Post on 26-May-2022
ARTICLE
Flavones with inhibitory effects on glycogen synthase kinase 3b
Yearam Jung1 . Soon Young Shin2 . Young Han Lee2 .
Yoongho Lim1
Received: 9 March 2017 / Accepted: 22 March 2017 / Published online: 30 March 2017
� The Korean Society for Applied Biological Chemistry 2017
Abstract Because glycogen synthase kinase-3 (GSK-3)
activity is linked to various human diseases, it has been
targeted in new drug development. Flavonoids, including
luteolin, apigenin, and quercetin, inhibit GSK-3b; however,the relationships between their structural properties and
inhibitory effects are unclear. We measured the inhibitory
effects of 34 flavonoid derivatives on GSK-3b and calcu-
lated hologram quantitative structure–activity relationships
to provide information on pharmacophores for designing
novel compounds with better activities. The in vitro bind-
ing effect of flavonoids was confirmed using Western
blotting for myricetin, which showed the best inhibitory
activity, and the binding mode between myricetin and
GSK-3b was elucidated using in silico docking.
Keywords Flavone � Glycogen synthase kinase 3b �Hologram quantitative structure–activity relationships �In silico docking � Myricetin
Introduction
Since the identification of glycogen synthase kinase-3
(GSK-3), its cellular multi-functions, including cell pro-
liferation, apoptosis, and development, have been identified
[1]. Because the activity of GSK-3 is linked to various
human diseases such as neurodegenerative disorders,
inflammation, and diabetes, it has been targeted in the
development of new drugs [2]. The first GSK-3b inhibitor
was lithium ion, which enhances serine phosphorylation
and competes with magnesium ion [3–5]. Its half-maximal
inhibitory concentration (IC50) is 1–2 mM [4]. Stau-
rosporine isolated from Streptomyces staurosporeus is a
potent GSK-3b inhibitor (IC50 = 15 nM) [6]. Flavonoids
have been reported to have inhibitory effects on GSK-3b[7]. Although the inhibitory effects of several flavonoids
against GSK-3b have been tested [8], the relationships
between their structural properties and inhibitory effects
have not been reported. Quantitative structure–activity
relationships (QSARs) can provide information on phar-
macophores for designing novel compounds with better
activities. Even the data reported previously contained the
IC50 values of several citrus flavonoids [8]; we tested the
inhibitory effects of 34 flavonoid derivatives on GSK-3bhere. The hologram QSARs (HQSARs) could be calculated
in this work. The aim of this study is to identify the
structural conditions of flavonoids that can enhance the
inhibitory effects on GSK-3b. To confirm the in vitro
binding effect of flavonoids, Western blot analysis was
carried out for myricetin, which showed the best inhibitory
activity in this research, and in silico docking was per-
formed to elucidate the binding mode between myricetin
and GSK-3b.
Materials and methods
The 34 flavones tested here included 13 hydroxyflavones,
two methoxyflavone, five hydroxymethoxyflavones, and 14
glycosylated flavones (Table S1). All compounds were
Electronic supplementary material The online version of thisarticle (doi:10.1007/s13765-017-0271-2) contains supplementarymaterial, which is available to authorized users.
& Yoongho Lim
yoongho@konkuk.ac.kr
1 Division of Bioscience and Biotechnology, BMIC,
Konkuk University, Seoul 05029, Republic of Korea
2 Department of Biological Sciences, Konkuk University,
Seoul 05029, Republic of Korea
123
Appl Biol Chem (2017) 60(3):227–232 Online ISSN 2468-0842
DOI 10.1007/s13765-017-0271-2 Print ISSN 2468-0834
used without further purification, and their purities were
determined using high-performance liquid chromatography
[9].
The GSK-3b kinase assay was carried out using the EMD
Millipore KinaseProfiler service assay protocol (EMD Mil-
lipore Corp., Billerica, MA, USA). GSK-3bwas supplied byEMD Millipore Corp. GSK-3b was incubated with 8 mM
3-(N-morpholino)propanesulfonic acid buffer (pH 7.0),
0.2 mM ethylenediaminetetraacetic acid, 20 lM substrate
for phosphorylation (YRRAAVPPSPSLSRHSSPHQS),
10 mM Mg acetate, and 10 lM ATP. The specific activity
of 33P-ATP was adjusted to approximately 500 cpm/pM.
The magnesium–ATP mixture was added for the initiation
of the enzyme reaction. The incubation was performed for
40 min at room temperature. And, 3% phosphoric acid was
added for the stop of the reaction. Ten lL of the reaction
solution was spotted on a P30 filtermat (PerkinElmer,
London, UK). It was washed with 50 mM phosphoric acid
three times and with methanol once. It was dried for
scintillation counting [10, 11]. All experiments were
repeated three times at five different concentrations (0, 5,
10, 20, and 40 lM). The IC50 values were obtained using
SigmaPlot software (Systat, Chicago, IL, USA). The sig-
moid curve fit method was used. The negative logarithmic
values (pIC50) were used as the biological data for the
HQSAR calculations (Table S1) [12]. The procedures for
HQSAR calculations followed the method reported previ-
ously [9, 13].
HCT116 cells were either left untreated or treated with
30 mM LiCl in the absence or presence of different con-
centrations of myricetin. After the indicated time periods,
cell lysates containing 20 lg protein were separated by
10% SDS-PAGE and transferred to nitrocellulose mem-
branes. The blots were probed with an antibody against
phospho-GSK-3b (Ser9; Cell Signaling Technology, Dan-
vers, MA, USA; 1:1000) for 4 h, followed by incubation
with a secondary antibody conjugated to horseradish per-
oxidase for 2 h, as described previously [13]. Glyceralde-
hyde 3-phosphate dehydrogenase (GAPDH) was used as an
internal control. The results were visualized using an
enhanced chemiluminescence detection system (GE
Healthcare, Piscataway, NJ, USA). The relative band
intensities were measured with a quantitative scanning
densitometer and image analysis software, Bio-1D ver
97.04 (Froebel, Wasserburg, Germany).
The three-dimensional (3D) structure of myricetin was
obtained from the crystal structure deposited in the protein
data bank (PDB) as a complex with human pancreatic
alpha-amylase (4gqr.pdb) [14]. Human GSK-3b consists of
420 amino acids. Sixty-nine 3D structures of human GSK-
3b are deposited in the PDB. All of these structures were
determined using X-ray crystallography. Because stau-
rosporine isolated from Streptomyces staurosporeus is
known to be a representative GSK-3b inhibitor, the crys-
tallographic structure (1q3d.pdb) containing staurosporine
as a ligand was chosen [15]. In silico docking experiments
followed the method reported previously [11]. The struc-
ture of 1q3d.pdb consists of two chains, A and B. Chain A
was used because its structure includes two more residues
than chain B. The 3D structure of 1q3d-A is composed of
35–119, 126–286, and 293–385 residues. The apo-protein
of 1q3d-A was prepared using the Sybyl program. To
obtain the solution structure of the apo-GSK-3b protein,
energy minimization was performed according to a method
reported previously [12]. The superposed result between
apo-1q3d-A and the crystallographic structure deposited in
the PDB produced a root-mean-square standard value of
0.29 A. The binding site of the substrate was determined
using the LigPlot program and consisted of the following:
Ile62, Gly63, Gly65, Val70, Ala83, Leu132, Asp133,
Tyr134, Val135, Gln185, Asn186, Leu188, Cys199, and
Asp200. The flexible docking method used the Sybyl
program, and the details of this method have been reported
previously [12]. To confirm that this docking method was
working well, an original ligand, staurosporine, was
docked to apo-1q3d-A. Of the 30 complexes of stau-
rosporine and 1q3d-A generated from the 30 iterations, the
5th complex showed the best docking pose and its binding
energy was -16.67 kcal/mol. Ten hydrophobic interac-
tions (Ile62, Ala83, Lys85, Leu132, Tyr134, Gln185,
Asn186, Leu188, Cys199, and Asp200) and two hydrogen
bonds (H-bonds, Asp133 and Val135) were observed in the
complex based on the LigPlot analysis (Fig. S5). Statistical
significance was analyzed using Student’s t test. A p value
of less than 0.05 was considered statistically significant.
All experiments were performed in triplicate [16].
Results and discussion
The hologram QSAR (HQSAR) studies generate fragment-
based structure–activity relationships using molecular
holograms [17]. The IC50 values on GSK-3b of the 34
flavonoid derivatives ranged from 0.90 lM (8, myricetin)
to 98.73 lM (20, apigetrin) (Fig. S1). The flavonoid
derivatives used here consist of 14 glycosylated flavones
and 20 aglycons (Table S1). The biological data for the
HQSAR calculations were the negative logarithmic IC50
(pIC50) values, as listed in Table S1. The derivatives were
divided into a training set and a test set. The former was
prepared to build up the HQSAR models, and the latter was
used to validate the models. For the test set, six derivatives,
3, 6, 7, 11, 12, and 26, were selected arbitrarily. This
selection was validated using hierarchical clustering anal-
ysis (HCA) [18]. The HCA results obtained from the Sybyl
7.3 program (Tripos, St. Louis, MO, USA) indicated that
228 Appl Biol Chem (2017) 60(3):227–232
123
the six derivatives are not structurally similar derivatives,
as shown in Fig. S2.
HQSAR calculations were carried out using the QSAR/
HQSAR module provided by Sybyl 7.3. The hologram
lengths were set to 53, 59, 61, 71, 83, 97, 151, 199, 257,
307, 353, and 401, which were provided as default values
in the Sybyl 7.3 program. The fragment sizes ranged
between 4 and 7. Fifteen HQSAR models were generated
based on the influence of the fragment distinction in terms
of atom (A), bond (B), connections (C), hydrogen atoms
(H), chirality (Ch), and donor, and acceptor (DA), as listed
in Table S2. Because the fragment distinction including A,
B, and DA and the fragment distinction including A, B, Ch,
and DA showed good cross-validated correlation coeffi-
cient (q2) values, 0.519 and 0.446, respectively, 12 models
were generated for each fragment distinction using six
different fragment sizes, 2–5, 3–6, 4–7, 5–8, 6–9, and 7–10,
as listed in Table S3. Although for the fragment distinction
including A, B, and DA the variation of the fragment size
did not improve the q2 value, for the fragment distinction
including A, B, Ch, and DA, the model with a fragment
size of 5–8 or 7–10 increased the q2 value to 0.556 or
0.661, respectively. The residual values between the
experimental data and the values predicted by the HQSAR
model using a fragment size of 5–8 showed better results
than those using a fragment size of 7–10. Therefore, the
HQSAR model with the fragment distinction of A, B, Ch,
and DA and the fragment size of 5–8 was chosen. Its sta-
tistical parameters are as follows: cross-validated correla-
tion coefficient (q2), 0.566; non-cross-validated correlation
coefficient (r2), 0.942; standard error of estimate, 0.163;
hologram length, 61; and optimal number of components
(N), 6 [19]. The pIC50 values calculated using this HQSAR
model are listed in Table S4. The experimental data were
plotted against the values predicted by this HQSAR model
(Fig. S3). Its Pearson R correlation coefficient was 0.9403.
The residuals between the experimental pIC50 values and
the predicted pIC50 values were obtained. The residuals for
the training set ranged from 0.12% to 21.26%, and for the
test set, they ranged from 4.07% to 21.21%. As a result,
this HQSAR model is reliable and can be used for the
development of novel GSK-3b inhibitors.
To visualize the results obtained from the HQSAR cal-
culations, contribution maps were generated using the
Sybyl program. Positive, neutral, and negative contributions
are expressed in green, white, and red colors, respectively.
In the case of 3,30,40,5,50,7-hexahydroxyflavone (8, myr-
icetin), which showed the best activity, hydroxyl groups at
the meta and para positions of the B-ring are shown in green
(Fig. 1A). This figure demonstrates that the electronegative
Fig. 1 Contribution maps generated from the HQSAR calculations.
Positive, negative, and neutral contributions are colored in green, red,
and white, respectively. (A) 3,30,40,5,50,7-hexahydroxyflavone (8,
myricetin), (B) apigenin-7-O-glucoside (20, apigetrin), (C) 5-hy-
droxy-30,40,50,6,7,8-hexamethoxyflavone (16, gardenin), and
(D) myricetin-3-O-rhamnoside (30, myricitrin)
Appl Biol Chem (2017) 60(3):227–232 229
123
fragments of the m-, p-positions of the B-ring increase the
inhibitory effects on GSK-3b. The bulky fragment at the
7-position of apigenin-7-O-glucoside (20, apigetrin), which
showed the worst activity, negatively affects the inhibitory
activity (Fig. 1B). The bulky fragments at the 6-position of
the A-ring of 5-hydroxy-30,40,50,6,7,8-hexamethoxyflavone
(16, gardenin) and at the 3-position of the B-ring of myr-
icetin-3-O-rhamnoside (30, myricitrin) also negatively
affect the inhibitory activity (Figs. 1C, D). These findings
provide information about the structural conditions of fla-
vonoids that can enhance the inhibitory effects on GSK-3b.Myricetin, which had the best inhibitory effect on GSK-
3b, is found in various plants such as tea, vegetables, fruits,
and berries [20]. Its antioxidative and anticancer activities
have been studied thoroughly [20, 21]. However, its inhi-
bitory effect on GSK-3b has not been reported. Unlike
most protein kinases, GSK-3b is active in resting condi-
tions and is inactivated by cellular responses. The activity
of GSK-3b is negatively regulated by phosphorylation at
the serine-9 residue in its regulatory N-terminal region
[22]. Thus, increased phosphorylation of this serine-9
residue in GSK-3b is functionally associated with the
inhibition of its kinase activity. To confirm the result
obtained from the in vitro GSK-3b kinase assay, HCT116
human colon cancer cells were treated with different con-
centrations of myricetin, and the phosphorylation status at
serine-9 was then assessed by Western blot analysis using a
phosphospecific antibody. Lithium, a known GSK-3binhibitor, was used as a reference compound. The level of
serine-9 phosphorylation of GSK-3b increased rapidly
within 30 min after LiCl treatment (Fig. 2A), indicating
the rapid inactivation of GSK-3b by lithium. HCT116 cells
were then treated with different concentrations (20, 40, and
80 lM) of myricetin for 30 min, and we measured the
serine-9 phosphorylation of GSK-3b (Fig. 2B). Densito-
metric analysis demonstrated that myricetin caused an
increase in the level of serine-9 phosphorylation of GSK-
3b in a dose-dependent manner. As a result, the data
obtained here suggest that the binding of myricetin to
GSK-3b is associated with the inhibition of GSK-3bactivity.
To elucidate the molecular binding mode between
myricetin and GSK-3b, in silico docking analysis was
carried out. Among sixty-nine 3D structures of human
GSK-3b deposited in the PDB, the crystallographic struc-
ture (1q3d.pdb) containing staurosporine (Fig. S4) as a
Fig. 2 Effect of myricetin on
the serine-9 phosphorylation of
GSK-3b. (A) HCT116 cells
were serum-starved for 24 h,
and 30 mM LiCl was then
added for various time periods.
Whole lysates were prepared,
and western blot analysis was
performed using a
phosphospecific antibody
against serine-9 of GSK-3b.GAPDH was used as an internal
control. (B) HCT116 cells were
serum-starved for 24 h, and
30 mM LiCl or different
concentrations (20, 40, and
80 lM) of myricetin were
added for 30 min. Whole lysates
were prepared, and western blot
analysis was performed as in
(A). The phosphorylated levels
at serine-9 of GSK-3b were
quantified using densitometry,
and the results are shown as the
fold change compared to
unstimulated control cells
(Cont). GAPDH was used as an
internal control
230 Appl Biol Chem (2017) 60(3):227–232
123
ligand was chosen [15]. This structure consists of two
chains, A and B. Chain A was used because its structure
includes two more residues than chain B. First, the ligand
contained in 1q3d.pdb, staurosporine, was docked into the
apo-GSK-3b protein (1q3d-A). The 5th complex showed
the best binding pose, and its binding energy was
-16.67 kcal/mol. Ten hydrophobic interactions and two
hydrogen bonds (H-bonds) were observed in the LigPlot
analysis (Fig. S5). Next, Myricetin was docked to apo-
1q3d-A. Among the 30 complexes of myricetin and 1q3d-
A generated from the 30 iterations, the second complex
showed the best docking pose and lowest binding energy
(-17.87 kcal/mol). The LigPlot analysis revealed five
hydrophobic interactions (Ile62, Val70, Ala83, Leu132,
and Leu188) and four H-bonds (Asp133, Tyr134, and
Val135) (Fig. S6). To compare the binding conditions of
myricetin to GSK-3b with those of staurosporine, 3D
images of the binding sites were generated using the
PyMOL program (The PyMOL Molecular Graphics Sys-
tem, Version 1.3, Schrodinger, LLC). In the complex of
myricetin docked to GSK-3b, four H-bonds were observed,which are expressed as yellow dot circles (Fig. 3A). The
distances between the oxygen in the ketone of Asp133 and
the hydrogen in the 5-hydroxyl group of myricetin, the
oxygen of the hydroxyl group of Tyr134 and the hydrogen
of the 30-hydroxyl group of myricetin, the hydrogen of the
amide of Val135 and the oxygen of the ketone in myricetin,
and the oxygen of the ketone of Val135 and the hydrogen
of the 3-hydroxyl group of myricetin were 2.93, 3.09, 2.81,
and 2.03 A, respectively. In the case of staurosporine, two
H-bonds were observed (Fig. 3B). The distances between
the oxygen of the ketone of Asp133 and the hydrogen of
the nitrogen of the pyrrolone of staurosporine and between
the hydrogen of the amide of Val135 and the oxygen of the
ketone of the pyrrolone of staurosporine were 2.62 and
3.01 A, respectively. The interactions between myricetin
and the binding site of GSK-3b were better than those
between staurosporine and GSK-3b. These results may
explain why the binding energy of myricetin is lower than
that of staurosporine.
Luteolin abolishes GSK-3 activation [23]. Apigenin
induces cell death by inhibition of GSK-3b in BxPC-3 and
PANC-1 human pancreatic cancer cells [24]. Pretreatment
of BxPC-3 human pancreatic cancer cells with luteolin and
apigenin inhibits cell proliferation by reducing the
expression of GSK-3b [24]. The inhibitory effects of 22
flavonoids were measured using the Kinase-Glo Lumines-
cent Kinase Assay. Of these flavonoids, luteolin, apigenin,
and quercetin showed good IC50 values of 1.5, 1.9, and
2.0 lM, respectively [8]. In the paper published previously
[8], the IC50 values of several flavonoids showed several
tens of micromolar level and others were over hundreds of
micromolar level, so that they could not obtain the
structure–activity relationships (SAR) data quantitatively.
Since the IC50 values of 34 flavonoids tested here ranged
between 0.9 and 98 lM, however, we could obtain the
SAR data quantitatively. While the electronegative frag-
ments of the m-, p-positions of the B-ring increase the
inhibitory effects on GSK-3b, the bulky fragments at the 6-
and 7-positions of the A-ring and at the 3-position of the
B-ring negatively affect the inhibitory activity. While the
paper published previously focused on in silico experi-
mental results, the current results showed in vitro biolog-
ical data including Western blot analysis as well as in silico
Fig. 3 (A) 3D image of the binding site surrounding myricetin
docked to GSK-3b generated using the PyMOL program and (B) thatsurrounding staurosporine docked to GSK-3b
Appl Biol Chem (2017) 60(3):227–232 231
123
experimental results. In addition, the structural conditions
of flavonoids that enhance the inhibitory effects on GSK-
3b were identified using hologram QSAR calculations.
These findings will help in the development of novel GSK-
3b inhibitors.
Acknowledgment This paper was supported by Konkuk University
in 2014.
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