of orobol with soluble metal-free A42, monitored by
ESI-MS. Inset: Zoom-in spectrum from 1860 to 1880 m/z
(indication of [2A42 + orobol]5+ at 1863 m/z). (b) Analysis of the
IM-MS spectra of [2A42]5+ (black) and [2A42 + orobol]5+
(purple). Conditions: [A42] = 20 M; [orobol] = 100 M; 100 mM
ammonium acetate, pH 7.4; 3 h incubation; no agitation.
and metal–A42 (Figure S3). Under metal-free conditions,
when preformed A42 aggregates were incubated with
orobol, a different MW distribution from orobol-free A42
aggregates with darker bands at ca. 50-240 kDa was
observed (Figure S3b). Orobol noticeably reduced the
band intensity of preformed Cu(II)–A42 aggregates
smaller than at ca. 100 kDa. As for Zn(II)–A42, the MW
distribution of preformed species was altered by orobol
with an increase in the intensity of the smearing band at
ca. 20-50 kDa. It should be noted that the A aggregate
species detected through gel/Western blot are small
enough to enter the gel matrix. In order to visualize the
larger A assemblies, transmission electron microscopy
(TEM) was used to examine the morphology of
the resultant metal-free A42 and metal–A42 aggregates
from the gel/Western blot experiments (Figures 2c and
S3c). According to the TEM images, orobol discernibly
modified the morphology of metal-free A42 and metal–
A42 assemblies from both the inhibition and
disaggregation experiments, inducing the formation of
smaller chopped fibrils or amorphous aggregates. The
modulative reactivity of orobol was also observed against
A42 aggregation in the presence of Cu(I) and Fe(III)
(Figure S5). Overall, the gel/Western blot and TEM results
support orobol’s modulative reactivity towards the
aggregation of both metal-free and metal-bound A42
species. Note that despite such reactivity, orobol could not
noticeably reduce the toxicity of metal-free and metal-
treated A42 in living cells under our experimental
conditions (Figure S6). The cytotoxicity of orobol was
minimal at concentrations up to 100 M.
Figure 4. Interactions of orobol with Cu(II) and Cu(II)–A42.
(a) Cu(II) binding of orobol in a buffered solution. UV–Vis
spectra of orobol (black) with up to 5 equiv of Cu(II) (blue).
Conditions: [orobol] = 25 M; [CuCl2] = 0, 12.5, 25, 50, and 125
M; pH 7.4; room temperature. Interaction of orobol with
soluble Cu(II)–A42, monitored by (b) ESI-MS and (c) IM-MS.
(d) Tandem mass spectrometric analysis in conjunction with
CID of the singly oxidized A42 (●, at 1511 m/z). Conditions:
[A42] = 20 M; [CuCl2] = 20 M; [orobol] = 100 M; 100 mM
ammonium acetate, pH 7.4; 3 h incubation; no agitation.
a 2A42–orobol complex was detected (Figure 3a; purple).
The complexation of orobol with A42 dimer was observed
to vary the size distribution of the peptide dimer indicating
a structural compaction, according to the ion mobility-
mass spectrometry (IM-MS) data (Figure 3b; purple). Such
changes could be responsible for the altered aggregation
pathways of A42.17,31
Interaction between Cu(II) and orobol was confirmed
via UV–Vis denoted by notable optical changes with the
addition of Cu(II) to an aqueous solution of the isoflavone
(Figure 4a). Under Cu(II)-present conditions, the mass
spectrometric analysis of orobol-treated A42 led to the
detection of singly oxidized A42 (Figure 4b; red circle).
The oxidation-induced conformational change of A42
monomer was monitored by IM-MS (Figure 4c), in which
the singly oxidized A42 exhibited a more compact
structure. Using ESI-MS2, the oxidation sites of A42 were
determined by analyzing the peptide fractions of the singly
oxidized A42, reported as b fragments (Figure 4d). Upon
collision-induced dissociation (CID) of [A42 + O]3+ at 1511
m/z, A fragments were found in their oxidized form from
b14, revealing H14 as a possible oxidation site. Based on the
indication of a mixture containing both the oxidized and
To elucidate the mechanistic details regarding orobol’s
ability to modulate the aggregation pathways of metal-free
A42 and metal–A42, spectrometric and spectroscopic
studies [i.e., electrospray ionization-mass spectrometry
(ESI-MS), tandem MS (ESI-MS2), and UV–Visible
spectroscopy (UV–Vis)] were performed to determine the
interactions of orobol with metal-free A42, Cu(II), and
Cu(II)–A42 (Figures 3 and 4). Under metal-free conditions,
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orobol’s inhibitory activity against AChE. The interactions
1
between orobol and AChE were further analyzed by H
NMR (800 MHz; Figure S7). The peak intensities of orobol
were noticeably decreased upon titration with AChE.
These NMR results suggest a significant intermolecular
interactions between orobol and AChE.
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CONCLUSION
9
Orobol, a naturally occurring isoflavone, is demonstrated
as a multifunctional small molecule able to modulate four
distinct pathological features found in the brains affected
by AD (i.e., metal-free and metal-induced A aggregation,
oxidative stress, and AChE-catalyzed ACh hydrolysis).
Aside from the properties tested herein, additional
neuroprotective properties of orobol have been previously
reported: (i) protection against 6-hydroxydopamine-
induced neurotoxicity by restoring proteasomal function;41
(ii) anti-inflammatory effects;42 (iii) inhibition of tyrosine-
specific protein kinase;43 phosphatidylinositol turnover;44
and 15-lipoxygenase;45 (iv) hypotensive effects.46 Based on
its functional versatility, the isoflavone framework of
orobol conferring multifunctionality could be useful
towards developing chemical reagents for advancing our
understanding of the multi-faceted pathology of
neurodegenerative disorders, including AD.
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Figure 5. Free radical scavenging and AChE inhibitory activities
of orobol. (a) TEAC values and IC50 values against AChE activity
of orobol and the corresponding standards (Trolox:
antioxidant; Tacrine: AChE inhibitor).32 (b,c) Docking studies of
orobol and AChE (PDB 1C2B33). (b) Top view perspective
looking into the active site gorge of AChE. (c) Side view of
orobol bound to AChE and zoom-in image of the interaction
between orobol and the amino acid residues of the active site
gorge. The dashed lines and the corresponding numbers
indicate the possible hydrogen bonding between orobol and
AChE and their predicted distances (Å).
non-oxidized fragments from b14 to b34 and the sole
detection of the oxidized A fragments starting from b35,
M35 was designated as another plausible oxidation site of
A. A noteworthy observation was that orobol noticeably
diminished the peak intensity of Cu(II)–A42 while
increasing that of metal-free A42 (Figure 4b), indicating
that the isoflavone may disrupt the interaction between
A42 and Cu(II), potentially through Cu(II) chelation.18
Together, our spectrometric and spectroscopic studies of
orobol demonstrate its ability to (i) interact with metal-
free A42 and Cu(II), (ii) disrupt the interaction between
Cu(II) and A42, and (iii) oxidize A42 in the presence of
Cu(II) at H14 or M35. Such molecular interactions and
alterations of A have previously been reported as
mechanistic strategies to modify the aggregation pathways
of A42 in both the absence and presence of metal ions.34–37
Note that Zn(II)–A42 could not be detected under our
experimental conditions for ESI-MS studies.
The free radical scavenging activity of orobol was
determined relative to Trolox, a vitamin E analog with
notable antioxidant activity, using the Trolox equivalent
antioxidant capacity (TEAC) assay (Figure 5a).38 Orobol
was observed to significantly scavenge free radicals with a
TEAC value of 3.39 (± 0.14). Moreover, orobol’s inhibitory
activity against AChE was measured by a fluorometric
AChE assay,39 indicating a nanomolar IC50 value [123 (± 32)
nM; Figure 5a]. In silico studies (Figure 5b and c) presented
that orobol could interact with multiple amino acid
residues lining the active site gorge (e.g., G120, Y124, F295,
Y337, and Y341). Orobol was shown to interact with S203
of the catalytic triad, responsible for initiating the
enzymatic hydrolysis of acetylcholine (ACh),40 via
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on
the ACS Publications website. Experimental details, Table
S1, and Figures S1-S7 (PDF).
AUTHOR INFORMATION
Corresponding Author
*E-mail: miheelim@kaist.ac.kr (M.H.L.)
ORCID
Mi Hee Lim: 000-0003-3377-4996
#G.N., Y.J., and H.J.L. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported by the National Research
Foundation of Korea (NRF) grant funded by the Korean
government
[NRF-2017R1A2B3002585
and
NRF-
2016R1A5A1009405 (to M.H.L.); NRF-PG2018123 and NRF-
PG2019046 (to Y.-H.L.)]; KAIST (to M.H.L.). J.K.
acknowledges the Global Ph.D. fellowship program through
the NRF funded by the Ministry of Education (NRF-
2015HIA2A1030823).
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