pharmaceuticals

Article
Molecular Docking and 3D-Pharmacophore Modeling
to Study the Interactions of Chalcone Derivatives
with Estrogen Receptor Alpha
Muchtaridi Muchtaridi 1, * ID , Hasna Nur Syahidah 1 , Anas Subarnas 2 , Muhammad Yusuf 3 ID
,
Sharon D. Bryant 4 and Thierry Langer 5
1 Department of Pharmaceutical Analysis and Medicinal Chemistry, Faculty of Pharmacy,
Universitas Padjadjaran, Jln. Raya Bandung Sumedang KM. 21, Jatinangor 45363, West Java, Indonesia;
hasnans20@gmail.com
2 Department of Pharmacology, Faculty of Pharmacy, Universitas Padjadjaran,
Jln. Raya Bandung-Sumedang KM. 21, Jatinangor 45363, West Java, Indonesia; aasubarnas@yahoo.co.id
3 Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran,
Jln. Raya Bandung-Sumedang KM. 21, Jatinangor 45363, West Java, Indonesia; m.yusuf@unpad.ac.id
4 Inte: Ligand GmbH, Mariahilferstrasse 74B/11, A-1070 Vienna, Austria; bryant@inteligand.com
5 Department of Pharmaceutical Chemistry, Faculty of Life Sciences, University of Vienna, Althanstraße 14,
A-1090 Vienna, Austria; thierry.langer@univie.ac.at
* Correspondence: muchtaridi@unpad.ac.id; Tel.: +62-22-7796200

Received: 21 August 2017; Accepted: 15 October 2017; Published: 16 October 2017

Abstract: Tamoxifen is the most frequently used anti-estrogen adjuvant treatment for
estrogen receptor-positive breast cancer. However, it is associated with an increased risk
of several serious side–effects, such as uterine cancer, stroke, and pulmonary embolism.
The 20 ,40 -dihydroxy-6-methoxy-3,5-dimethylchalcone (ChalcEA) from plant leaves of Eugenia aquea,
has been found to inhibit the proliferation of MCF-7 human breast cancer cells in a dose-dependent
manner, with an IC50 of 74.5 µg/mL (250 µM). The aim of this work was to study the molecular
interactions of new ChalcEA derivatives formed with the Estrogen Receptor α (ERα) using computer
aided drug design approaches. Molecular docking using Autodock 4.2 was employed to explore the
modes of binding of ChalcEA derivatives with ERα. The 3D structure-based pharmacophore model
was derived using LigandScout 4.1 Advanced to investigate the important chemical interactions of
the ERα-tamoxifen complex structure. The binding energy and the tamoxifen-pharmacophore fit
score of the best ChalcEA derivative (HNS10) were −12.33 kcal/mol and 67.07 kcal/mol, respectively.
The HNS10 interacted with Leu346, Thr347, Leu349, Ala350, Glu353, Leu387, Met388, Leu391, Arg394,
Met421, and Leu525. These results suggest that the new ChalcEA derivatives could serve as the lead
compound for potent ERα inhibitor in the fight against breast cancer.

Keywords: chalcone; molecular docking; structure-based 3D pharmacophore modeling; anti-breast

cancer; estrogen receptor α; MCF-7

1. Introduction
Breast cancer accounts for more than 1.2 million new cases and 500,000 deaths annually. It is
the leading cause of cancer death among females (23% of total cancer and 14% of cancer mortality
cases) [1]. Mortality in women due to this cancer in both developed (189,000 deaths) and emerging
(269,000 deaths) economies was reported to reach 15.5% and 12.7% of total breast cancer cases,
respectively in 2012 [2]. In 2013, the prevalence of breast cancer in Indonesia was reported to afflict
1.4% of the total population (approximately 347,792 persons) [3].

Pharmaceuticals 2017, 10, 81; doi:10.3390/ph10040081 www.mdpi.com/journal/pharmaceuticals

Pharmaceuticals 2017, 10, 81 2 of 12

Estrogen plays a critical role in the growth and development of bone, breast, and uterine pathology.
There are two subtypes of estrogen receptor, ERα (Estrogen Receptor-α) and ERβ (Estrogen Receptor-β).
ERα plays a role in cell proliferation and has been found in the endometrial, breast cancer and ovarian
stromal cells, as well as in the hypothalamus [4]. The most commonly used anti-estrogen adjuvant
treatment for ERα-positive (ERα+) premenopausal women is tamoxifen, a selective estrogen receptor
modulator (SERM). The active metabolite of tamoxifen is 4-hydroxytamoxifen (4-OHT) [5]. Tamoxifen
is also often prescribed for postmenopausal patients with ERα+ tumors. It acts as an antagonist to ERα
and inhibits its signaling pathway in ERα+ breast cancer cells. Tamoxifen therapy significantly reduces
the risk of breast cancer recurrence. The tamoxifen-bound ER complex inhibits the genes from being
switched on by estrogen, leading to the prevention of the estrogenic effects responsible for cancer cell
proliferation [6].
Despite the obvious benefit of tamoxifen in breast cancer treatment, this drug also has serious
risks. For example, the risk of endometrial malignancy and hyperplasia varies from 1.5- to 6.9-fold [7]
after cumulative and long duration usage [8] due to its agonistic effect in the uterus. In addition,
the potential for endometrial cancer increased significantly with overweight postmenopausal females.
To further complicate matters, many ER+ patients, regardless of high levels of ER, demonstrated
intrinsic resistance to hormonal therapies. Thus, alternative treatments are needed.
One promising natural anti-breast cancer adjuvant compound is chalcone, a flavonoid group
secondary metabolite found in many plants. The 20 ,40 -dihydroxy-6-methoxy-3,5-dimethylchalcone
(ChalcEA) from the leaves of Eugenia aquea inhibited the cell proliferation of MCF-7 human breast
cells, in a dose-dependent manner, with an IC50 of 74.5 µg/mL (250 µM). It has been hypothesized
to promote apoptosis via the activation of poly(adenosine diphosphate-ribose)polymerase (PARP).
Further investigations have provided a basis for its use in breast cancer disease management [7].
However, its IC50 value was considered as a moderate level of inhibition compared to tamoxifen [8].
Therefore, further efforts to improve the inhibitory activity of ChalcEA through structural modifications
guided by computer-aided drug design (CADD) methodologies, such as molecular docking method
and 3D-pharmacophore modeling, were explored in this study.
The biological activity of a compound defined by the affinity of a small-molecule ligand towards
the macromolecular receptor can be explored using in silico methods and compared to the experimental
methods. Molecular docking scoring functions can compute binding affinities that are useful for
predicting bioactive poses of compounds and prioritizing compounds for experimental assessment.
These structure-based drug design methods are useful for drug discovery research. The molecular
docking approach can be used to model an interaction between a small molecule (ligand) and
protein at the atomic level. The docking process involves two basic steps: prediction of multiple
structural conformations in a binding pocket (pose), and scoring the pose in order to rank the multiple
solutions [9].
A pharmacophore represents the key interaction features of a molecule responsible for eliciting or
blocking biological activity [10]. 3D-pharmacophore models not only describe the chemical feature
types but also define the 3D geometry of the features of a bioactive compound. Ligand-based (LB)
pharmacophore modeling involves a set of known active molecules without including information from
the macromolecular target. Structure-based (SB) pharmacophore models are composed of features
derived from interactions between the binding pocket and the ligand and require a target ligand
complex binding pocket. Pharmacophore models resulting from SB and LB approaches are used to
understand the key interaction features of a set of active molecules and how a bioactive ligand interacts
with the target-binding site. The 3D-models can be used to search for bioactive molecules using
virtual screening methods or to provide medicinal chemistry decision support during hit expansion
and lead optimization [11]. The pharmacophore modeling study involved the evaluation of the key
pharmacophore interaction features of 4-OHT, ChalcEA and the derivatives reported herein.
Pharmaceuticals 2017, 10, 81 3 of 12

Pharmaceuticals 2017, 10, 81 3 of 12

2. Results
2. Results
The X-ray derived
Pharmaceuticals 2017, 10, 81structure of ERα in complex with 4-OHT (PDB code: 3ERT) was selected 3 of 12 for
molecularThe X-ray derived
docking studiesstructure
of ChalcEAof ERαdueinto
complex with 4-OHT (PDB
good parameters code: 3ERT) was
for experimental selected (1.9
resolution for Å),
molecular
2. Results docking studies of ChalcEA due to good parameters for experimental resolution (1.9 Å),
R-value free and R-value work of 0.262 and 0.229, respectively [12]. The interactions derived from the
R-value free and R-value work of 0.262 and 0.229, respectively [12]. The interactions derived from the
X-ray derived
The structure
X-ray derived revealed
structurethat ERα
4-OHT formedwith
hydrophobic interactions predominantly for with
X-ray derived structure revealedof in complex
that 4-OHT 4-OHT (PDB
formed hydrophobic code: 3ERT)
interactions was selectedwith
predominantly
the butenyl group
molecular and
docking aromatic
studies of rings,
ChalcEAa positive
due to ionizable
good interaction
parameters for with primary
experimental the tertiary
resolution
the butenyl group and aromatic rings, a positive ionizable interaction with primary the tertiary amine (1.9 amine
Å),
R-value
nitrogen free and
and hydrogen
nitrogen R-value
and hydrogen bond work of 0.262
interactions
bond and
interactions 0.229,
with
withthe respectively
thephenoxy [12]. The
andhydroxyl
phenoxy and interactions
hydroxyloxygens
oxygens derived from
(Figure
(Figure the
1a,b).
1a,b).
X-ray derived structure revealed that 4-OHT formed hydrophobic interactions predominantly with
the butenyl group and aromatic rings, a positive ionizable interaction with primary the tertiary amine
nitrogen and hydrogen bond interactions with the phenoxy and hydroxyl oxygens (Figure 1a,b).

(a) (b)

Figure 1. (a) Pharmacophore-Molecular Docking Based of 4-OHT with ERα derived from the X-ray
Figure 1. (a) Pharmacophore-Molecular Docking Based of 4-OHT with ERα derived from the X-ray
derived structure (PDB code: 3ERT). Hydrophobic, positive ionizable,(b)
(a)3ERT). hydrogen bond donor and
derived structure (PDB code: Hydrophobic, positive ionizable, hydrogen bond donor and
acceptor interactions are depicted as yellow spheres, blue star, green and red arrows, respectively. (b)
acceptorThe interactions
Figure 2D-depiction are depicted
1. (a) Pharmacophore-Molecular
illustrates as yellow
a hydrophobic spheres,
Docking
pocketBased blue
with star, green
ofhydrophobic
4-OHT andderived
red arrows,
with interactions
ERα from
with respectively.
the
the X-ray
binding
(b) The site2D-depiction
derived structure
residues. illustrates
(PDB code:
Interactions a 3ERT).
hydrophobic
derived pocket
andHydrophobic,
depicted using with
positivehydrophobic
ionizable,
LigandScout interactions
hydrogen
4.1 Advanced. bond with
Hydrogen the
donor binding
and
atoms
acceptor
site residues. interactions
on the ligandInteractions arederived
and excluded depicted as yellow
and
volumes spheres,
depicted
(restricted using
areas blue star, green
LigandScout
that define theand4.1red
shape ofarrows,
Advanced. respectively.
the binding pocket) (b)
Hydrogen atoms
are
on theThe 2D-depiction
ligand
not illustrates
and excluded
displayed. a hydrophobic
volumes (restrictedpocket
areaswiththathydrophobic
define the shapeinteractions
of thewith the binding
binding pocket) are
site residues. Interactions derived and depicted using LigandScout 4.1 Advanced. Hydrogen atoms
not displayed.
on
The theligand
ligandbinding
and excludeddomainvolumes
(LBD) (restricted
of ERα is areas
mainlythat define the shape of
a hydrophobic the binding
cavity composed pocket) are
of residues
not displayed.
from helices 3, 6, 7, 8, 11 and 12 [13]. Helix-12 of the ER (residues 536–544) plays a major role in
The ligand binding domain (LBD) of ERα is mainly a hydrophobic cavity composed of residues
determining the agonist or antagonist activity of a ligand. When 4-OHT, an antagonist, binds to the
from helices 3, 6, 7,binding
8, 11 and 12 [13].
(LBD)Helix-12 is of the ER (residues 536–544) plays aofmajor role in
ERαTheLBD, ligand
helix-12 domain
occludes of ERα
the co-activator mainly
recognition agroove
hydrophobic
resultingcavity composed
in antagonist residues
activity. This
determining
from the agonist
3, 6, 7, 8, or
11 antagonist
and 12 [13]. activity
Helix-12 of
of a ligand.
the ER When
phenomenon occurs due to the absence of hydrogen bonding with His524 when 4-OHT is bound.in
helices (residues 4-OHT,
536–544) an
plays antagonist,
a major rolebinds
In to
determining
the ERα LBD, the
helix-12 agonist or
occludes antagonist
the activity
co-activator of a ligand.
recognition
contrast, hydrogen bonding occurs with His524 and the ER agonist estradiol [13]. When 4-OHT,
groove an
resultingantagonist,
in binds
antagonist to the
activity.
ERα LBD,
Beforehelix-12
This phenomenon occurs
performingoccludes
duethetothe
the co-activator
molecularabsence ofrecognition
docking hydrogen
simulation, groove
bonding
we firstresulting
with in antagonist
His524
validated our when
docking activity.
4-OHT
method This
is bound.
by
phenomenon
In contrast,
extractinghydrogen occurs
4-OHT bonding due to the
from the occurs absence of hydrogen
with His524
crystallographic ERα and bonding
the ER
structure with
andagonistHis524
docking when
estradiol 4-OHT
it into the[13].binding site In
is bound. to
contrast,
verify thathydrogen bonding
the docking occurs with His524 and thethe ERantagonist
agonist estradiol [13]. conformation of 4-OHT.
Before performing theprogram
molecular could reproduce
docking simulation, we firstbioactive
validated our docking method
The Before
best-docked
by extracting
performing the
4-OHT ligandfrom the
molecular docking
conformation
crystallographicshown in simulation,
ERαFigure we first
2 had
structure aand validated
root mean square
docking
our docking
deviation
it into
method
(RMSD)
the binding
by
site to
extracting
ofthat
0.893 4-OHT
Å compared from the crystallographic
to the original X-ray ERα structure
derived conformation. and docking it into the binding site to
verifyverify the docking program could reproduce the antagonist
that the docking program could reproduce the antagonist bioactive conformation of 4-OHT. bioactive conformation of 4-OHT.
The best-docked
The best-docked ligand conformation
ligand conformation shown
shown ininFigure
Figure22had hadaa rootroot mean squaredeviation
mean square deviation(RMSD)(RMSD) of
0.893 of
Å 0.893
comparedÅ comparedto theto original X-ray
the original derived
X-ray derivedconformation.
conformation.

Figure 2. Best docked pose of 4-hydroxytamoxifen (4-OHT) with estrogen receptor-alpha (ERα) using
AutoDock 4.2.

2. Best
Figure
Figure docked
2. Best pose
docked of of
pose 4-hydroxytamoxifen (4-OHT)with
4-hydroxytamoxifen (4-OHT) withestrogen
estrogen receptor-alpha
receptor-alpha (ERα)
(ERα) usingusing
AutoDock
AutoDock 4.2. 4.2.
Pharmaceuticals 2017, 10, 81 4 of 12
Pharmaceuticals 2017, 10, 81 4 of 12
Pharmaceuticals 2017, 10, 81 4 of 12

2.1. Modification of ChalcEA Derivatives

2.1. Modification of ChalcEA Derivatives
The best-docked conformation of ChalcEA (Figure 3) within the LBD of ERα revealed hydrogen
The best-docked conformation of ChalcEA (Figure 3) within the LBD of ERα revealed hydrogen
bonds with the 22′0 and 4′
40 hydroxyl
hydroxyl groups,
groups, whereas
whereas the carbonyl
carbonyl group
group did
did not form
form any
any interaction.
interaction.
bonds with the 2′ and 4′ hydroxyl groups, whereas the carbonyl group did not form any interaction.
Comparison of the predicted binding poses of ChalcEA and 4-OHT showed that two aromatic rings
Comparison of the predicted binding poses of ChalcEA and 4-OHT showed that two aromatic rings
molecule were
from each molecule were positioned
positioned similarly
similarly in
in the
the LBD
LBD (Figure
(Figure4).
4).
from each molecule were positioned similarly in the LBD (Figure 4).

Figure 3. Best docked pose of ChalcEA with ERα. One hydrogen

One hydrogen bond donor, one hydrogen acceptor
hydrogen bond
Figure 3. Best docked pose of ChalcEA with ERα. One donor, one hydrogen acceptor
and hydrophobic black,
and three
three hydrophobic
hydrophobic (pi-alkyl)
(pi-alkyl) interactions
interactions are
are represented
represented with
with green,
green, red,
red, and
and black,
black, colored
colored
respectively. This
dashed lines, respectively. This interaction
interaction was
was visualized
visualized by
by LigandScout
LigandScout4.1.
4.1.
dashed lines, respectively. This interaction was visualized by LigandScout 4.1.

Figure 4. Overlay of the docked pose of ChalcEA (gray) and 4-hydroxy-tamoxifen (4-OHT) (blue) in
Figure 4.
Figure 4. Overlay
Overlay of ofthe
thedocked
dockedpose
poseofofChalcEA
ChalcEA (gray)
(gray) and
and 4-hydroxy-tamoxifen
4-hydroxy-tamoxifen (4-OHT)
(4-OHT) (blue)
(blue) in
in the
the binding site of estrogen receptor alpha (ERα). Hydrogen bond and pi-alkyl interactions are
the binding
binding site ofsite of estrogen
estrogen receptor
receptor alphaHydrogen
alpha (ERα). (ERα). Hydrogen
bond andbond and
pi-alkyl pi-alkyl interactions
interactions are
are represented
represented in green and pink colored dashed lines, respectively.
represented
in green andin green
pink and pink
colored colored
dashed lines,dashed lines, respectively.
respectively.
However, the dimethylaminoethoxy group of 4-OHT extended further than the carbonyl group
However, the dimethylaminoethoxy group of 4-OHT extended further than the carbonyl group
However,
of ChalcEA. thedifference
This dimethylaminoethoxy
might accountgroup of 4-OHT
for the higher extended
computedfurther than the
free energy carbonyl(∆G)
of binding group
of
of ChalcEA. This difference might account for the higher computed free energy of binding (∆G) of
of ChalcEA. This difference might account for the higher computed
ChalcEA (−8.23 kcal/mol) compared to a ∆G of −11.04 kcal/mol for 4-OHT. free energy of binding (∆G) of
ChalcEA (−8.23 kcal/mol) compared to a ∆G of −11.04 kcal/mol for 4-OHT.
ChalcEA (−8.23the
Therefore, kcal/mol) compared
design of to a ∆G of
ten new ChalcEA −11.04 kcal/mol
derivatives forTable
shown in 4-OHT.
1 focused on modifications
Therefore, the design of ten new ChalcEA derivatives shown in Table 1 focused on modifications
at the position of the carbonyl group and dihydroxy substituted aromatic ring. All modifications were
at the position of the carbonyl group and dihydroxy substituted aromatic ring. All modifications were
Pharmaceuticals 2017, 10, 81 5 of 12

Therefore,
Pharmaceuticals the
2017,
Pharmaceuticals 10,design
2017, 8110, 81 of ten new ChalcEA derivatives shown in Table 1 focused on modifications 5 of 12
5 of 12
at the position of
Pharmaceuticals
Pharmaceuticals the
2017,
Pharmaceuticals 2017, carbonyl
2017,
2017, 10,10,
10, 81
81
10, 81
81 group and dihydroxy substituted aromatic ring. All modifications 5 5 of
of were
12
12
Pharmaceuticals
Pharmaceuticals 2017, 10, 81 555 of
of 12
of 12
12
guided
guided by the
guided
by the
by keykey key
the ERαERα
ERα interactions
interactions
interactions withwith
with 4-OHT
4-OHT 4-OHT(Figure 1), and
(Figure
(Figure 1), and
1), andLipinski’s
Lipinski’s
Lipinski’s RuleRule
Rule of Five
of Five (molecular
of Five (molecular
(molecular
weight guided
guided
guided
less lessby the
by
by
than the
the key
500,key
key ERα
logERα
ERαPor interactions
interactions
interactions
or coefficient with
with
with 4-OHTbetween
4-OHT
4-OHT
partition (Figure 1),
(Figure
(Figure 1),−5and
1), and Lipinski’s
and Lipinski’s
Lipinski’s
less Rule five
Rule
Rule
than of Five
of
of Five
Five (molecular
(molecular
(molecular
hydrogen bond
weight
weight less
guided than
by than
500,
the 500,
log
key Plog
ERα Pcoefficient
or coefficient
interactions partition
partition
with 4-OHT between
between
(Figure −5and
1), −5Lipinski’s
and and
5, less5, less
thanthan
Rule five
of five hydrogen
hydrogen
Five (molecular bond bond
weight
weight
weight less
less
lessthanthan
than
thanthan 500,
500,
500, log
log
log P P
PP or
or coefficient
coefficient
or coefficient
coefficient partition
partition
partition between
between
between −5
−5
−5 and
and
and 5,
5,
5, less
less
less than
than
than five
five
five hydrogen
hydrogen
hydrogen bond
bond
bond
donors,
donors, andless
donors,
weight
and less
and
less than
less
than tenhydrogen
500,
ten hydrogen
ten
log hydrogen
or bond
bond acceptors
bond partition
acceptors [14]
acceptors (Table
[14]
between
[14] (Table (Table
−5
2).and2). Therefore,
2).less
5,
Therefore, we we
Therefore,
than five
aimed aimed
we aimed
hydrogen
to to increase
to increase
bond
increase the
donors, and
donors,
donors, and less
and less than
less than ten
than ten hydrogen
ten hydrogen bond
hydrogen bond acceptors
bond acceptors [14]
acceptors [14] (Table
[14] (Table 2).
(Table 2). Therefore,
2). Therefore, we
Therefore, we aimed
we aimed to
aimed to increase
to increase
increase
thedonors,
the hydrophobicity
hydrophobicity
the
and
hydrophobicity less
around
hydrophobicity
than
around around
the ten
thehydrogen
dihydroxyl
the
dihydroxyl
around the
bond
dihydroxyl acceptors
substituted
substituted
dihydroxyl
[14]
substituted
aromatic
substituted
(Table
ring, 2).
aromatic
aromatic
aromatic
ring,
by Therefore,
ring,
ring,
replacing
by
we
by replacing aimed
by replacing
hydroxyl
replacing
to
hydroxylincrease
hydroxyl
groups
hydroxyl
groups
groups
groups
with
the hydrophobicity
the hydrophobicity
hydrophobicity around around
around the the dihydroxyl
the dihydroxyl substituted
dihydroxyl substituted aromatic
substituted aromatic
aromatic ring, ring,
ring, byby replacing
by replacing hydroxyl
hydroxyl groups
replacing hydroxyl groups
withwiththe
methoxy groups and introducing extended substitutions at the R5 position of ChalcEA groups
(Table
methoxy withmethoxy
groups
with methoxy andgroups
methoxy groups
with methoxy groups and
methoxy groups
groups and
and
introducing introducing
extended
and introducing
and introducing
extended
introducing extended
introducing extended
substitutions
substitutions at
extended substitutions
extended substitutions
substitutions at
the
substitutions at
at the
the R5
R5 position
position of
R5 at
at the the
position
the R5
of ChalcEA
R5
R5 position
ChalcEA (Table
position
of ChalcEA
position of
(Table 1).
1).
of
of ChalcEA ChalcEA
(Table
ChalcEA (Table
(Table 1). 1). 1). 1).
(Table
1).
with
Table 1.Derivatives
TableDerivatives
1.1. 0 ,4
of0 -dihydroxy-6-methoxy-3,5-dimethylchalcone
of22′,4′-dihydroxy-6-methoxy-3,5-dimethylchalcone (ChalcEA).
Table 1.
Table
Table
Table 1.Derivatives
1. of
Derivatives
Derivatives
Derivatives of
of 2′,4′-dihydroxy-6-methoxy-3,5-dimethylchalcone
2′,4′-dihydroxy-6-methoxy-3,5-dimethylchalcone
2′,4′-dihydroxy-6-methoxy-3,5-dimethylchalcone
2′,4′-dihydroxy-6-methoxy-3,5-dimethylchalcone (ChalcEA).
(ChalcEA).
(ChalcEA).
(ChalcEA).
(ChalcEA).
Table 1. Derivatives of 2′,4′-dihydroxy-6-methoxy-3,5-dimethylchalcone (ChalcEA).
ChalcEA
ChalcEA
ChalcEA
ChalcEA
ChalcEA ChalcEA
ChalcEA
ChalcEA
ChalcEA Derivatives
Derivatives
ChalcEA (HNS1-10)
(HNS1-10)
Derivatives
Derivatives
Derivatives (HNS1-10)
(HNS1-10)
(HNS1-10)
ChalcEA
ChalcEA ChalcEA Derivatives (HNS1-10)
ChalcEADerivatives
ChalcEA Derivatives (HNS1-10)
(HNS1-10)
ChalcEA

No.
No.
No. Molecule
Molecule Name
Molecule Name
Name R1
R1
R1 R2
R2
R2 R3
R3
R3 R4
R4
R4 R5
R5
R5
No. Molecule Name R1 R2 R2 R3 R4 R5 R5
No.
No. No. Molecule
Molecule Name
Molecule NameR1 R1 R1
Name R2 R2
R2 R3R3 R3 R4 R4
R4 R5R5 R5
111 HNS1
HNS1
HNS1 OCH333
OCH
OCH H
H
H H
H
H H
H
H ---
11 HNS1
HNS1 OCH 333
OCH3 HH H
H H
H --
1 1 HNS1
HNS1 OCHOCH
3 3 H H H H H H - -
222 HNS2
HNS2
HNS2 H
H
H H
H
H H
H
H H
H
H
22 HNS2
HNS2 H H H
H H
H H
H
2 2 HNS2
HNS2 H H H H H H H H
33 HNS3
HNS3 H
H H
H H
H H
H
3 33 HNS3
HNS3
HNS3 H
H H H
H
H H
H
H H
H
H
3 3 HNS3
HNS3 H H H H H H H H
44 HNS4
HNS4 H
H H
H H
H H
H
4 44 HNS4
HNS4
HNS4 H
H H H
H
H H
H
H H
H
H

4 4 55 HNS4
HNS4
HNS5
HNS5 H H
H
H H H
HH HH
H H H
H H
H
5 55 5 HNS5
HNS5
HNS5 HNS5 H
H H H
H
H H H
H
H H
H
H

5 5 6 6666 HNS5 HNS6

HNS6
HNS6
HNS5
HNS6
HNS6 H H
H
H
H
H H H
H H
HH
H
H
H H
HH
H
H
H
H
H
H H
H
H ----

77 HNS7 CH H
6 6 7 777 HNS6 HNS7
HNS7
HNS7
HNS6
HNS7
CHCH
CH
H CH 33
H 3333 3
H
HH
H
H H H ----- H H
H
H H
H
H - -

7 7 HNS7
HNS7 CH3 CH3 H H - - H H
8 8888 HNS8
HNS8
HNS8
HNS8
HNS8
H
H
H
H
H H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
8

8 8 HNS8
HNS8 H H H H H H H H

9 9999 HNS9
HNS9
HNS9
HNS9 CHCH
CH
CH 33
3 3 OH
OH
OH
OH H
H
H
H OH
OH
OH
OH
9 HNS9 CH 333 OH H OH

9 9 HNS9
HNS9 CH3 CH3 OH OH H H OH OH

1010
10
10
10
HNS10
HNS10
HNS10
HNS10 --- - H
H
H
H --- H
H
H
H
10 HNS10 - H - H

10 10 HNS10HNS10 - - H H - - H H
Table 2.
Table
Table 2. Computed
2. Computed standard
Computed standard properties
standard properties of
properties of ChalcEA
of ChalcEA derivatives
ChalcEA derivatives based
derivatives based on
based on Lipinski’s
on Lipinski’s Rule
Lipinski’s Rule of
Rule of Five.
of Five. HB
Five. HB
HB
Table 2. Computed standard properties of ChalcEA derivatives based on Lipinski’s Rule of Five. HB
indicates
indicates hydrogen
hydrogen
indicates hydrogen bond.
bond.
hydrogen bond.
bond.
indicates
No. Derivative
No.
No. Derivative Name
Derivative NameMolecular
Name Molecular Weight
Molecular WeightLog
Weight Log P
Log PPNumber
Number of
Number of HB
of HB Donors
HB DonorsNumber
Donors Number of
Number of HB
of HB Acceptors
HB Acceptors
Acceptors
No. Derivative Name Molecular Weight Log P Number of HB Donors Number of HB Acceptors
111 HNS1
HNS1
HNS1 284.355
284.355
284.355 3.60
3.60
3.60 111 333
1 HNS1 284.355 3.60 1 3
Table
Table 222
2. Computed
2. Computed HNS2
HNS2
standard
standard
HNS2 374.501
374.501
properties
properties
374.501 2.27 derivatives
2.27
of ChalcEA
of ChalcEA
2.27 333 based
derivatives based 444 RuleRule
on Lipinski’s
on Lipinski’s of Five. HB
of Five. HB
2 HNS2 374.501 2.27 3 4
333 HNS3
HNS3
HNS3 328.408
328.408
328.408 4.03
4.03
4.03 2 2
2 4 4
4
indicates hydrogen
indicates hydrogen
33 bond.bond.
HNS3
HNS3 328.408
328.408 4.03
4.03 22 44
444 HNS4
HNS4
HNS4 330.484
330.484
330.484 3.07
3.07
3.07 333 333
4 HNS4 330.484 3.07 3 3
5 5 HNS5
HNS5 330.424
330.424 3.82
3.82 3 3 4 4
No. No. 555 Derivative
Derivative HNS5Name
Name
HNS5
HNS5 330.424
Molecular Weight
Molecular
330.424
330.424Weight 3.82
Log3.82
PLog
Number
3.82 333 ofDonors
of HB
P Number Number
HB Donors of444HB
Number ofAcceptors
HB Acceptors
666 HNS6
HNS6
HNS6 399.531
399.531
399.531 4.22
4.22
4.22 222 444
1 1 6 HNS1 HNS1
HNS6 284.355
284.355 3.60
399.531 3.60
4.22 12 1 43 3
2 2 HNS2
HNS2 374.501
374.501 2.27 2.27 3 3 4 4
3 3 HNS3
HNS3 328.408
328.408 4.03 4.03 2 2 4 4
4 4 HNS4
HNS4 330.484
330.484 3.07 3.07 3 3 3 3
5 5 HNS5
HNS5 330.424
330.424 3.82 3.82 3 3 4 4
6 6 HNS6
HNS6 399.531
399.531 4.22 4.22 2 2 4 4
Pharmaceuticals 2017, 10, 81 6 of 12

Table 2. Computed standard properties of ChalcEA derivatives based on Lipinski’s Rule of Five.
HB indicates hydrogen bond.

No. Derivative Name Molecular Weight Log P Number of HB Donors Number of HB Acceptors
1 HNS1 284.355 3.60 1 3
2 HNS2 374.501 2.27 3 4
3 HNS3 328.408 4.03 2 4
4 HNS4 330.484 3.07 3 3
5 HNS5 330.424 3.82 3 4
6 HNS6 399.531 4.22 2 4
7 HNS7 372.529 2.88 2 3
8 HNS8 427.585 5.00 2 4
9 HNS9 436.572 3.75 4 4
10 HNS10 450.599 4.05 3 4

The docking results of the ten derivatives, HNS1-HNS10 is summarized in Table 3.

Table 3. Results of molecular docking simulation of ChalcEA derivatives in ligand binding domain of
estrogen receptor alpha (ERα).

Interactions with Amino Acids

Molecule Chemical ∆G Number Calculated
No. van der Waals
Name Formula (kcal/mol) in Cluster Ki (nM) Hydrogen Bond
(Hydrophobic)
Leu525, Met421,
1 HNS1 C18 H18 O3 −9.30 35 153.41 Glu353, Arg394 Leu387, Ala350,
Leu349
Leu391, Met388,
Leu384, Leu525,
2 HNS2 C22 H29 NO4 −9.25 29 66.92 Leu346, Leu387
Met421, Met343,
Asp351, Thr347
Gly521, Val418,
Leu346, Met343,
Met421, Leu525,
3 HNS3 C22 H29 NO4 −9.01 64 249.32 Gly420 Ala350, Thr347,
Trp383, Leu387,
Leu391, Asp351,
Leu348
Leu349, Ala350,
Thr347, Leu346, Leu387, Leu391,
4 HNS4 C20 H23 NO3 −9.26 53 162.64
Glu353, Arg394 Met388, Met421,
His524, Leu525
Leu387, Ala350,
Thr347, Leu346, Leu349, Leu391,
5 HNS5 C20 H24 O4 −9.44 75 120.52
Glu353, Arg394 Met388, Met421,
Leu525
Leu387, Leu349,
Leu346, Glu353,
6 HNS6 C24 H31 NO4 −9.76 30 70.51 Leu391, Ala350,
Arg394
Leu525
Leu346, Leu349,
Arg394, Leu387, Ala350, Asp351,
7 HNS7 C23 H31 NO3 −9.93 49 52.67
Thr347 Leu525, Met421,
Met388, Leu391
Met421, Leu525,
Met388, Leu391,
8 HNS8 C26 H35 NO4 −10.96 43 9.27 Glu353 Leu346, Leu349,
Leu387, Ala350,
Thr347, Trp383
Met421, Leu525,
Met343, Asp351,
Leu346, Glu353,
9 HNS9 C27 H31 NO4 −12.15 83 1.25 Thr347, Ala350,
Arg394, Leu387
Leu349, Leu391,
Leu384, Met388
Met421, Leu525,
Leu346, Glu353, Thr347, Ala350,
10 HNS10 C28 H33 NO4 −12.33 42 0.91
Arg394 Leu387, Leu349,
Met388, Leu391
Pharmaceuticals 2017, 10, 81 7 of 12

The ∆G calculated the free energy of binding ranged from −12.33 to −9.30 kcal/mol. The best
pose from each derivative exhibited better ∆G than the best-docked pose of ChalcEA (−8.23 kcal/mol).
Seven out of ten ChalcEA derivatives formed a hydrogen bond with Arg394, similar to the 4-OHT.

2.2. Structure-Based 3D Pharmacophore Modeling

The 3D structure-based pharmacophore model was created using LigandScout 4.1 Advanced.
LigandScout automatically derives key chemical features, such as hydrogen bond donors and acceptors,
hydrophobic, positive and negative ionizable, aromatic interactions along with their 3D-geometries
of a bioactive molecule. During virtual screening, it aligns ligands based on their pharmacophore
features in 3D space using an advanced algorithm [15]. The validation of the structure-based 3D
pharmacophore model involved virtual screening of 626 molecules active at ERα and 20,773 ERα
decoys taken from the enhanced Database of Useful Decoys (DUDe) using the derived 3D-model.
The early enrichment factor (EF1% ) was 32.4 with an excellent AUC (area under the ROC curve) value
of 1.00 (Figure 5) indicating that the pharmacophore model was able to distinguish successfully true
active from decoy ERα molecules in accordance with methods described by Kirchmair et al. [16].
The key pharmacophore interaction features of 4-OHT and ERα included hydrophobic, a positive
ionizable and hydrogen bond donor and acceptors (Figure 1). Virtual screening results of ChalcEA the
ten derivatives are reported in Table 4 in the form of a pharmacophore fit score which measures the
geometric fit2017,
Pharmaceuticals of the10,features
81 of a molecule to the 3D-structure-based pharmacophore model. A higher 7 of 12
fit score indicates a better fit to the model, and therefore molecules that fit to the pharmacophore model
not all of
should theshow
also features of the
activity at model could be
ERα because notmatched
all of theany two features
features could could
of the model be omitted during any
be matched the
virtual
two screening
features couldprocess.
be omitted In this case,the
during features
virtual that could process.
screening not be matched would
In this case, resultthat
features in lower
could
pharmacophore
not be matched would fit scores. Interestingly,
result the pharmacophore
in lower pharmacophore fit scores
fit scores. of all ofthe
Interestingly, thepharmacophore
derivatives (46.61fit
to 67.07)
scores of were
all ofall
thehigher than the
derivatives parent
(46.61 compound,
to 67.07) were ChalcEA
all higher(45.90).
than the HNS9
parentandcompound,
HNS10 hadChalcEA
the best
pharmacophore
(45.90). HNS9 and fitHNS10
scores had
meaning thatpharmacophore
the best their chemical fitfeatures
scores aligned
meaningbestthattotheir
the chemical
features of the 4-
features
OHT SBbest
aligned pharmacophore
to the features model.
of theMoreover,
4-OHT SB apharmacophore
strong correlation between
model. pharmacophore
Moreover, fit scores
a strong correlation
and docking
between scores was indicated
pharmacophore fit scores from its R-squared
and docking scores(R indicated from its R-squared (R2 = 0.75).
2 = 0.75).
was

5. Receiver
Figure 5. Receiver operating
operating characteristic
characteristic (ROC)
(ROC) curve
curve validation
validation ofof the 3D structure-based
using aa set
pharmacophore model using set of
of 626
626 estrogen
estrogen receptor
receptor alpha
alpha active
active and
and 20,773
20,773decoy
decoymolecules.
molecules.

Table 4. LigandScout pharmacophore fit score of ChalcEA derivatives retrieved using the 3D-
structure-based pharmacophore derived from 4-hydroxytamoxifen (4-OHT) bound to the estrogen
receptor alpha (ERα). A higher fit score indicates a better geometric alignment of the features of the
compound to the 3D-pharmacophore model. The docking score of each compound is provided for
comparison.
No. Compound Pharmacophore-Fit Score Docking Score (kcal/mol)
Pharmaceuticals 2017, 10, 81 8 of 12

Table 4. LigandScout pharmacophore fit score of ChalcEA derivatives retrieved using the
3D-structure-based pharmacophore derived from 4-hydroxytamoxifen (4-OHT) bound to the estrogen
receptor alpha (ERα). A higher fit score indicates a better geometric alignment of the features of
the compound to the 3D-pharmacophore model. The docking score of each compound is provided
for comparison.

No. Compound Pharmacophore-Fit Score Docking Score (kcal/mol)

1 ChalcEA 45.90 −8.23
2 HNS1 46.76 −9.30
3 HNS2 46.62 −9.25
4 HNS3 56.57 −9.01
5 HNS4 46.61 −9.26
6 HNS5 56.68 −9.44
7 HNS6 55.33 −9.76
8 HNS7 55.00 −9.93
9 HNS8 56.20 −10.96
10 HNS9 66.50 −12.15
11 HNS10 67.07 −12.33

2.3. Interpretation of Molecular Docking Results and Pharmacophore Modeling

HNS9 and HNS10 displayed the best pharmacophore fit scores compared to the other derivatives
and ∆G free energy of binding (−12.01 kcal/mol and −12.33 kcal/mol, respectively) better than 4-OHT
(−11.04 kcal/mol). HNS9 formed ten hydrophobic contacts with Met421, Leu525, Met343, Asp351,
Thr347, Ala350, Leu349, Leu391, Leu384, and Met388, and four hydrogen bonds with Leu346, Glu353,
Pharmaceuticals 2017, 10, 81 8 of 12
Arg394, and Leu387 (Figure 6a), whereas HNS10 formed eight hydrophobic contacts with Met421,
Leu525, Thr347,Leu525,
with Met421, Ala350,Thr347,
Leu387,Ala350,
Leu349, Leu387,
Met388, and Leu391,
Leu349, and three
Met388, and hydrogen bonds
Leu391, and with
three Leu346,
hydrogen
Glu353, andLeu346,
bonds with Arg394.Glu353, and Arg394.

(a) (b)

Figure 6.
Figure 6. Interaction
Interactionofof
(a)(a)
HNS9
HNS9andand
(b) (b)
NHS10 within
NHS10 binding
within site ofsite
binding ERα.
of Hydrogen bond, ion-ion
ERα. Hydrogen bond,
interaction, and pi-alkyl interactions are represented in green, blue and purple colored dashed
ion-ion interaction, and pi-alkyl interactions are represented in green, blue and purple colored dashedlines,
respectively.
lines, respectively.

Both HNS9 and HNS10 formed hydrogen bonds with Leu346, Glu353, and Arg394 (Figure 6b).
Both HNS9 and HNS10 formed hydrogen bonds with Leu346, Glu353, and Arg394 (Figure 6b).
The formation of hydrogen bonds with Glu353 and Arg394 is essential for binding to ERα [17], which
The formation of hydrogen bonds with Glu353 and Arg394 is essential for binding to ERα [17], which is
is in agreement with the previous docking studies involving chalcone [18]. Hydrophobic interactions
also contributed to the overall binding of ChalcEA derivatives with ERα. The aromatic rings of HNS9
and HNS10 formed CH-pi hydrophobic interactions with Ala350, Met421, and Leu525 (Figure 6).
Figure 7 shows that the hydroxyl groups in ortho position on HNS9 hindered the complete
mapping with all four hydrophobic features of 4-OHT (Figure 7a). In contrast, the meta position of
hydroxyl groups on the aromatic ring of HNS10 enabled a better alignment with the center of the
 (a) (b)

Figure 6. Interaction of (a) HNS9 and (b) NHS10 within binding site of ERα. Hydrogen bond, ion-ion
interaction, and pi-alkyl interactions are represented in green, blue and purple colored dashed lines,
respectively.
Pharmaceuticals 2017, 10, 81 9 of 12
Both HNS9 and HNS10 formed hydrogen bonds with Leu346, Glu353, and Arg394 (Figure 6b).
The formation of hydrogen bonds with Glu353 and Arg394 is essential for binding to ERα [17], which
in agreement with the previous docking studies involving chalcone [18]. Hydrophobic interactions
is in agreement with the previous docking studies involving chalcone [18]. Hydrophobic interactions
also contributed to the
also contributed overall
to the binding
overall bindingofofChalcEA
ChalcEA derivatives withERα.
derivatives with ERα. The
The aromatic
aromatic ringsrings of HNS9
of HNS9
and HNS10
and HNS10formed
formedCH-pi
CH-pihydrophobic
hydrophobicinteractions
interactions with Ala350,Met421,
with Ala350, Met421,andand Leu525
Leu525 (Figure
(Figure 6). 6).
Figure 7 shows
Figure 7 shows thatthat
thethe
hydroxyl
hydroxylgroups
groups inin ortho positionononHNS9
ortho position HNS9 hindered
hindered the the complete
complete
mapping
mappingwithwith
all four hydrophobic
all four hydrophobicfeatures
featuresofof 4-OHT (Figure7a).
4-OHT (Figure 7a).InIn contrast,
contrast, thethe
meta meta position
position of of
hydroxyl groups on the aromatic ring of HNS10 enabled a better alignment with
hydroxyl groups on the aromatic ring of HNS10 enabled a better alignment with the center of the the center of the
hydrophobic
hydrophobic features,
features, thusthus resulting
resulting inina ahigher
higherfit
fit score
score (Figure
(Figure7b).
7b).

Pharmaceuticals 2017, 10, 81 9 of 12

Figure
Figure 7. Fit7.of
Fitthe
of the
(a) (a) HNS9
HNS9 (b)(b) HNS10totothe
HNS10 the structure-based
structure-based pharmacophore
pharmacophore model derived
model from from
derived
4-OHT with ERα from PDB code: 3ERT. The models were generated using LigandScout
4-OHT with ERα from PDB code: 3ERT. The models were generated using LigandScout 4.1 Advanced. 4.1 Advanced.
Virtual screening was performed leaving at least two features out. The ligands fit six of the eight
Virtual screening was performed leaving at least two features out. The ligands fit six of the eight
features and all of the excluded volumes.
features and all of the excluded volumes.
3. Discussion
3. Discussion
To address the issue related the agonistic effect of 4-OHT in the uterus [19], Elwood et al.
To address
reported the
that issue related
a shorter thebetween
distance agonistic effectwould
Asp351 of 4-OHT
likelyintothe uterus the
decrease [19], Elwoodeffect
agonistic et al.ofreported
4-
that aOHT to uterus
shorter [20]. We
distance measured
between and compared
Asp351 the distances
would likely of the the
to decrease dimethylaminoethoxy
agonistic effect ofgroups
4-OHT to
uterusof [20].
4-OHT,
WeHNS9, and HNS10
measured to be 3.3 the
and compared Å, 3.2 Å, and 2.8
distances Å respectively,
of the indicating that
dimethylaminoethoxy HNS9of
groups and4-OHT,
HNS10 might form stronger interactions with Asp351 and potentially a decreased
HNS9, and HNS10 to be 3.3 Å, 3.2 Å, and 2.8 Å respectively, indicating that HNS9 and HNS10 agonistic effect in
the uterus. However, further theoretical studies related to the agonistic effects of 4-OHT would need
might form stronger interactions with Asp351 and potentially a decreased agonistic effect in the
to be conducted to verify the hypothesis that the modified ChalcEA might not have the same adverse
uterus. However, further theoretical studies related to the agonistic effects of 4-OHT would need to be
effects as observed with tamoxifen. Future studies will elaborate the synthesis and biological
conducted to verify
evaluation of thethe
besthypothesis
predicted that the modified
ChalcEA derivativesChalcEA
to assessmight not have the
their interaction same
with ERαadverse
and their effects
as observed
potentialwith tamoxifen.anti-breast
for modulating Future studies will elaborate the synthesis and biological evaluation of
cancer activity.
the best predicted ChalcEA derivatives to assess their interaction with ERα and their potential for
4. Materials
modulating and Methods
anti-breast cancer activity.
The general scheme of methods in this work is presented in Figure 8.
4. Materials and Methods
The general scheme of methods in this work is presented in Figure 8.
effects as observed with tamoxifen. Future studies will elaborate the synthesis and biological
evaluation of the best predicted ChalcEA derivatives to assess their interaction with ERα and their
potential for modulating anti-breast cancer activity.

4. Materials and Methods

Pharmaceuticals 2017, 10, 81 10 of 12
The general scheme of methods in this work is presented in Figure 8.

Figure 8.
Figure 8. The general scheme of methodologies
methodologies in
in the
the present
present work.
work.

4.1. Molecular Docking Simulation

4.1.
The positive
The positive control
control system
system was
was the
the X-ray
X-ray crystallography
crystallography derived
derived ERα
ERα in
in complex
complex with
with 4-OHT
4-OHT
taken from Protein Data Bank (PDB ID: 3ERT) [21]. The macromolecule and ligand structures
taken from Protein Data Bank (PDB ID: 3ERT) [21]. The macromolecule and ligand structures were were
separatedusing
separated usingDiscovery
Discovery Studio
Studio Visualizer
Visualizer 4.0.structures
4.0. 3D 3D structures of were
of ligands ligands were and
prepared prepared and
optimized
optimized by Hyperchem (HyperCube, Inc., Gainesville, FL, USA) and LigandScout
by Hyperchem (HyperCube, Inc., Gainesville, FL, USA) and LigandScout 4.1 Advanced (Inte:Ligand 4.1 Advanced
(Inte:Ligand
GmbH, GmbH,
Vienna, Vienna,
Austria). TheAustria).
molecularThedocking
molecular docking methods
simulation simulation methods
were were
carried outcarried out
according
to a previously reported study [13]. All ligands and the ERα receptor were prepared for docking
using AutoDockTools (ADT) 1.5.6. The ligands and receptor were protonated. The default Kollman
charges [22] and solvation parameters were allocated to the protein atoms. Gasteiger charges were
added to each ligand atom [23]. A grid box comprised of 40 × 40 × 40 points spaced by 0.375 Å was
centered on the ERα active site (x = 30.282, y = −1.913, and z = 24.207). The pre-calculated binding
affinity of each ligand’s atom type was prepared using Autogrid [24].
AutoDock 4.2 was utilized for the molecular docking simulation. The parameters of the
Lamarckian Genetic Algorithm (LGA) were: 100 runs, elitism of 1, the mutation rate of 0.02,
the population size of 150, a crossover rate of 0.80 and 5,000,000 energy evaluations [25]. The resulting
docked conformations were clustered using a root-mean-square deviation (RMSD) tolerance of 1.0 Å.
The ligand conformation with the lowest free energy of binding (∆G), chosen from the most favored
cluster, was selected for the further analysis. The docking results were visualized using Accelrys
Discovery Studio Visualizer 4.0, and the ligand-interaction features for each pose within the binding
pocket were determined automatically using LigandScout Advanced 4.1.

4.2. Structure-Based 3D-Pharmacophore Modeling

A 3D SB-pharmacophore model was derived automatically from the X-ray derived structure of
ERα in complex with 4-OHT, (PDB code: 3ERT) using Ligandscout 4.1 Advanced [15]. The resulting
3D-interaction feature model was validated for its ability to distinguish true active compounds from
decoys by screening a set of 626 known active and 20,773 decoy compounds obtained from the
enhanced Database of Useful Decoys (DUDe: http://dude.docking.org) [26]. The libraries from DUDe
were converted into 3D multi-conformational databases for virtual screening using the LigandScout
4.1 Advanced algorithm, idbgen, which computes conformations and annotates each conformation
with pharmacophore features. All of the ChalcEA derivatives were screened virtually using the
validated 3D-SB pharmacophore model and the LigandScout 4.1 Advanced VS algorithm, iscreen with
a maximum of 2 omitted features to identify and rank ligands in the library that could fit the geometry
and features of the 3D-model. The pharmacophore fit score measured the fit of features of each hit
compound to the pharmacophore model features and was used to rank the hit molecules retrieved by
the pharmacophore model.
Pharmaceuticals 2017, 10, 81 11 of 12

5. Conclusions
Essential interactions of ChalcEA derivatives with ERα involves hydrogen bond and hydrophobic
non-covalent features. The best derivatives among the ten designed candidates identified using the
molecular docking and pharmacophore modeling studies were HNS9 and HNS10. HNS9 contained
a modification at R1 (methyl), R2 (hydroxyl), R4 (hydroxyl) and R5 (dimethylaminoethoxyphenyl),
while HNS10 only at the R5 with a similar group with HNS9. Both HNS9 and HNS10 still complied the
Lipinski’s Rule of Five. The free energy of binding (∆G) of HNS9 and HNS10 were −12.15 kcal/mol
and −12.33 kcal/mol, respectively. Both contained key interaction features that could be geometrically
aligned with a validated SB pharmacophore model of 4-OHT (pharmacophore fit score of 67.07).
The two derivatives represent rational computationally designed compounds prioritized for further
biological investigation and hit optimization targeted for future development of chalcone based
derivatives as new anti-breast cancer therapies with ERα inhibitor activity and better side effect
profiles compared to tamoxifen.

Acknowledgments: This study was supported by The Directorate General of Higher Education of The Ministry
of Research and Technology of Indonesia through International Research Collaborations and Publications
Grants 2016.
Author Contributions: Hasna Nur Syahidah and Muhammad Yusuf performed the experiments. Muchtaridi
Muchtaridi and Anas Subarnas conceived and designed the experiments. Muhammad Yusuf, Hasna Nur Syahidah,
and Sharon D. Bryant analyzed the data. Hasna Nur Syahidah, Muhammad Yusuf, and Muchtaridi wrote the
paper. Sample Availability: Samples of the compounds HNS9 and HNS10 are available from the authors.
Conflicts of Interest: The authors declare no conflict of interests.

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