Journal of Steroid Biochemistry and Molecular Biology 213 (2021) 105966

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Journal of Steroid Biochemistry and Molecular Biology

journal homepage: www.elsevier.com/locate/jsbmb

In silico identification of novel inhibitors targeting the DNA-binding domain

of the human estrogen receptor alpha
Huiming Cao 1, Yuzhen Sun 1, Ling Wang, Yu Pan, Zhunjie Li, Yong Liang *
Hubei Key Laboratory of Environmental and Health Effects of Persistent Toxic Substances, Institute of Environment and Health, Jianghan University, Wuhan, 430056,
China

A R T I C L E I N F O A B S T R A C T

Keywords: The human estrogen receptor alpha (ERα) is an important regulator in breast cancer development and pro­
Estrogen receptor alpha gression. The frequent ERα mutations in the ligand-binding domain (LBD) can increase the resistance of anti­
DNA binding domain estrogen drugs, highlighting the need to develop new drugs to target ERα-positive breast cancer. In this study, we
Docking
combined molecular docking, molecular dynamics simulations and binding free energy calculations to develop a
Molecular dynamics simulation
Antiestrogen drug
structure-based virtual screening workflow to identify hit compounds capable of interfering with the recognition
Inhibition of proliferation of ERα by the specific response element of DNA. A druggable pocket on the DNA binding domain (DBD) of ERα
was identified as the potential binding site. The hits binding modes were further analyzed to reveal the structural
characteristics of the DBD–inhibitor complexes. The core structure of the lead molecules was synthesized and was
found to inhibit the E2-induced cell proliferation in MCF-7 cell lines. These findings provide an insight into the
structural basis of ligand-ERα for alternate sites beyond the LBD-based pocket. The core structure proposed in
this study could potentially be used as the lead molecule for further rational optimization of the antiestrogen
drug structure with stronger binding of DBD and higher activity.

1. Introduction
Breast cancer (BC) is the second leading cause of female death after
ovarian cancer. About 70 % of postmenopausal breast cancer patients
express estrogen receptor alpha (ERα) [1]. Currently, all antiestrogen
drugs in clinical use target the ligand-binding domain (LBD) of the ERα,
thereby competing with endogenous estrogen for the receptor binding
[2]. However, recent studies identified several ERα mutations that
frequently occur in the LBD of BC patients resistant to antiestrogen
therapy [3–5]. These high rates of polymorphisms are associated with
acquired drug resistance, disease recurrence and increased mortality,
and therefore represent the main clinical challenge for the current use of
antiestrogen therapy [6–9]. The significant mutations occur within the
L536Q, Y537S, and D538 G genes, which lead to stabilization of the
active conformation of key helix 12 (H12) and constitutive activity of
ERα in the absence of endogenous ligands [10,11]. Moreover, previous
studies have also shown that these mutations decreased the binding
affinities of ERα antagonists by reshaping the LBD architecture [12–14].
These findings suggest that to overcome ERα-LBD mutations, new ERα
antagonists need to be designed to target the new druggable site beyond
the LBD of the receptor.
The ERα DNA-binding domain (DBD) plays an essential role during
transcriptional activation through the specific recognition of the estro­
gen response element (ERE) [15,16]. This suggests that new anties­
trogen drugs that can disrupt the interactions between the DBD and the
ERE could be used to overcome antiestrogens resistance. The architec­
ture of the DBD–ERE complex reveals that each DBD monomer contains
two independent zinc finger domains [17]. The first zinc finger domain
includes the so-called "recognition helix", which is mainly responsible
for the ERE binding by its interactions with the major groove of each
half-site in the ERE, while the second zinc finger domain forms a contact
surface for DBD dimerization [18]. Previous studies [19,20] have indi­
cated that a series of hairpin polyamides could mainly bind to the minor
groove of the ERE with allosteric interference with the receptor–DNA
contacts. Alternatively to polyamides, a DNA bis-intercalator XR5944
[21] was designed to inhibit ERα transcription through competitively
binding to the DNA major groove, thereby directly impeding the loca­
tion of the DBD in this site. Furthermore, disulfide benzamide and

* Corresponding author at: Hubei Key Laboratory of Environmental and Health Effects of Persistent Toxic Substances, Institute of Environment and Health,
Jianghan University, Wuhan, China.
E-mail address: ly76@263.net (Y. Liang).
1
These authors contributed equally.

https://doi.org/10.1016/j.jsbmb.2021.105966
Received 5 May 2021; Received in revised form 29 July 2021; Accepted 11 August 2021
Available online 17 August 2021
0960-0760/© 2021 Elsevier Ltd. All rights reserved.
H. Cao et al.
benzisothiazolone derivatives have been demonstrated to disturb the
ERα binding to the ERE via an electrophilic reaction in the zinc finger
domains of DBD, which in turn inhibits the proliferation of the Michigan
cancer foundation-7 (MCF-7) cell lines [22,23]. However, the afore­
mentioned molecules do not selectively disturb the transcriptional
activation of ERα, but rather universally interact with the specific
response element within the DNA or the zinc finger domains of nuclear
receptors.
Additionally, by using a high throughput screening based on the
fluorescence anisotropy microplates, theophylline, 8-[(benzylthio)
methyl]-(7Cl,8Cl) (TPBM) was identified to disrupt the E2-bound ERα
complex binding to the ERE [24]. Nevertheless, the underlying struc­
tural basis of the DBD-TPBM complex is still unclear, which limited the
understanding of the binding mechanism of DBD inhibitor and the
further structural optimization based on the structure-based drug
design. Therefore, utilizing structural information on the DBD–ERE
complex will provide a feasible way to identify the lead DBD inhibitor
compounds and hence optimize the structure of the drug.
In this study, we combined molecular docking [25,26], molecular
dynamics (MD) simulations [27,28], and binding free energy calcula­
tions [29] to develop a structure-based virtual screening workflow to
identify hit compounds capable of interfering with the DBD–ERE in­
teractions and their corresponding binding sites. The computational
model has been demonstrated to be effective for the virtual screening of
potential antagonists of Y537S mutation to block the mutant-induced
metastatic progression of breast cancer [30]. The binding modes of li­
gands were elaborated to explore the structural characteristics of po­
tential DBD inhibitors. The structural optimization of lead molecules
was further carried out and the promising core structure (CS) was syn­
thesized to evaluate the antiproliferative activity of MCF-7 cell lines.
Generally, these findings provide an insight into the structural basis of
ligand-ERα for alternate sites beyond the LBD-based pocket.
2. Materials and methods
2.1. Structure-based virtual screening
The coordinates of the ERα-DBD structure were extracted from the
crystal structure of the DBD-DNA complex (PDB ID: 1HCQ) [17]. The
missing hydrogen atoms were added, and incomplete residues were
repaired using the protein preparation module of the Discovery Studio
2017 software. Virtual screening was carried out using an online tool
based on the idock docking engine (http://istar.cse.cuhk.edu.hk/idock/
). A total of 23,129,083 compounds were first identified from the ZINC
database and filtered according to nine physicochemical molecular
properties including; molecular weight (g/mol): [250, 500], partition
coefficient xlogP: [1, 3], rotatable bonds: [0, 6], hydrogen bond donors:
[0, 6], hydrogen bond acceptors: [0, 6], net charge: [-1, 0], apolar
desolvation (kcal/mol): [0, 10], polar desolvation (kcal/mol): [-40, 0],
and polar surface area tPSA (Å2): [60, 80]. Only the compounds satis­
fying all the nine filtering conditions were submitted to the following
docking protocol. The whole surface of DBD was defined as the potential
binding site of compounds screened in this work. During the docking
calculations, the DBD structure was set to be rigid, while the structures
of the tested compounds were flexible for conformations samplings. The
top 100 compounds with the highest binding scores and an idock score
greater than -10 kcal/mol were selected. The LeDock program was
further adapted to perform the re-docking for the above compounds,
which was found to give a good performance on the ligand-protein
systems [23,31]. The best-scoring docking poses of the top 20 ligands
were kept as the initial structures for the stability evaluations of sub­
sequent MD simulations.
2.2. MD simulations
The virtual screening of docking-based calculations has intrinsic
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Journal of Steroid Biochemistry and Molecular Biology 213 (2021) 105966
defects in assessing the binding affinity of a compound because the
protein flexibility and explicit solution environment have not been
considered in the docking scores of ligand conformations. Furthermore,
in the current complex system, the screened compounds may have a
trend of free diffusion from the surface site of the ERα-DBD to the so­
lution or exchange between the two states. This means that the top-
scored hits predicted by the docking calculations may be false-positive
results. Therefore MD simulations were performed to identify poten­
tial false positive inhibitors among the top 20 scored compound and to
explore dynamic binding behaviors of these compounds based on the
initial docking poses of DBD-ligand complexes.
The AMBER ff14SB force field [32] and GAFF force field were
adopted to construct the topology structures of the ERα-DBD and li­
gands, respectively. The partial charges of ligands were generated using
the AM1-BCC method in the antechamber module of the AmberTools17
[33]. The DBD–ligand complexes were solvated in a cubic water box of
the TIP3P model [34] and were then neutralized by adding the appro­
priate sodium (Na+) counterions. The zinc ion (Zn2+) was treated using
the cationic dummy atom (CaDA) approach [35] in which the topology
structures of 16 cysteines were specially set to residue type "CYM" for
Zn2+ chelation. The particle mesh Ewald method [36] and periodic
boundaries were used to estimate the long-range electrostatic in­
teractions with a cutoff value of 10 Å. All of the hydrogen-containing
bonds were constrained using the SHAKE algorithm [37], and the inte­
gration time step was set to 2 fs using the Verlet leapfrog algorithm. A
constant temperature was maintained using the Langevin thermostat
with a 2 ps− 1 collision frequency. The Berendsen barostat with isotropic
position scaling was used to maintain constant pressure [38]. For
relaxing the entire system, 5000 step minimizations (2500 steps at the
steepest descent and a 2500 step conjugate gradient) were restricted
with a force constant (k) of 100 kcal mol− 1 Å-2 on the solute, followed by
full 5000 step minimizations without any constraints. Subsequently, the
entire systems with a weak restraint on the solute (k = 10 kcal mol− 1
Å-2) were gradually heated in the canonical ensemble (NVT) from 0 to
300 K over 500 ps. Likewise, the same weak restraint was applied using
500 ps MD simulations in the NPT ensemble for the systems’ density
equilibrium. Finally, 200 ns MD production simulations were performed
at 1 atm and 300 K in the isothermal-isobaric (NPT) ensemble NPT
ensemble. All conventional MD simulations were carried out using a
mixed single-precision or fixed precision pmemd.CUDA module [39] in
the AMBER16 package. Trajectory coordinates were saved every 10 ps
for subsequent analysis, ultimately resulting in 10,000 structures per
trajectory. The distance between the two mass centres of the compound
and the DBD structure was monitored during the whole simulation. The
distance parameter was adopted to reflect the binding stabilities and
residence time of the tested compounds on the surface site of the
ERα-DBD.
2.3. Binding free energy calculations
The binding free energies of the studied DBD–ligand complexes were
calculated using the molecular mechanics generalized Born surface area
(MM/GBSA) approach [30,40], with the MMPBSA.py.MPI module [41]
of the parallel version in AmberTools 17 [33]. The calculated binding
free energy (ΔGcal) was determined as a composition of the corre­
sponding enthalpy (ΔH) and entropy (TΔS), using the Eq. (1):
ΔGcal = ΔH – TΔS (1)
whereby ΔH can be further described using the Eq. (2):
ΔH = ΔEMM + ΔGsol = ΔEvdw + ΔEele + ΔEint + ΔGGB + ΔGSA (2)
The term ΔEMM represents the molecular mechanical (MM) energy,
which includes the contribution of van der Waals interactions (ΔEvdw),
electrostatic forces (ΔEele) and the internal energy (ΔEint). The term
ΔGsol symbolizes the solvation free energy, including both polar (ΔGGB)
H. Cao et al.
and nonpolar contribution (ΔGSA). The ΔGGB can be calculated using the
Onufriev–Bashford–case model (igb = 2), while ΔGSA is evaluated using
the solvent-accessible surface area (SASA) using the equation ΔGSA =
γSASA + β, where the correction constants γ and β are 0.0072 and 0,
respectively. The remaining parameters were set to the default values. A
python script, ante-MMPBSA.py, was used to build the topology struc­
tures for dry DBD–ligand complex, DBD and ligand from their original
solvated topology files for the implicit solvent calculations. All of the
energy components were calculated using 500 snapshots extracted from
the 100–200 ns trajectories. The key residues responsible for the
recognition process were identified using the enthalpy contributions
were decomposed into each residue. Due to the high computational cost,
only 100 snapshots from the last 100 ns trajectories were extracted for
the entropy contribution calculations (TΔS) using the NMODE module
of AmberTools17 [42].
2.4. Cytotoxicity assays
The MCF-7 breast cancer cell line was grown in Dulbecco’s modified
eagle’s medium with 10 % FBS and cultured in a 5% carbon dioxide
(CO2) enriched atmosphere at 37 ◦ C. Exponentially growing cells were
seeded into 96-well plates at a density of 8000 cells per well and allowed
to grow for 24 h. Following removal of the cell culture medium, MCF-7
cells were incubated for 48 h and treated with various concentrations of
the newly synthesized compound (from 1 to 10,000 nM) with or without
E2 (1 nM). Subsequently, MCF-7 cells were treated with the cell counting
kit-8 (CCK-8) reagent at 37 ◦ C for two hours, and the solution was
converted to a quantifiable yellow dye using the mitochondrial de­
hydrogenases present within viable cells. Absorbency was measured at
450 nm using a microplate reader.
Fig. 1. Schematic representation of the work-flow in the current study. The virtual screening of ERα-DBD inhibitors from the ZINC database.
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Journal of Steroid Biochemistry and Molecular Biology 213 (2021) 105966
3. Results and discussion
The potential ERα-DBD inhibitors identified according to structure-
based virtual screening, MD simulations and binding free energy cal­
culations are summarized in Fig. 1. A stepwise filtered strategy was
performed to reduce the ZINC database size and facilitate the identifi­
cation of the lead-like molecules with a high binding affinity for the
DBD. After a physicochemical property-based filtration, the multistep
docking approaches (idock and Ledock) were used to rank the binding
affinities of ligands with the active pocket of DBD. Subsequently, the
stability of the complex formations was evaluated by the MD simulations
in the explicit solvent environment. Further interaction pattern analysis
was conducted based on the binding free energy calculations of the MM/
GBSA method. Finally, the scaffold structure of lead compounds was
synthesized, and its antiproliferative activity was validated in MCF-7
cell lines.
3.1. Screening of the top-scored hits
A total of 1,848,620 compounds were screened from the ZINC
database and subsequently filtered to discover the potential ERα-DBD
inhibitors with high binding affinities. On the basis of high-throughput
virtual screening, the top scored compounds were retained as possible
lead molecules. We also predicted the docking score of TPBM as a pos­
itive control using the same idock docking protocol. The result showed
that these top-ranked compounds had more favorable docking scores
than TPBM (-6.07 kcal/mol), suggesting that these compounds have
higher binding affinities with ERα-DBD. Additionally, we refined the
binding modes of compounds using the Ledock program. The superim­
positions of binding poses with best docking scores between idock and
Ledock displayed the low root-mean-square deviation (RMSD) values (<
H. Cao et al.
2.5 Å) for heavy atoms of the compounds, which means that the current
docking results were reliable and could therefore be used in the post-
docking analysis.
Following an evaluation of the binding modes used by these com­
pounds, we found that most compounds were located on the surface
pocket of the DBD monomer and showed similar binding patterns
(Fig. 2, left panel). Interestingly, it should be noted that this surface
pocket could be recognized by the phosphate backbones and base pairs
of the ERE sequence (Fig. 2, right panel). Based on the above results, we
speculated that these lead molecules might competitively bind in the
DBD recognition helix, thereby directly obstructing the loading of DBD
on the special DNA fragment and inhibiting the formation of ERα–DNA
complex and subsequent transcriptional activation.
3.2. Evaluations of binding stability for the top-scored hits
MD simulations were performed to identify potential false positive
inhibitors among the top 20 scored compounds. Based on the distance
between two mass centers of the compound and DBD structure, the
binding stabilities and residence time of the tested compounds on the
surface site of ERα DBD were identified and illustrated in Fig. S1. Two
binding behaviors were observed for the compounds and divided into
cluster 1 and 2. For cluster 1, the distance was lower than 25 Å for most
compounds, suggesting that the compounds have relatively stable
binding on the DBD surface. For example, ZINC32180841 maintained a
slight structural fluctuation relative to the initial docking pose (Fig. 3A).
Moreover, a relatively large distance of approximately 15 Å was found
for some compounds in cluster 1 (such as ZINC49089886), suggesting
that the ligands attempted to accommodate the surface pocket of the
DBD via local conformational adjustments within the solution environ­
ment (see red dashed box in Fig. 3A).
All the compounds in cluster 2 had a distance larger than 35 Å and
showed a trend of escaping from the surface of the ERα-DBD ligand, even
completely entering into the solution and eventually reaching a distance
greater than 45 Å, such as in the case of ZINC57521965 (Fig. 3B). The
dynamic behaviors meant that these compounds were forming unstable
complexes and only binding on the DBD surface for a short residence
time. Based on these findings, we concluded that these compounds could
not interfere with the ERα-DBD interactions and were therefore classi­
fied as false positive hits to be excluded from further analysis.
3.3. Evaluations of binding free energy for potential DBD inhibitors
The RMSD values of the DBD backbone Cα atoms of the compounds
in cluster 1 were further assessed, as shown in Fig. S2. Most complex
systems showed an increase in the RMSD value in the initial phase of
simulations and reached the equilibrium after 10 ns simulations with an
Fig. 2. The DNA-binding domain (DBD) dimer was shown by monomer structures in blue and cyan (PDB id: 1HCQ). The top 20 score compounds obtained from the
docking-based virtual screening were showed as stick models. The binding poses of compounds were aligned with the ERE on the surface site of DBD structure (red
dashed box).
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Journal of Steroid Biochemistry and Molecular Biology 213 (2021) 105966
RMSD value around 2.0 Å. The small amplitude indicated that the
studied complexes rapidly reached the dynamic equilibrium after the
initial simulation stage. Thus, the last 100 ns trajectories were extracted
to assess the binding affinity of the protein-ligand complex by means of
the binding free energy calculations. TPBM was used as a positive con­
trol to screen potential DBD inhibitors with stronger binding free en­
ergies. Among the tested compounds, ZINC49047307, ZINC49089886,
and ZINC68979694 displayed lower binding free energies when
compared with TPBM, suggesting that the three hits have relatively
stronger binding affinities on the surface pocket of DBD (Table 1 and
S1).
Subsequent investigations on the nature of dominant binding forces
for the DBD-ligand interactions were performed based on the energy
decomposition calculations for the stable binding simulation systems
(Table 1). Noticeably, the negative values of ΔEvdw and ΔEele indicate
that the van der Waals and electrostatic interactions of the tested ligands
favor the DBD recognition. Specifically, the electrostatic interactions
(ΔEele) play a significant role in the binding ability of ZINC49047307,
ZINC49089886, and ZINC68979694 at the surface site of the DBD,
which was counteracted by unfavorable polar contributions of the sol­
vation free energy (ΔGGB). Conversely, the contributions of ΔEvdw were
observed as the most important contribution for stabilizing the DBD-
TPBM complex. This meant that relative to the neutral molecule
TPBM, the top hits have the negatively charged carboxylic acid groups to
mimic the phosphate backbone of DNA structure, thus leading to the
appropriate electrostatic attraction between the interfaces of the
protein-ligand complex. In addition, the entropy contribution (TΔS)
displayed slight changes from -21.79 to -21.54 kcal/mol among these
compounds, suggesting that the numerical values of TΔS did not influ­
ence the relative order rank of binding affinities when screening for
potential DBD inhibitors. This implies that the calculating only the
enthalpy contribution (ΔH) could accelerate the virtual screening pro­
cess due to the high computational cost of TΔS.
3.4. Structural characteristics in the recognition process of potential DBD
inhibitors
The clustering analysis method was used to gain a better under­
standing of the molecular basis involved in recognition of the DBD-
ligand complex and its binding characteristics. The respective poses of
ligands from the largest cluster from the last 100 ns trajectories were
extracted to elaborate the binding modes of the complexes. As expected,
three of them (ZINC49047307, ZINC49089886, and ZINC68979694)
occupied the same binding site of the DBD surface and shared a similar
interaction pattern. Analysis of the binding modes revealed that the
positively charged center consisted of the residues Arg234 and Arg241
and provided the important electrostatic attractions with the carboxyl
H. Cao et al. Journal of Steroid Biochemistry and Molecular Biology 213 (2021) 105966

Fig. 3. The distance between mass centers of compound and DBD structure were plot during the 200 ns MD simulations for the stable binding molecules (A) and false
positive molecules (B) from the molecular docking results. The representative complex conformations for ZINC49089886 and ZINC57521965 were shown in
red frame.

The interaction contacts showed that Arg234 and Arg241 could form the
Table 1
electrostatic interactions with the phosphate backbone of the G31 ERE
Binding free energy components of different simulation systems based on the
base (Fig. 4E). Next, we built the DBD-ERE complex system to identify
MM/GBSA method (kcal/mol).
the important residues in the DBD surface for the ERE recognition by
Systems ZINC49047307 ZINC49089886 ZINC68979694 TPBM
using the same MD protocol. The energy decomposition results indicated
ΔEvdw − 22.21 ± 4.20 − 21.83 ± 3.66 − 17.26 ± 3.19 − 29.01 ± that Arg234 and Arg241 rendered the top energy contributions among
2.50 all the DBD residues, thus playing a significant role in the ERE binding.
− 272.32 ± − 257.76 ± − 290.98 ± − 13.17 ±
Meanwhile, the energy decompositions of the DBD-ligand systems sug­
ΔEele
20.38 17.46 20.21 6.49
ΔGGB 254.97 ± 16.96 241.13 ± 15.72 272.73 ± 18.83 21.81 ± gested that the two residues have relatively stronger contributions
4.16 compared to other residues, which were comparable with that in the
ΔGSA − 3.65 ± 0.39 − 3.43 ± 0.41 − 3.31 ± 0.33 − 3.42 ± DBD-ERE complex system (Fig. 4F). Consequently, based on the above
0.23
energy analysis, we proposed that the positively charged center con­
ΔH − 43.22 ± 5.81 − 41.89 ± 4.37 − 38.82 ± 4.33 − 23.79 ±
3.53
sisting of Arg234 and Arg241 could attract the carboxyl group of po­
TΔS − 21.74 ± 3.08 − 22.79 ± 3.64 − 22.54 ± 3.41 − 21.65 ± tential DBD inhibitors, which mimics the function of the phosphate
2.50 backbone ERE to form the strong electrostatic recognition, eventually
ΔGcal − 21.48 ± 6.93 − 19.10 ± 6.03 − 16.28 ± 5.82 − 2.14 ± leading to an increase in the binding affinity of DBD-ligand complex,
4.72
thereby blocking the loading of special DNA motifs on the surface
ΔH =ΔEvdw + ΔEele + ΔGGB + ΔGSA, ΔEnon-polar = ΔEvdw+ ΔGSA, ΔEpolar = ΔEele binding pocket of DBD.
+ ΔGGB, ΔGcal = ΔH – TΔS.

3.5. Structural optimization of potential DBD inhibitors

group of the compounds (Fig. 4A-C). On the other hand, the residues
Phe208, Ala207, and Gly204 also stabilized the protein-ligand com­
The compound ZINC49047307 and its isomer ZINC49089886 were
plexes by means of the hydrophobic interactions with the benzene rings
retained as the final candidates of the DBD inhibitors because of their
of ligands. Conversely, TPBM acted as the positive control by adopting
relatively lower binding free energies. Following the superimposition of
an alternative binding pose in the DBD surface, where Tyr219 and
the last 100 ns trajectories, unstable binding behaviour was observed in
Asn217 provided the H-bonding interactions for the ligand binding.
region B of the two compounds, which implies that this molecular region
Compared to the hits, the TPBM molecule moved away from the posi­
could be replaced or modified for further structural optimization, as
tively charged center, leading to a decrease in the binding affinity. This
indicated in Fig. 5. Considering the strong H-bonding interactions of the
might be due to a reduction in the electrostatic attractions during the
carbonyl group in region A, we therefore hypothesized that this region
ligand recognition caused by the neutrality of the TPBM form.
might serve as the CS for the DBD recognition (Fig. 5). The major sta­
Additionally, we scrutinized the crystal structure of the DBD-ERE
bilization energies for the complex formation had been derived from this
complex to investigate the structural features of complex formation.
CS.

5
H. Cao et al.
Fig. 4. Binding modes of potential DBD inhibitors (A-D). The key residues for binding were shown in stick mode. The interaction contacts between the residues
Arg234 and Arg241 and the phosphate backbone of G31 base of ERE (E). The energy contributions of Arg234 and Arg241 for the DBD-ligand complex systems (F).
To verify this hypothesis, we implemented the MD simulations to
assess the binding capacity of CS based on the MM/GBSA calculations.
The result indicated that the binding free energy was -23.41 kcal/mol for
the CS, which was relatively superior to that of ZINC49047307 (-21.48
kcal/mol) (Fig. 5). On the basis of the energy decomposition analysis
(Table 1 and S2), we found that the enthalpy contribution of
ZINC49047307 was more favorably than that of CS (-43.22 vs -40.52
kcal/mol), whereas the entropy contribution for ZINC49047307 was
-21.74 kcal/mol lower when compared with that for CS (-17.11 kcal/
mol). The results signified that the remove of unstable fragment of
ZINC49047307 decreased the molecular size, thereby leading to a
reduction in the entropic penalty during the ligand binding and
impairing the enthalpy contribution simultaneously. This delicate
enthalpy-entropy balance finally improved the total binding free energy
of the CS, and eventually, the stably binding on the DBD surface.
Therefore, we selected the CS as the initial point for structural
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Journal of Steroid Biochemistry and Molecular Biology 213 (2021) 105966
optimization with some modification to the different functional groups.
A series of substituted CS analogues have been designed to enhance
the binding affinity of the molecules with DBD. Among these molecules,
the CS analogue 30 (CS30) showed a relatively more favorable Ledock
score compared to other newly designed molecules (Fig. S3). The MD-
based binding free energy calculations displayed a stronger binding af­
finity for CS30 when compare with CS, suggesting that it is more likely
to disturb the binding between DBD and ERE. Subsequently, we
compared the different compositions of binding free energy to identify
which factors are more likely to improve the binding affinity of CS30
(Table S2). The results indicated that the van der Waals and electro­
static forces are more favorable for the binding of CS30 relative to CS,
which means that the substitution of the phenol ring increased the hy­
drophobic and H-binding interactions by stabilizing the binding of this
functional group on the surface pocket of DBD. Although the increased
molecular size of CS30 aggravated the entropic penalty (-22.54 kcal/
H. Cao et al.
Fig. 5. Parent structure of potential DBD inhibitors was identified and optimized for the more stable binding and favorable binding free energy.
mol), this adverse effect was compensated by the above-mentioned
favorable intermolecular interactions.
3.6. Predicted pharmacokinetic properties of potential DBD inhibitors
The pharmacokinetic properties of compounds play a significant role
in drug development processes. By structural optimizations, an ideal
drug should be non-toxic as well as outstanding pharmacokinetic
properties. To evaluate the adverse effects of potential DBD inhibitors
proposed by our computational study, the in silicon predictions of ab­
sorption, distribution, metabolism, excretion, and toxicity were per­
formed for the screened and structurally optimized molecules. All the
tested molecules showed the high human intestinal absorption above 70
% (Table 2). In distribution properties, the blood-brain barrier perme­
ability was evaluated as logBB, a logarithmic ratio of brain to plasma
drug concentrations. The numerical values of logBB indicated that
ZINC49047307 is poorly distributed to the brain, while other molecules
were more likely to cross the blood-brain barrier. Moreover, the
assessment of the metabolic behaviour indicated that CYP3A4, an iso­
form of cytochrome P450, might be the ZINC49047307 and TPBM
target. The drug excretion was measured by the predictions of the he­
patic and renal clearances. The results indicated a rapid clearance for all
the molecules due to their drug-likeness behaviour (Table 2).
For toxicity predictions, the standard lethal dosage 50 value (LD50)
was used to estimate the relative toxicity of the different compounds.
The LD50 was defined as the quantity of a compound given all at once
that lead to the death of 50 % of a group of test animals. The results
suggested that the designed molecules had low toxicity levels. Addi­
tionally, the Ames test, a common method used to assess mutagenic
potentials of compounds using bacteria, was negative for all designed
compounds. This means that these compounds are not mutagenic and
will therefore not act as a carcinogen. Furthermore, a compound is
defined as hepatotoxicity if at least one pathological or physiological
liver event is associated with the drug. The results indicated that both
Table 2
ADMET properties of potential DBD inhibitors.
Absorption Distribution Metabolism
Molecules Intestinal absorption BBB permeability CYP3A4 substrate
% log BB Yes/No
TPBM 89.356 − 0.501 Yes
ZINC49047307 70.215 − 1.168 Yes
CS 95.007 0.008 No
CS30 93.646 − 0.247 No
7
Journal of Steroid Biochemistry and Molecular Biology 213 (2021) 105966
TPBM and CS are non-hepatotoxic (Table 2).
3.7. Antiproliferative activities of the newly synthesized compound
The CS structure was ordered and synthesized by the WuXi AppTec
(Shanghai) Co., Ltd. and synthetic schemes were summarized as sup­
plementary data (Fig. S4). The antiproliferative activities of the CS were
tested against the MCF-7 (human breast cancer cell) cell lines using CCK-
8 assay, which was used to assess the estrogenic or anti-estrogenic ac­
tivity of a compound. Inhibition of cell proliferation by the CS with or
without E2 (an endogenous ligand of ERα, 10− 9 M) was measured from
10-9 to 10-5 M concentrations. The results showed that the CS could
cause significant cytotoxicity of MCF-7 cell lines, in a dose-dependent
manner (Fig. 6, cyan bar). Co-exposure of CS with E2 indicated that
CS could inhibit the E2-induced cell proliferation, especially at the low
concentration (10− 9 M) when compared to that of only E2-exposed MCF-
7 cell lines (Fig. 6, grey bar), which means that the CS could cause anti-
estrogenic effects through ERα-mediated signaling pathway. To further
explore the expression alterations in estrogen-responsive genes, we
carried out the real-time PCR experiments to determine whether the CS
treatment resulted in a disruption in ERα-mediated transcription. The
details of method were summarized in the Supporting Information (SI).
By the comparison of the expression of estrogen-regulated genes (TFF1
and EGR3), we found that the mRNA levels of TFF1 and EGR3 were
decreased in a dose-dependent manner after the treatment of CS with or
without E2 (10-9 M). The results suggested that CS could attenuate
endogenous ERα signaling, thus resulting in the disruption of estrogen-
modulated endogenous gene expression in MCF-7 cell lines (Fig. S5).
Moreover, we speculated that the other potential targets or inhibition
mechanisms of CS for the MCF-7 cell lines may provide the multiple
antagonism effects on the proliferation and progression breast cancer as
well. Based on the above results, we confirmed that CS, as the scaffold
structure of potential DBD inhibitors, could potentially be used to
develop new antiestrogens therapy for breast cancer.
Excretion Toxicity
Total Clearance AMES toxicity LD50 Hepatotoxicity
log ml/min/kg Yes/No mol/kg Yes/No
0.671 No 2.629 No
0.248 No 2.427 Yes
0.725 No 2.169 No
0.55 No 2.577 Yes
H. Cao et al.
Fig. 6. Anti-proliferative activity of synthesized compound CS in MCF-7 human
breast cancer cells. Cells were exposed to 0.1 % DMSO (vehicle control), 1 nM
E2 (positive control), 10− 9–10-5 M CS, or CS + E2 for 2 days. Results are
expressed as the mean ± SD of triplicate measurements in three replicate
samples (* means p ≤ 0.05 and ** means p ≤ 0.005).
In a previous report, anacardic acid (C24:1), known as histone ace­
tyltransferase inhibitor [43], was shown to inhibit breast cancer cell
proliferation regardless of their endocrine or tamoxifen sensitivity [44].
This study also demonstrated that C24:1 could inhibit the interactions
between the ERα and pS2 gene in MCF-7 cell lines. Based on the mo­
lecular docking prediction, the authors speculated that C24:1 might bind
on the surface of the zinc DBD fingers rather than in the LBD. Compared
to C24:1, the potential DBD inhibitors tested in this study displayed an
alternative mechanism of action for the CS compounds, whereby the
exposed surface pocket could bind on the ERα-DBD and act as a
drug-target site with high binding affinity, thereby blocking the tran­
scriptional activation of E2-induced in MCF-7 cells. Furthermore, the
screened potential small molecule inhibitors for the druggable binding
site on the AR-DBD surface have been demonstrated to be effective in
inhibiting the transcriptional activity of androgen receptor in the pre­
vious studies [45,46]. However, these surface sites of the DBD for the
nuclear receptors are generally shallow with transient pockets. This
makes it difficult to design a drug that can stably bind in these sites of
DBD. Thus, the current computational workflow combining molecular
docking, MD simulations and binding free energy could provide a
feasible strategy for the development of potential DBD inhibitors of ERα.
4. Conclusion
In the current study, potential antiestrogens capable of inhibiting the
binding of DBD to the target DNA were identified through the use of an
integrated computational approach. The CS molecule of these DBD-ERE
inhibitors was synthesized, and antiproliferative activities of the newly
designed drug were validated against MCF-7 cell lines. The experimental
results suggested that the CS clearly inhibits E2-induced cell prolifera­
tion. Thus, we believe that the CS proposed in this study could be used as
the lead molecule for further structural optimization and further
experimental DBD binding affinities and pharmacokinetic properties of
CS and its analogues will be evaluated in our future work.
Author statement
Huiming Cao: Conceptualization, Methodology, Writing-Original
draft.
Yuzhen Sun: Data preparation and analysis, Software.
Ling Wang: Experimental validation.
Yu Pan: Experimental validation.
8
Journal of Steroid Biochemistry and Molecular Biology 213 (2021) 105966
Zhunjie Li: Visualization, Investigation.
Yong Ling: Reviewing and Editing.
Declaration of Competing Interest
The authors declare no conflict of interest, financial or otherwise.
Acknowledgements
This work was supported by the National Natural Science Foundation
of China (21806058, 21607060, and 21777061).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.jsbmb.2021.105966.
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