Review

Discovery of estrogen receptor

modulators: a review of virtual
screening and SAR efforts
1. Introduction Xue Xu, Wei Yang, Yan Li & Yonghua Wang†
†
2. Overview of the approaches of Dalian University of Technology, School of Chemical Engineering, Dalian, 116012, Liaoning,
VS and SAR China and Northwest A&F University, Center of Bioinformatics, Yangling, Shaanxi, 712100, China
3. Expert opinion
Importance of the field: Virtual screening (VS) coupled with structural biology
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is a significantly important approach to increase the number and enhance the

success of projects in lead identification stage of drug discovery process.
Recent advances and future directions in estrogen therapy have resulted in
great demand for identifying the potential estrogen receptor (ER) modulators
with more activity and selectivity.
Areas covered in this review: This review presents the current state of the art
in VS and structure–activity relationship of ER modulators in recent discovery,
and discusses the strengths and weaknesses of the technology.
What the reader will gain: Readers will gain an overview of the current
platforms of in silico screening for discovery of ER modulators; they will learn
which structural information is significantly correlated with the bioactivity of
ER modulators and what novel strategies should be considered for the
For personal use only.

creation of more effective chemical structures.

Take home message: With the goal of reducing toxicity and/or improving
efficacy, challenges to the successful modeling of endocrine agents are
proposed, providing new paradigms for the design of ER inhibitors.

Keywords: drug discovery, estrogen receptor modulator, SAR, selectivity, virtual screening

Expert Opin. Drug Discov. (2010) 5(1):21-31

1. Introduction

Estrogen receptor (ER), as a member of the steroid family of nuclear receptors, is a

ligand-dependent transcriptional activator mainly accumulated in the mammary and
uterine cytosol [1]. When binding to an agonist, such as estradiol, the state of the ER
is altered, allowing the receptor dimer to interact with specific DNA sequences
located within the regulatory region of target genes, and then regulating the target-
gene transcription, either positively or negatively [2]. Through the recognition of the
binding between the ER and its ligand, the ER serves as the basis for the hormone
replacement therapy of symptoms of menopause and chronic influence such as
cardiovascular disease and osteoporosis [3].
As a ligand-activated transcription factor, the ER is comprised of two subtypes
ERa and ERb. ERa is a predominant ER in the female reproductive tract and
mammary glands, whereas ERb primarily presents in vascular endothelial cells, bone
and male prostrate tissues. The amino-acid sequence identity between the two
subtypes is ~ 97% in the DNA-binding domain and ~ 56% in the ligand-binding
domain (LBD), whereas the N terminus is poorly homologous at 24% [4]. The main
difference of binding pockets of ERa and ERb is determined by two amino acids as
revealed by the X-ray crystal structures of receptor–ligand complexes (Leu and Met
in ERa are replaced by Met and Ile in ERb) [5].
Given the specific targeting and structural features of ERa and ERb, alternative
therapeutic opportunities concentrating on the selectivity for ER subtypes have been

10.1517/17460440903490395 © 2010 Informa UK Ltd ISSN 1746-0441 21

All rights reserved: reproduction in whole or in part not permitted
 Discovery of estrogen receptor modulators: a review of virtual screening and SAR efforts
Article highlights.
. Alternative therapeutic opportunities concentrating on
the selectivity for estrogen receptor (ER) subtypes have
been developed, thereby, reducing undesired side effects
on breast, uterine tissues and the cardiovascular system.
. Virtual screening methods have gained recent acceptance
in the regulatory community for both human health and
ecological end points.
. Approaches discovering estrogenic drugs that can exert a
desired therapeutic effect have been divided into two
main techniques, namely, the ligand-based (SAR,
pharmacophore) and structure-based (Docking) modeling
techniques.
Expert Opin. Drug Discov. Downloaded from informahealthcare.com by The University of Manchester on 11/02/14
. Previous rigid docking simulations have failed to provide
reasonable scores due to the neglect of interactions of the
flexible residues in the binding pocket.
. By using the flexible docking method, the present
simulations show different binding modes from previous
work with the high evaluation scores.
. It is necessary to model the mutated ERs interacting with
binders and to develop novel ER modulators to overcome
the increasing drug resistance.
. In order to improve the efficiency of the discovery of ER
modulators, the inclusion of ADME in the future modeling
practice is of significance.
This box summarises key points contained in the article.
For personal use only.
developed, thereby, reducing undesired side effects on breast,
uterine tissues and the cardiovascular system. These novel ER
modulators, tailored to have specific and selective effects on
ER function, are now collectively known as selective estrogen
receptor modulators (SERMs), providing beneficial estrogen-
like effects on bone and lipid metabolism, while antagonizing
estrogen in reproductive tissue, particularly the uterus [6,7].
However, despite the obvious usefulness of the SERMs, their
exposure to estrogen mimics can still cause a diverse series of
toxicological and pharmacological responses [8], which pre-
sents a challenge to structure–activity relationship (SAR)
approaches aimed at identification, activity and understanding
of mechanisms of action.
Numerous naturally occurring and man-made estrogen-like
compounds, named endocrine disrupting chemicals (EDCs),
have also aroused attention due to their large impact on
human health and wide life clinical applications [9], which
are able to potentially interact with and disrupt the endog-
enous hormone pathways, consequently interfering with
genetic functions such as sexual development and reproductive
fecundity. Due to the deleterious effects of EDCs, the US
Environmental Protection Agency has run a screening and
testing program to identify such compounds [10].
To facilitate this, virtual screening (VS) methods have
gained recent acceptance in the regulatory community for
both human health and ecological end points [11]. It is known
to be a complementary approach to experimental screening,
which when coupled with SAR promises to filter the
combinatorial libraries of the drug candidates, and enhances
22 Expert Opin. Drug Discov. (2010) 5(1)
the success of agent exploration in the lead identification stage
of the discovery process [12]. The method for computer-aided
drug discovery is, therefore, set priority to overcome the
difficulty and improve the speed of the developmental process
of ER modulators.
A previous review describing the prediction of ER binding
affinity before 2003 has been reported [13]. In this paper, the
recent discovery (since 2003) of ER modulators involving the
application of SAR and VS methods is summarized, with
the purposes of disclosing the efforts of certain research groups
to: i) identify descriptors representing the most important
structural information that is correlated significantly with
bioactivity and ii) explore novel models with the simplest
process and the highest speed.
2. Overview of the approaches of VS and SAR
To highlight the importance of VS and SAR that enable the
researchers to investigate the potential and new ER modula-
tors, antagonists or agonists; all the computational methods
involved in the discovery of estrogenic drugs that can exert a
desired therapeutic effect are summed up here. In general, they
can be divided into two classes, namely, the ligand-based
(SAR, pharmacophore) and structure-based (Docking)
modeling techniques.
2.1 QSAR models
By integrating experimentation and modeling, a series of
quantitative SAR (QSAR) models have been developed and
validated in the prediction of ER modulators. This type of
modeling is to quantitatively correlate a set of molecular
descriptors that encode the chemical structures or properties
with a dependent variable that represents the biological
activity of the studied molecules by using certain statistical
techniques. Currently, the tested data sets are mainly derived
from the National Center for Toxicological Research (NCTR,
http://edkb.fda.gov/databasedoor.html) endocrine disruptor
knowledge base (EDKB), including a large series of com-
pounds with their ER binding affinity data. For QSAR
models, the mathematical tools involved are categorized as
regression methods (e.g., multiple linear regression (MLR),
partial least squares (PLS), artificial neural networks (ANN))
and classification approaches (e.g., linear discriminant analy-
sis, Decision Forest (DF)). These modeling approaches based
on 2D- or 3D-SARs have been presented in Table 1 and
discussed as follows.
2.1.12D-QSAR models
The estrogen activity (IC50) of 25 compounds involving the
industrial phenolic chemicals, phytoestrogens, natural and
man-made steroids were collected by Hu and Aizawa [14] to
establish a QSAR model by a quantum chemistry method.
A proper regression was obtained using volume (Vm)
combined with the energies of the highest occupied molecular
 Xu, Yang, Li & Wang

Table 1. Information of the 2D-QASR models from the recent literature.

Model Data set* Bioassay Descriptor Method Results‡ Reference

Regression 25 Recombinant eHOMO, MLR R2 > 0.92 [14]

hERa eLUMO, MV, S < 0.62
logPow
131 Rat uterine TSAR MLR, PLS, MLR [15]
cytosol ERa FIRM (R2 = 0.732, S = 0.965);
PLS (R2 > 0.524,
S < 1.151);
FIRM (R2 > 0.636, S < 1.07)
132 Rat uterine CODESSA PLS RX2 = 0.74, RY2 = 0.64, [26]
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cytosol ERa Q2 = 0.62

81 hERa and hERb Fragment HQSAR R2 = 0.91, Q2 = 0.73 [33]
distinctions
245 Rat uterine DRAGON Consensus hERa (R2 = 0.89, [23]
cytosol, mouse, KNN Q2 = 0.79),
calf and human mouse
ERs (R2 = 0.88, Q2 = 0.77),
rat (R2 = 0.89, Q2 = 0.75),
calf (R2 = 0.86, Q2 = 0.73),
hERb (R2 = 0.83, Q2 = 0.69)
127 Rat, hERa and MolconnZ MLR MLR (R2 = 0.52, Q2 = 0.62) [31]
hERb PCA-BRNN BRNN (R2 = 0.83,
Qcv2 = 0.76, Qext2 = 0.81)
108 Rat, human DRAGON PCA-MLR R2 = 0.944, Q2= 0.898 [16]
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and mouse ERs

45 Rat uterine From Olah and PLS R2 = 0.589, Q2= 0.557 [17]
cytosol ER Rekker’s
132 Rat uterine CODESSA CP-ANN CP-ANN [26]
cytosol ER BP-ANN (R2 = 0.88, Q2 = 0.62)
BP-ANN
(R2 = 0.922, Q2 = 0.78)
93 hERa Molecular BRNN R2 = 0.83 – 0.91 [28]
Modeling Pro Q2 = 0.62 – 0.86
Classification 232 Rat uterine Cerius DF Accuracy: 0.7 – 1.0 [19]
cytosol ERa
232 Rat uterine MolconnZ DF ER232: 96.6% [20]
cytosol ERa ER1092: 95.4%
232 Rat uterine MolconnZ DF, DT DF: 82.94% [21]
cytosol ERa DT: 80.99%
311 Calf, mouse, rat, DRAGON DT, LVQ DT (> 69.6%), [22]
hERa and hERb k-NN LVQ (> 68.8),
k-NN (> 73.5%)
706 Rat uterine Topological SVM, SVM + RFE, ER agonists [32]
cytosol ER k-NN, PNN, SVM: 78.8%, SVM + RFE:
C4.5 DT 86.2%, C4.5 DT: 66.3%,
PNN: 83.6%,
k-NN: 72.7%
ER non-agonists
SVM: 86.2%,
SVM + RFE: 91.1%,
C4.5DT: 83.8%, PNN:
76%, k-NN: 85.9%
553 Rat uterine CODESSA, LogP CP-ANN Accuracy: 0.75 – 0.8 [25]
cytosol ER

*The data were collected from literature.

‡
Classification accuracy for classification model.
ER: Estrogen receptor; hER: Human estrogen receptor; QSAR: Quantitative structure–activity relationship.

Expert Opin. Drug Discov. (2010) 5(1) 23

 Discovery of estrogen receptor modulators: a review of virtual screening and SAR efforts
orbital (eHOMO) (R2 > 0.92). However, this model might
not be applicable for prediction of those compounds with
structures dissimilar to the data set used in the work, and as
suggested by the authors that other molecular descriptors were
still needed to improve the model performance. Considering
the quality of a QSAR model depending heavily on the
descriptors, another work was concentrated on the selection
of key descriptors associated with bioactivity by using different
mathematical methods. Ghafourian and Cronin [15] applied
four statistical methods including the stepwise regression,
variable clustering, PLS and formal inference-based recursive
modeling for variable selection on the basis of the ER binding
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affinity data for 131 compounds. The hierarchical PLS model
was observed to have a reasonable predictive ability, while the
stepwise-MLR model had a better predictive power than the
former. The results showed that the most important molecular
features for ER binding affinity were the molecular size and
shape, hydrophobicity, the presence of a phenol moiety and
double bonds. More recently, Liu et al. [16] have also reported a
MLR model for 108 compounds with estrogenic activity data
from different bioassays. Principal components analysis (PCA)
was used to condense the bioassay data into an estrogen
activity index. The process could preliminarily identify che-
micals most likely possessing estrogen activity for early entry
For personal use only.
into screening, following with the modeling with the MLR
method. Using an improved minimal topologic difference
method, a QSAR model was developed by Kurunczi et al. [17]
for predicting the binding affinities of 45 ER agonists based on
PLS. This work found the receptor site maps using the
spatially assigned analysis of fragment properties, and also
the benefic hydrophobic and van der Waals interactions
between ligand and receptor.
In recent researches for VS of potential ER modulators,
Decision Tree (DT) method as a powerful tool has been
introduced by several authors. This approach builds QSAR
models through a series of rules based on selection of descrip-
tors, and these rules are operated using IF-THEN expres-
sions [18]. For improving the performance of DT in both
training and validation steps, Tong et al. [19] applied another
approach, named DF, that combined a set of individually
trained classification trees which were developed using unique
sets of descriptors. With the aim of demonstrating the supe-
riority of DF over the DT, 232 structurally diverse chemicals
derived from NCTR were classified into estrogen and non-
ER-binding categories predicted by the two methods. This
work showed that the DF comprising seven trees eliminated
about two-thirds of the misclassification of the initial DT,
utilizing more descriptors (88 vs 10) than the DT.
In the subsequent studies, DF was also applied: i) For
seeking a better priority-setting tool for EDCs, Tong et al. [20]
compared predictive capability of two classification models
developed from DF based on different training sets, and
applied 2000 runs of 10-fold cross-validation to assess the
prediction confidence and extrapolation sensitivity of the
models. They found that the model with a larger training
24 Expert Opin. Drug Discov. (2010) 5(1)
set of 1092 compounds was more precise in predicting
chemicals than that with the small set (232 compounds),
and could be particularly useful for rapidly prioritizing poten-
tial endocrine disruptors from large chemical universe. ii)
Hong et al. [21] also performed a DF model using the
NCTR data set. Both internal Leave-10%-out cross-validation
and external validation were used to assess the model perfor-
mance, resulting in a high prediction accuracy of 81.9%. To
further investigate the effectiveness of this model, the authors
screened 92,964 chemicals to identify potential estrogens. The
high confidence bin for active prediction containing 1384
compounds, to some extent, demonstrated the reliability of
the model. iii) For proposing a comparison of this DF with
other methods, learning vector quantization (LVQ) and
k-nearest neighbor (k-NN) were also introduced with the
aim of classifying active and inactive estrogenic compounds
based on a data set of 311 compounds from NCTR by
Asikainen et al. [22]. PCA combined with a feature selection
procedure was used to reduce the dimensionality of the
descriptor pool. The results showed that the k-NN (98.3%
for accuracy) had the best classification ability while the DT
exhibited the weakest power, but all the three approaches were
suitable for classification of estrogenic compounds.
As mathematical algorithms ANNs consist of many artifi-
cial neurons and are designed to mimic the information
processing and knowledge acquisition of the human brain.
In the discovery of ER modulators, several ANNs have been
used to build different SAR models as follows: i) Asikainen
et al. [23] evaluated the predictive ability of the consensus
Kohonen neural networks (consensus KNN) using five subsets
of 245 compounds from NCTR data set which was assayed for
their relative binding affinities to the ER of four species:
human (ERa and ERb), calf, mouse and rat. The consensus
KNN was derived from the average performance of several
k-NN models to gain a final model with R2 ranging from 0.69
to 0.79, and Q2 from 0.62 to 0.77. This model had an
advantage of high speed but was limited in its narrow feature
to physicochemical interpretation. ii) Counter-propagation
neural network (CP-ANN) is a supervised network with
two layers, a Kohonen and an output layer [24]. Using this
method, Roncaglioni et al. [25] developed a model for the
classification of 553 potential endocrine disrupters collected
from European Union Commission. AM1 and the PM3 semi-
empirical methods were applied for the minimization of total
molecular energy to obtain 3D structure coordinates. This
model was a promising tool for preliminary assessment of
potential endocrine disrupters, despite the weakly defined end
point, which was the degree of distribution of the compounds
among categories. iii) Back-propagation neural network
(BPNN) refers to a multi-layer feed-forward network based
on error algorithm, trained with back-propagation learning,
which is also a supervised learning method. Marini et al. [26]
applied the two neural network methods (CPNN and BPNN)
and PLS based on 132 compounds for predicting their
binding affinities to the ER. Genetic algorithm (GA) was

applied as a variable selection to avoid the noise generated by
the descriptors. By comparison of the prediction ability of the
three methods, the authors found that the BPNN was the best
approach (with R2 = 92.2% and Q2 = 70.8% of the corre-
sponding model). iv) Probabilistic neural network (PNN) or
Bayesian network is a one-pass learning algorithm with a
highly parallel structure [27]. This method was introduced by
Agatonovic-Kustrin et al. [28] to predict the relative binding
affinities of ERa and ERb for 93 structurally diverse EDCs.
Two models were constructed with different data division of
73/25/25 and 63/30/30 data subsets for training, internal
testing and external validation. Suitable nonlinear relation-
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ships among the data were built with high correlation
coefficients (~ 0.8) for both models for ERa and ERb.
In recent years, researchers have paid great interest in the
design of new modulators capable of retaining the beneficial
effects of estrogens while avoiding most of their undesired
effects. Therefore, the requirement for novel modulators with
selectivity for ER increased considerably [29,30]. More recently,
several QSAR approaches have been applied in some studies to
facilitate the identification of selective ER agonists or antago-
nists. Wang et al. [31] performed a Bayesian-regularized neural
network (BRNN) to investigate multiple ligand–receptor
interactions by predicting binding affinities (pIC50) of 127
For personal use only.
ERa ligands. To reduce the descriptor space and the chance of
correlation among descriptors, in this work, a principal
component analysis was performed. The authors indicated
that the BRNN model was reliable to predict the binding
affinities for the training and test sets with coefficients
~ = 0.90. The resultant BRNN model showed obvious
superiority over the MLR model (R = 0.72, Q = 0.79),
although both models were adequate for screening and design
of more potent ERa modulators. For the purpose of predict-
ing ER agonists, Li et al. [32] selected four widely used
statistical learning methods, that is, support vector machines
(SVM), k-NN, PNN and C4.5 DT to study the SARs of a
large number of ER agonists and non-agonists primarily
derived from NCTRER and EDKB. Fivefold cross-validation
and an independent evaluation set were used in building
models in which a recursive feature elimination (RFE) method
was applied for selecting molecular descriptors. As a result,
their SVM system presented good prediction ability with
respect to ER agonists. With the similar aim of discovering
new selective ERb-agonists, Salum et al. [33] applied the
hologram QSAR (HQSAR) method to investigate 81
6-phenylnaphthalene and 2-phenylquinoline derivatives as
ERb modulators. The best model for binding affinity was
derived with R2 = 0.91 and Q2 = 0.73, demonstrating the use
of this model for the rational design of novel ERb modulators.
The MultiCASE program, QSAR-ES, is able to learn
automatically from data and organize this knowledge into
an expert system [34]. In 2003, the MultiCASE program was
proposed and applied by Klopman and Chakravarti [35] to
analyze 313 diverse molecules with ER binding potential.
Their simulation results turned out to be that a phenolic OH
Expert Opin. Drug Discov. (2010) 5(1)
Xu, Yang, Li & Wang
group was the most statistically significant biophore, and those
compounds which possess a phenolic ring and a lipophilic
carbon chain should exhibit stronger ER binding affinity.
Subsequently in 2004, using MCASE algorithm (MultiCASE)
parameters, another QSAR model was developed by
Cunningham et al. [36] based on the relative effect (RPE)
and relative proliferative potency for 122 compounds assayed
for estrogenicity, resulting in an prediction accuracy of 72%
for the testing compounds.
2.1.2 3D-QSAR models
As compared with 2D-QSAR methods, 3D-QSAR models are
more capable of describing ligand–receptor interactions for
their consideration on the properties of the ligands at 3D
level [37,38]. Therefore, great attention has been paid to
3D-QSAR approaches applied in modeling of ER
modulators recently.
As a novel 3D descriptor, the local intersection volume
(LIV) was developed by Cunha da et al. [39] to explore the SAR
of 2-arylbenzothiophene analogues. On a combination of GA
and PLS methods, an optimal model was derived with
R2 = 0.76, Q2 = 0.68, proving the effectiveness of the LIV
descriptor. Concerning with 3D-QSAR studies, several meth-
ods have been widely used in which the comparative molecular
field analysis (CoMFA) approach has almost became the most
frequently used one. In 2005, Demyttenaere-Kovatcheva
et al. [40] applied this method on a study of a number of
diphenolicazoles derivatives to identify the most favorable
steric and electrostatic structural regions contributing to the
binding affinity to ERa and ERb receptors, and their obtained
R2 was 0.91 with Qcv2 of 0.60 for ERa model, and R2 of 0.95
and Qcv2 of 0.40 for ERb model, respectively. Another widely
used 3D-QSAR technique is the comparative molecular sim-
ilarity indices analysis (CoMSIA), which is capable of calcu-
lating not only the steric and electrostatic features, but also the
hydrogen bond (H-bond) parameters (expressed by the
H-bond donor and acceptor fields) and hydrophobic variables
(expressed by the hydrophobic field) for the compounds of
interest. During the same time, Coleman et al. [41] imple-
mented both the CoMFA and CoMSIA as well as HQSAR
models to describe the SARs of bisphenol analogues. Their
results showed that the optimal model structures of bisphenol
analogues contained two unencumbered phenolic groups in
the para orientation and multiple alkyl substituents extending
from the carbon linking phenolic rings. FRED/SKEYS algo-
rithm is another 3D-QSAR paradigm that utilizes Molecular
Design Ltd substructure keys (SKEYS) as descriptors in
combination with an evolutionary algorithm, Fast Random
Elimination of Descriptors (FRED). For comparison, the
statistical robustness and predictive ability of the QSAR
models built by this FRED/SKEYS method, by CoMFA
and by HQSAR were studied individually by Waller in
2004 [42] using a set of specific compounds including phenols,
phthalates, phytoestrogens, DDTs, PCBs, pesticides, DESs
and steroids with the ER binding affinities. As a result, the
25
 Discovery of estrogen receptor modulators: a review of virtual screening and SAR efforts

F
H
OH

OH
N
N
HO

O
C3H7
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OH
N

4-propyl-1,3,5-triphenolpyrazole (4g) Benzoxepin (26b)

OH

N
HO S

O O
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O O

OH

HO S

67 Raloxifene

HO
OH OH

OH HO

F CI HO CH2CH(CH3)2

Modulator 1 Estradiol-17β DMB Bis A

Figure 1. Representative compounds in the review.

CoMFA and HQSAR methods based on the training set
presented similar results (R ~ = 0.50), while the FRED/
SKEYS model obtained a relatively higher value of R
(0.70). Therefore, Waller concluded that the FRED/SKEYS
method was superior to the other two for this particular data
set. As a novel 3D-QSAR model, a template-based molecular
mechanistic superposition algorithm FLUFF (flexible ligand
unified force field) and an accompanying local coordinate
QSAR method BALL (boundless adaptive localized ligand)
were also validated by Korhonen et al. [43] against the bench-
mark techniques steric and electrostatic alignment and
CoMFA using 245 xenoestrogens extracted from the

26 Expert Opin. Drug Discov. (2010) 5(1)


Human ERα
GLU353 ARG394 LEU391
1.89 1.98
1.87
1.89
LEU349
MET388
LEU387
LEU402
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ALA350
THR347
LEU525
1.82
1.96
2.35
MET343 HIS524
Figure 2. Binding modes of the three small potent ERa, b modulators (red, modulator1; green, DMB Bis A; yellow,
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estradiol-17b). The red square: hydroxyl group of Thr347 (ERa) and of Thr299 (ERb). H-bond: dotted pink line. The docking was
performed using Autodock 4.0 (www.autodock.com) with default parameters. (Readers are referred to the full-colour version, available
at http://informahealthcare.com/loi/edc)
ER: Estrogen receptor.
EDKB. The study showed that FLUFF-BALL could be used
as a superposition technique for BALL and CoMFA to
generate reasonable models. For providing an alternative
and supplement to 3D-QSAR model, Asikainen et al. [44]
performed three alignment-free QSAR methods, named
‘spectroscopic’ QSAR methods (eigenvalue, electronic eigen-
value and comparative spectra analysis (CoSA)) for predicting
the activity of 36 estrogenic compounds. Their predictive
ability was compared with that of self-organizing molecular
field analysis (SOMFA). The authors suggested that both the
CoSA and SOMFA models were suitable for the estrogenic
chemicals. More recently, two CoMFA models have been
developed by Salum et al. [45] to 81 hER-ligands, and the
models presented both internal (hERa, Q2 = 0.76; hERb,
Q2 = 0.70) and external validation (hERa, R2 = 0.80; hERb,
R2 = 0.88) between actual and predicted pIC50 values.
Meanwhile, the influences caused by structural differences
between hERa and hERb were also investigated by the GRID/
PCA based on five hER crystal structures to generate molecular
interaction fields maps. Salum et al. [46] used CoMFA
and HQSAR to predict the binding affinities of 127 ERa
modulators. The two models generated were significantly
consistent with the experimental values (CoMFA, R2 = 0.93
and Q2 = 0.79; HQSAR, R2 = 0.92 and Q2 = 0.71).
Pharmacophore modeling, as another important tech-
nique in drug design to investigate the common molecular
structural features, is also introduced into this field.
Xu, Yang, Li & Wang
Human ERβ
ARG346
GLU305 1.73
LEU343
LEU301
ALA302
MET340
THR299 LEU380
ILE424
LEU476 ILE376
3.33 2.94
1.95 HIS475
MET295
A receptor-independent pharmacophore mapping model
was proposed by Mukherjee et al. [47] to visualize the
pharmacophores of ER a and b based on the binding
affinities of 14 bridged bicyclic-1 and 20 1-diarylethylene
derivatives. The models for ER subtypes demonstrated
the importance of the critical inter-feature distances
among H-bond acceptor, hydrophobic and ring aromatic
features, along with steric influence primarily governing the
ER-subtypes specific binding of scaffolds containing the
1,1-diaryl compounds of SERMs.
As a comparison with the ligand-based models as shown
above, docking is a structure-based method and its success for
VS has been determined by two components, the search
algorithm and the scoring function. GEMDOCK (generic
evolutionary method for molecular docking) combined with a
new pharmacophore-based scoring function was applied by
Yang and Shen [48] for hER and a ligand database. The new
scoring function consisting of a empirical binding score and a
pharmacophore-based score was able to simultaneously serve
as the scoring function for both molecular docking and the
ranking of screened compounds for postdocking analysis,
resulting in enhancing the accuracy during flexible docking
and improving the screening utility.
Another docking study (coarse-grain molecular docking
procedures plus fine-grain all-atom force field optimization
strategies) was conducted by Kekenes-Huskey et al. [49] to
investigate the binding mechanism of the SERMs. The
Expert Opin. Drug Discov. (2010) 5(1) 27
 Discovery of estrogen receptor modulators: a review of virtual screening and SAR efforts

Human ERα Human ERβ

ARG346
ARG394

GLU353 1.70
2.13 1.88
LEU391 2.00
2.17
1.86
GLU305 1.73

PHE356
LEU387 LEU301

PHE404
LEU402
LEU339
LEU384

LEU298 ILE380
LEU428
Expert Opin. Drug Discov. Downloaded from informahealthcare.com by The University of Manchester on 11/02/14

VAL487
PHE425

ILE424 ILE376
1.99
2.05 GLY472
LEU525
GLY521 1.98
2.55 2.39
MET421 VAL484 MET295 ILE373
HIS524 2.14
LEU476
HIS475

Figure 3. Binding modes of four big potent ERa, b modulators (blank green, raloxifene; yellow, 26b; purple, 67; green, 4g).
Flexible residues are LEU391, LEU387, LEU384, LEU525 for ERa, and leu301, leu339, leu298, leu476 for ERb. The docking was performed
using Autodock (www.autodock.com) with default parameters. The binding energy is -9.97, -15.73, -7.19 and -13.69 for ERa modulators,
respectively, while -8.89, -15.45, -9.8 and -4.72 for ERb modulators, respectively. (Readers are referred to the full-colour version, available
For personal use only.

at http://informahealthcare.com/loi/edc)
ER: Estrogen receptor.

binding mode of type I agonistic estrogens (estradiol and
diethylstilbesterol) was deduced from the crystal structure of
the ligand–protein complexes, while that for type II antago-
nistic estrogens (4-hydroxytamoxifen and raloxifen) was
hypothesized based on the relative binding activity and
luciferase activation data.
Recently, the combination of docking technique with
other QSAR methods has become a trend in the study of
the ER binders, presenting more informative data for the drug
design. A docking and 2D-QSAR modeling were introduced
by Barrett et al. [50] to a series of benzoxepin-derived com-
pounds as potent selective hER modulators. The QSAR model
obtained good performance (R2 = 0.84 and Q2 = 0.72) using a
set of standard descriptors such as logP. The docking analysis
showed that the ERb selectivity was achieved by a ligand
that could differentiate between ERa Met421 and ERb
Ile328 residues.
Wolohan and Reichert [51] reported the combined appli-
cation of docking and 3D-QSAR (CoMFA) for investigating
36 chemicals interacting with ERa and ERb with the tools of
Gold and Flexidock. The original CoMFA models for the two
subtypes were reasonable (ERa: R2 = 0.79 and Q2 = 0.44;
ERb: R2 = 0.93 and Q2 = 0.63). The inclusion of the CScore
and Gold scoring parameters failed to improve the 3D-QSAR
model performance. But with the addition of solvation
energy of the isolated ligand, the predictive nature of the
ERb model was improved (R2 = 0.96; Q2 = 0.70); yet, the
ERa did not change.
Flexible docking (software Yeti) and 6D-QSAR
(software Quasar) were used by Vedani et al. [34] to
investigate 106 structurally diverse ER ligands. The
Quasar, a receptor-modeling concept, allowed the 6D-
QSAR modeling of solvent effects associated with ligand
binding in an unbiased fashion. The receptor surrogate
could effectively identify the characteristics including
H-bond acceptors mimicking Glu 353 and His 524, an
H-bond donor mimicking Arg 394, and a larger hydro-
phobic pocket representing Leu 346, Ala350, Trp 383,
Leu 384, Phe 404, Ile 424, Phe 425 and Leu 540.
The obtained results (Q2 = 0.903; R2 = 0.885) suggested
that this method was effective for identification of an
endocrine-disrupting compound.
3. Expert opinion
ERb has a dissimilar degree of plasticity in its LBD cavity
compared with ERa [52], indicating that the receptor confor-
mational change may be induced by the binding of a ligand,
thus, affecting the binding mode of compounds. However,
such a pheromone has not been taken into consideration in
most docking studies (rigid docking) previously. The rigid
docking might be suitable for those relatively small molecules
such as estradiol, but some interesting phenomena involved in
this are up to now still neglected. Considering the selectivity of
ER subtypes, for example, the residue THR347 (pdb: 1ERE),
presents an H-bond for DMB Bis A (a very potent estrogenic

28 Expert Opin. Drug Discov. (2010) 5(1)

 Xu, Yang, Li & Wang

compound [41]) in ERa, while for ERb no hydrogen bonding
is found for the same residue THR299 (pdb: 1QKM),
thus, affecting the specificity of ERa and ERb to binders
(Figures 1 and 2).
For those potent and relatively big modulators, such as
4-propyl-1,3,5-triphenolpyrazole (4g) [51], benzoxepin
compound (26b) [50], Raloxifene [39] and compound
67 [33], previous rigid docking simulations have failed to
provide reasonable scores due to the neglect of interactions
of the flexible residues in the binding pocket. By using the
flexible docking method, the present simulations show
different binding modes from previous work with the
Expert Opin. Drug Discov. Downloaded from informahealthcare.com by The University of Manchester on 11/02/14
be taken to consideration for improving the modeling
accuracy of relatively big molecules.
In addition, both the mutant forms of ERa and ERb could
alter the effects of bound antiestogens [53], which has not been
paid attention to by researchers in the areas of designing ER
modulators. Therefore, it is necessary to model the mutated
ERs interacting with binders and to develop novel ER mod-
ulators to overcome the increasing drug resistance. And also,
the evaluation of the ADME/T (absorption, distribution,
metabolism, excretion and toxicity) properties of drug candi-
dates is an important stage in drug discovery [54], but up to
now, no special concerns about in silico ADME have been

high evaluation scores (Figures 1 and 3). For example,
the H-bonds in ERa are formed by the residues of Gly
A521, Arg A394 and Glu A353 for 4g; Thr A347 and Gly
A521 for 26b; Glu A353 for compound 67; Thr A347 and
Gly A521 for raloxifene, respectively, while in ERb the
H-bonds are formed by the residues of His A524 and Gly
A521 for 4g, and His A475 and Gly A472 for 26b,
respectively. Therefore, it is suggested that great concern
about the flexible residues in the ER binding pocket should
placed in computer-aided discovery of ER modulators. There-
fore, in order to improve the efficiency of the discovery of ER
modulators, the inclusion of ADME in the future modeling
practice is of significance.
Declaration of interest
This work is supported by the National Natural Science
Foundation of China (Grants No. 108010235).

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Affiliation
Xue Xu1, Wei Yang1, Yan Li2 &
Yonghua Wang†1,2
†
Author for correspondence
1
Northwest A&F University,
Center of Bioinformatics,
Yangling, Shaanxi,
712100, China
2
Dalian University of Technology,
For personal use only.

School of Chemical Engineering,

Dalian, 116012,
Liaoning, China
Tel: +86 029 87092262;
E-mail: yh_wang@nwsuaf.edu.cn

Expert Opin. Drug Discov. (2010) 5(1) 31