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www.rsc.org/chemicalscience MINIREVIEW
Molecular docking for virtual screening of natural product databases
Dik-Lung Ma,*a Daniel Shiu-Hin Chana and Chung-Hang Leung*b
Received 16th March 2011, Accepted 3rd June 2011
DOI: 10.1039/c1sc00152c
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/C1SC00152C

Molecular docking enables the extraordinary structural diversity of natural products to be harnessed in
an efficient manner. In this mini-review, we highlight recent examples of the use of molecular docking in
virtual screening for the identification of bioactive molecules from natural product databases.

Introduction a new golden age of drug discovery. However, medicinal chem-

The 1990’s heralded a paradigm shift in the way pharmaceutical
research was conducted around the world. With new tools and
techniques supplied by molecular biology, millions of research
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ists were soon confronted with painfully disappointing results
and the realization dawned that the chemical space was too vast
to be systemically explored experimentally.8 Today, despite the

increasing financial investment into research and development,

dollars were invested into the development of highly efficient
high-throughput screening (HTS) in vitro and in vivo methodol-
ogies that were capable of screening hundreds of thousands of
compounds each year.1–4 This growth was accompanied by
exciting advances in the field of combinatorial chemistry.5–7
Whereas a typical bench chemist could be expected to possibly
make a few hundred novel compounds each year, the new
strategies afforded by combinatorial chemistry easily drove that
number into the thousands. The pharmaceutical industry eagerly
adopted these new technologies, which promised to usher in
the number of new drugs approved for the market has fallen to
an all-time low. Bereft of promising leads, large pharmaceutical
companies have increasingly shifted towards mergers and
acquisitions in order to fill their pipelines.9,10 Thus, researchers
are continually seeking to develop novel methodologies for the
efficient discovery of new chemical scaffolds for pharmaceutical
investigations. This review focuses on the emerging use of
computer-aided high-throughput molecular docking methodol-
ogies to identify bioactive NPs against important therapeutic
targets, and aims to summarize the recent successes in this area.

a
Department of Chemistry, Hong Kong Baptist University, Kowloon Tong,
Hong Kong. E-mail: edmondma@hkbu.edu.hk Role of natural products in drug research
b
Centre for Cancer and Inflammation Research, School of Chinese
Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong. Nature has provided us with a fascinating cornucopia of complex
E-mail: duncanl@hkbu.edu.hk molecular scaffolds unparalleled in function and diversity. These

Dik-Lung Ma completed his Daniel Shiu-Hin Chan

PhD in 2004 at the University of
Hong Kong under the supervi-
sion of Prof. C.-M. Che. He
spent the years 2005–09 at the
University of Hong Kong, the
Hong Kong Polytechnic
University, and the Scripps
Research Institute with Prof. C.-
M. Che, Prof. K.-Y. Wong, and
Prof. R. Abagyan. His research
mainly focuses on luminescent
sensing for biomolecules and
Dik-Lung Ma metal ions, computer-aided drug
discovery, and inorganic medi-
cines. In 2010 he was appointed
Assistant Professor at the Hong
Kong Baptist University.
completed his BSc degree in
Chemistry at the University of
New South Wales, Sydney,
Australia. He is currently
appointed as a Research Assis-
tant at the Department of
Chemistry at the Hong Kong
Baptist University. His research
interests include development of
luminescent oligonucleotide-
based assays for biomolecules
and metal ions.
Daniel Shiu-Hin Chan

1656 | Chem. Sci., 2011, 2, 1656–1665 This journal is ª The Royal Society of Chemistry 2011

natural products (NPs) have been refined over evolutionary time
scales for optimal interactions with biomolecules. As the
production of any new compound by a living organism entails
increased costs, the natural selection hypothesis predicts that
each natural product retained by an organism must be of value to
the producer. Before the advent of modern synthetic medicinal
chemistry, natural products were the only source of medicinal
compounds available to mankind. Pharmacological investiga-
tions into traditional and herbal remedies have yielded many
significant discoveries for modern medicine.11–16 For example,
the herb Artemisia annua has been used in traditional Chinese
medicine for thousands of years for treating malaria and other
skin diseases, but it was only several decades ago that the active
ingredient, artemisinin, was isolated.17–19 Huperzine A, isolated
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/C1SC00152C
from the plant moss Huperzia serrta, is an acetylcholinesterase
inhibitor investigated for Alzheimer’s disease, though the plant
itself has been used for centuries by Chinese herbalists for
treating swelling, fever and blood disoders.20–22 Another highly
important drug of natural origin is the anti-cancer paclitaxel
(taxol), isolated from the tree bark of Taxus brevifolia, and used
for the treatment of breast, ovarian, lung, head and neck
cancer.23 The total number of natural compounds produced by
plants has been estimated to be over 500 000.24 However, this is
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believed to represent only a small fraction of the total number of
existing NPs, considering the huge number of undocumented
species in the vast unchartered regions of the biosphere. Marine
organisms, in particular, are a tremendous source of secondary
metabolites possessing unique structural diversities yet to be
tapped by the medicinal chemist.25–27
Until recently, NPs were still regarded as a fundamental
cornerstone of pharmaceutical research.28–32 However, amidst
the rapid developments and promises of HTS and combinatorial
chemistry, the realm of natural products fell somewhat by the
wayside. Many major pharmaceutical companies have halted or
significantly cut natural product research. A survey of new
chemical entities by Newman and Cragg between the years 1981–
2006 shows that NPs and drugs derived directly from natural
compounds occupied only 5% and 23% of this class, respectively,
compared to 30% for synthetic compounds.33 It has also been
Chung-Hang Leung completed
his PhD in 2002 at City
University of Hong Kong under
the supervision of Prof. W.-F.
Fong. After completing a five-
year post-doctoral fellowship at
the Department of Pharma-
cology, Yale University with
Prof. Y.-C. Cheng, he was
appointed Research Assistant
Professor at the University of
Hong Kong and then at the
Hong Kong Baptist University.
Chung-Hang Leung His primary research interests
are in anticancer and anti-
inflammatory drug discovery,
molecular pharmacology and
immunology.
This journal is ª The Royal Society of Chemistry 2011
View Article Online
noted that the number of NP-based drugs in clinical trials has
fallen 30% between 2001 and 2008.34
Tellingly, the shift away from NP research in pharmaceutical
corporations was engendered not by any intrinsic faults with the
class of natural compounds as a whole, but by the incompati-
bility of NP with the highly automated drug discovery method-
ologies of HTS. The reasons are many-fold. Firstly, NPs are
often regarded as highly structurally complex, containing any
number of chiral centres, fused ring systems, and reactive func-
tional groups like the hydroxyl. Pursuing the synthesis of
a complicated NP or NP derivative is hardly a trivial task,
requiring the construction of intricate ring systems together with
a laborious strategy of protection and deprotection steps that are
not readily amenable to combinatorial methods. Secondly, the
extraction and purification of NPs from plant, marine and
microbial sources can be very difficult, and the precious
compounds are often obtained in only minute quantities. Issues
with supply can become a major bottleneck in NP drug discovery
especially if the NP itself is to be utilized as a benchmark or as
a synthetic intermediate for lead optimization and further in vivo
testing. Furthermore, structural elucidation still has to be per-
formed on any assay hit obtained from a natural source, whether
from bioassay-guided fractionation or the isolation of a single
active compound, and its structure would have to be compared
to the synthetically-generated NP. Thirdly, the HTS screening of
crude NP extracts can sometimes be problematic due to highly
polar or lipophilic components in the sample matrix, leading to
false positives or false negatives in the assay result. The screening
of crude extract libraries also entails a stringent identification
and dereplication program to prevent the expenditure of
resources on known or otherwise uninteresting compounds. A
further complication arises due to the fact that individual
compounds in an extract are likely to be present at vastly
different concentrations, such that levels of trace components
may be too low for detection, while inactive, highly abundant
compounds may be registered due to non-specific inhibition.
Additionally, the intrinsic optical properties of uncharacterized
NPs in the extract may interfere with fluoresence-based assay
methodologies. Lastly, the synergistic or antagonistic effects of
two or more components may vanish upon isolation of the
primary constituent. By comparison, the screening of large
libraries of pure, characterized, synthetic compounds present in
known quantities is regarded as a much easier endeavour.
However, the failure of combinatorial chemistry to yield
a significant increase in the number of drug candidates despite
the powerful testing capabilities of HTS prompted a rethink of
the nature and quality of the chemical libraries themselves. The
major drawback to combinatorial compounds lies in the implicit
requirement for efficient reaction and purification methodologies
that whilst able to produce large numbers of compounds at high
yield and low cost, severely limits the structural diversity of the
chemical library. According to a study performed by Feher and
co-workers, combinatorial compounds tend to contain fewer
chiral centers compared to natural compounds.35 They also
contain more rotable single bonds and aromatic rings, an artifact
of the construction process that involves joining many building
blocks together in simple reactions. On the other hand, NPs tend
to contain more rigid scaffolds comprising fused, unsaturated
ring systems, potentially exploiting the entropic advantages
Chem. Sci., 2011, 2, 1656–1665 | 1657

conferred by rigidity in order to achieve the high biochemical
affinities and specificities characteristic of NPs.35
Today, HTS is rarely performed using purely combinatorial,
unfocused libraries. Instead, efforts have focused on ‘‘privileged’’
synthetic databases that seek to occupy a more biologically
relevant space within the vast universe of possible chemical
structures. Such libraries include databases of existing drugs
(drug repurposing),36–38 as well as those based upon the principles
of ‘‘diversity-orientated synthesis’’ (DIOS)39–41 and ‘‘biology-
orientated synthesis’’ (BIOS).42–44 A comprehensive review of all
these methodologies is outside the scope of this work, and the
reader is directed to the excellent reviews that have been pub-
lished on these topics.36–44 However, despite the best of these
efforts, HTS screening of such carefully constructed libraries can
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/C1SC00152C
still realistically only sample but a small portion of the entire
chemical space at a time. This is where computer-aided drug
discovery can make an important contribution.
Virtual screening in drug discovery
Virtual screening has recently emerged as a powerful technique
complementing traditional HTS technologies. Virtual screening
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can be broadly defined as the use of computational analysis of
a database of chemical structures to identify possible drug
candidates for a specific pharmaceutical target, often a particular
enzyme or receptor. The major advantage derived from virtual
screening is the tremendous reduction in time and resources
required to screen a chemical library of known compounds in
a drug discovery project. By identifying non-binding compounds
in silico, the number of compounds to be tested in vitro can be
dramatically reduced, sometimes by orders of magnitudes. Due
to this ‘‘weeding out’’ or elimination of inactive chemical struc-
tures, the hit rates in the in vitro assays are often much higher
compared to conventional HTS without preliminary virtual
screening. Furthermore, the number of compounds in even the
largest chemical library is but a small fraction of the total
possible number of compounds that could be synthesized in
principle; estimates range up to 1020–24 for the total number of
molecules accessible using known synthetic methods.45
Computer-aided screening is thus a valuable tool to help
medicinal chemists decide what to synthesize, an advantage over
the somewhat random nature of combinatorial libraries used
previously.
The use of virtual screening methodologies in drug discovery
and development has been widely reviewed.46–51 Today, compu-
tational chemistry and chemoinformatics play a key role in early
phase drug research, by identifying the most promising candi-
dates for experimental investigations. Two major strategies have
been employed for virtual screening: pharmacophore modeling
and molecular docking (Fig. 1). Within the realm of pharmaco-
phore modeling, the two techniques of structure-based phama-
cophore modeling and ligand-based pharmacophore modeling
can be distinguished. In the former, the knowledge of the three-
dimensional (3D) structure of the biomolecular target is analyzed
to identify features of the binding site that are important for
ligand binding affinity and selectivity. The 3D structure may be
solved by X-ray crystallography, NMR studies or constructed by
homology with related proteins, and over 73 000 of such struc-
tures are freely available at the Protein Data Bank (PDB).52 An
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X-ray structure of the biomolecular target that is co-crystallized
with a ligand is especially advantageous since specific features of
the ligand–target interaction can be readily detected. Some
computational softwares such as LIGANDSCOUT are able to
automatically construct a pharmacophore model by identifying
and performing calculations on the relevant interactions between
the small molecule and the receptor. These ligand–targets are
classified into features such as hydrogen bonding, charge trans-
fer, and lipophilic interactions to construct at 3D pharmaco-
phore model. In ligand-based pharmacophore modeling, a 3D
structure of the biomolecular target is not required. Rather,
a collection of known ligands is assembled in order to derive
representative electrostatic and steric features that are then used
to construct the pharmacophore model. After the construction of
the 3D pharmacophore model, a chemical database is screened
against the model in order to extract ligands that possess the
complementary functional groups in the correct spatial
arrangement. Since the affinity calculation is based only on the
geometric fit of ligand atoms and groups to the chemical features
of the model, the strain on computational resources is signifi-
cantly lower compared to molecular docking. This makes phar-
macophore modeling suitable as a pre-filter for more demanding
virtual screening methodologies.53 However, a disadvantage is
that the calculated affinity values are often not accurate,
however, they are still broadly useful for eliminating non-
binders. Ligand-based pharmacophore modeling also requires
prior knowledge of a set of active compounds, which makes it use
unsuitable for novel biomolecular targets. An additional draw-
back of the pharmacophoric method is that in the absence of
structural or mechanistic information about the target, it is not
always possible to anticipate which particular structural features
of the ligands is important for the receptor–ligand interaction.
Lastly, due to the inherent nature of pharmacophore modeling,
screening usually reveals hits that are structurally similar to the
known ligands, rather than leads with novel modes of binding.
The use of pharmacophore modeling in NP drug research is not
the focus of this review, and the reader is directed to excellent
reviews published recently by the groups of Langer54 and
Wolber.55
Compared to the relatively established technique of pharma-
cophore modeling, 3D molecular docking is considered more
complex and computer-intensive. However, exponential
advances in computing processing power and capabilities have
increased the popularity of molecular docking methods (also
called structure-based or receptor-based virtual screening) in
drug discovery and development. The use of molecular docking
avoids some of the aforementioned drawbacks involved with
pharmacophore modeling. Molecular docking requires knowl-
edge of the 3D structure of the biomolecular target with or
without a bound ligand, at atomic resolution. Use of a co-crystal
structure of a biomolecular target with a ligand entails several
advantages. Firstly, the search area for the in silico docking can
be restricted to the binding site only (alternatively, the binding
site may be predicted from homology with similar proteins). An
unnecessarily large search area can increase the rate of false
positives due to the binding of virtual molecules outside the
actual binding site. Furthermore, this can also lead to wastage in
computer resources. Secondly, prior knowledge of critical
ligand–target interactions can help identify false positives in the
This journal is ª The Royal Society of Chemistry 2011

Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/C1SC00152C
Fig. 1 Schematic flowchart outlining the two major strategies employed in in silico virtual screening.
virtual screening. Third, more accurate docking calculations can
be performed since the target is in its active or induced confor-
mation. A computational model of the target is constructed from
the 3D structure, and ligands from the chemical database are
sequentially docked against the receptor. Most docking
programs incorporate ligand flexibility into the docking calcu-
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lations so that the binding geometry of the ligand can be cor-
rected predicted. However, the target is usually assumed to be
mostly rigid, as the explicit inclusion of receptor flexibility in the
docking calculations would be too computationally demanding.
Some docking algorithms attempt to model receptor flexibility in
an indirect way, for example by tolerating some degree of steric
clashing with the ligand without the explicit repacking of the
receptor side chains, or by using several alternative receptor
binding site conformations for docking and then merging the
results. Some modern docking algorithms are able to explicitly
model receptor flexibility, but this is usually constrained to the
ligand binding domain in order to conserve computing
resources.56,57 A RMSD of <2 A
from the predicted binding pose
to the X-ray crystal structure is considered satisfactory.
After the optimal binding poses have been predicted for each
compound in the database, the next step is to rank or score the
structures to determine their relative binding affinities. Scoring
functions perform calculations involving statistical potentials or
weighted interaction terms that have been previously calibrated
with ‘‘training sets’’ of known binders and non-binders. However,
it has been noted that scoring functions represent the major
weakness in docking programs; while the docking algorithm is
relatively accurate at predicting the preferred binding mode, the
ability of the scoring functions to distinguish strong and weak
binders still leads to a significant number of false positives in the
virtual screen. Consensus scoring, the use of multiple scoring
functions in concert, has been found to significantly improve hit
rates compared to the use of a single scoring function.58–61
As discussed in the previous section, despite the astonishing
structural diversity and fascinating molecular architecture
exhibited NPs, their use in HTS has been somewhat neglected
compared to purely synthetic libraries based on drug-likeness,
DIOS or BIOS. The screening of isolated natural compounds
and NP extracts has often been regarded as too ‘‘dirty’’ and too
expensive, and involving additional time and labour-consuming
efforts in isolation, fractionation, characterization, and
dereplication. Computational methods open up new possibilities
This journal is ª The Royal Society of Chemistry 2011
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for screening natural compounds by bypassing many of these
tedious steps. For example, non-binders can be predicted in silico,
avoiding the wastage of scarce NPs. Additionally, practical
difficulties associated with bioassay-guided fractionation and
dereplication are made irrelevant. One minor drawback is that
it is no longer possible to isolate totally new bioactive
compounds, as from a NP extract.
It is common knowledge that the results of any HTS exercise is
ultimately predicated upon the quality of the compound collec-
tion itself. Poorly designed libraries lacking sufficient diversity
may result in few hits. Fortunately, NP libraries possess an
intrinsic advantage in diversity of molecular scaffolds due to
their exquisite relationships with biomolecules that have been
streamlined over evolutionary time scales. Furthermore, many
NP compound collections also contain NP-like structures that
are based upon naturally occurring scaffolds. One should also
note that NPs often contain more than one violation of Lip-
inski’s ‘‘rule-of-fives’’,62 especially with regards to molecular
weight and hydrogen bond acceptors, and too-strict adherence to
these rules in a screening campaign would certainly act to negate
the inherent advantages of structural diversity conferred by the
use natural compounds. A summary of some available NP
databases is presented in Table 1.
Recent successes of molecular docking of natural
products for drug discovery
We discuss here some recent examples of the use of molecular
docking for the discovery of bioactive NPs in the last decade. For
ease of access, we have grouped docking targets into three
classes: enzyme–substrate interactions, receptor–ligand interac-
tions (including protein–protein interactions) and DNA
interactions.
Natural products targeting the enzyme–substrate interaction
In 2004, Toney and co-workers screened the National Cancer
Institute (NCI) diversity set of 1853 compounds of both natural
and synthetic origin against 3CLpro proteinase of the severe acute
respiratory syndrome coronavirus (SARS-CoV), the causative
agent for a pandemic that swept Southeast Asian regions in
2003.63 The compounds were docked against the X-ray crystal
structure of 3CLpro (PDB entry: 1P9S) using the AutoDock
Chem. Sci., 2011, 2, 1656–1665 | 1659

Table 1 Some available natural product databases for virtual screening
Database Company
ZINC Bioinfomatics and Chemical Informatics
Research Center (BCIRC)
MEGabolite Analyticon Discovery
NatDiverse Analyticon Discovery
IBS Database InterBioScreen Ltd
Super Natural Database Charite, Medical Faculty of the
Humboldt-University
CHEMnetBASE (Dictionary of Natural Chapman & Hall
Products)
CHEMnetBASE (Dictionary of Marine Chapman & Hall
Natural Products)
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/C1SC00152C
Natural Products Database (NPD) Molecular Diversity Preservation
International (MDPI)
Natural Compound Library (NCL) TimTec
Natural Derivative Library (NDL) TimTec
SPECS Natural Products SPECS, Inc.
CNPD NeoTrident Technology Ltd
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program. The highest scoring compound was found to be the
natural product sabadinine, which showed a binding energy of

11.6 kcal mol
1 and a clustering of 9 out of 10 docked
conformers within 0.5 A.
However, sabadinine did not affect
murine coronavirus replication at 100 mM, as gauged by syncy-
tium formation and cytopathic effects.
Sangma and co-workers (2005) utilized a combined approach
of molecular docking and neural networks to identify inhibitors
of HIV-1 reverse transcriptase (RT) and HIV-1 protease (PR)
from a Thai Medicinal Plants Database.64 2684 compounds were
docked against the X-ray co-crystal structures of HIV-1 RT with
nevirapine or calanolide A (PDB entry: 1VRT) and HIV-1 PR
with XK-263 (PDB entry: 1HVR) using AutoDock, and then the
results were applied to a neural network based on a self-orga-
nizing map (SOM) in order to reduce the size of the hit list. In the
SOM approach, the reference structures are analyzed to find
potential pharmacophoric groups, and for easier analysis, a map
was generated that contained only the distances between three
points of certain pharmacophoric groups. The same maps were
generated from the docking hit list and then superimposed onto
the reference map, and only the compounds having their features
represented in the common regions were reselected. This
successive SOM screening could be repeated as many times as
necessary (by using different features) to reduce the size of the
candidate pool. For example, AutoDock identified 562 (out of
2684 compounds) docking hits for HIV-1 PR, but this was
reduced to 135 and then 13 after successive rounds of SOM
screening. However, most of these compounds had already been
reported to show anti-HIV activity; no biological validation was
performed in this work. This study demonstrates that using
a pharmacophoric SOM neural network after molecular docking
is a computationally inexpensive method of reducing the size of
the virtual screening hit list.
In 2006, the group of Moro identified ellagic acid, a naturally
occurring derivative of tannic acid, as an inhibitor of casein
kinase 2 (CK2), a putative oncogene in animal and cellular
models.65 Using an in-house database of around 2 000 NPs, they
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View Article Online
Number of
compounds Link
89 425 http://zinc.docking.org/vendor0/npd/
index.html
4 700 http://www.ac-discovery.com
>20 000 http://www.ac-discovery.com
>45 000 http://www.ibscreen.com/natural.
shtml
45 917 http://bioinformatics.charite.de/
supernatural/
>226 000 http://www.chemnetbase.com/
>34 000 http://www.chemnetbase.com/
22 048 http://www.mdpi.org/
640 http://www.timtec.net/
3 000 http://www.timtec.net/
400 http://www.specs.net/
57 000 http://www.neotrident.com/newweb/
index.asp
utilized a consensus docking and scoring approach that includes
four docking algorithms (MOE-Dock, Glide, Fred and Gold)
and five scoring functions (MOE-Score, GlideScore, GoldScore,
ChemScore and Xscore) to dock and rank the NP database
against the X-ray crystal structure of CK2 (PDB entry: 1JWH).
Significantly, ellagic acid ranked in the top 5% of all five scoring
functions. Furthermore, all four docking programs predicted the
same lowest energy binding conformation for ellagic acid. This
promising in silico result was validated by the biological verifi-
cation experiments, which showed that ellagic acid represented
the most potent CK2 inhibitor reported at that time (Ki ¼
20 nM). Kinetic analysis revealed ellagic acid to be a competitive
inhibitor with respect to ATP, which was consistent with the
molecular docking results that showed binding of the small
molecule to the ATP-binding domain. Importantly, ellagic acid
was shown to be at least 72-fold more potent against CK2 kinase
(IC50 ¼ 0.04 mM) compared to a panel of 11 other kinases (IC50 >
2.9 mM), a remarkable result considering the ostentatiously
promiscuous structural features of ellagic acid (a planar
compound containing four hydroxyl moieties) and the fact that
this virtual screening hit was not optimized at all. This early work
helped to demonstrate the power of molecular docking for
identifying potent and selective NPs against therapeutic biomo-
lecular targets.
In 2008, Fu et al. identified Jadomycin C as an inhibitor of
Aurora B kinase by employing molecular docking of the
Microbial Natural Products Database containing about 15 000
natural compounds of microbial origin against the X-ray co-
crystal structure of Aurora-B cocrystallized with the Hesperadin
(PDB entry: 2BFY).66 Aurora kinases play key roles in cell
division through the regulation of centrosome maturation and
separation, microtubulue-kinetochore attachment, and chro-
mosome alignment and segregation. Consequently, Aurora
kinases have been found to be overexpressed in a variety of
cancers, and inhibitors of such kinases, including Hesperadin,
ZM447439 and VX-680, have been showed significant anticancer
activity in vitro and in vivo. Using the docking program FlexX,
This journal is ª The Royal Society of Chemistry 2011

a hit list of the 150 top scoring structures were obtained from the
virtual screening campaign, of which 22 were procured for
experimental testing. The most promising candidate, Jadomycin
B, could inhibit the activity of Aurora-B in vitro with an IC50
value of 10.5 mM and a Ki value of 6.8 mM. Jadomcyin was also
able to inhibit the proliferation of a variety of cancer cell lines
with IC50 values in the range of 10–26 mM. However, because
Jadomycin B induced apoptosis without blocking the cell cycle,
the putative target mechanism of Aurora-B inhibition, the
authors concluded that Jadomycin B could potentially inhibit
other kinases more effectively resulting in apoptosis. Due to the
highly conserved nature of the kinase ATP-binding domain,
achieving selectivity amongst the 518 protein kinases in the
human genome is often the paramount concern in kinase inhib-
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/C1SC00152C
itor discovery projects. It would have therefore been interesting
for the authors to investigate the activity of Jadomycin B against
panel of different kinases using both in silico and in vitro
methods, in order to evaluate the ability of molecular docking to
predict the selectivity profile of their NP screening hit.
In 2011, our research group reported the discovery of the NP-
like 6,600 -biapigenin as only the second inhibitor of NEDD8-
activating enzyme (NAE) using molecular docking.67 NAE is an
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analogue of the ubiquitin E1 enzyme that is involved with
regulating the ubuiqitination and degradation of the subset of
proteins regulated by E3 cullin-RING ligases, including cancer-
related substrates such as p-IkBa and c-myc. The first inhibitor of
NAE reported, MLN4924, displayed efficacy against both solid
and hematological human cancer cell lines.68 We performed high-
throughput molecular docking of the Analyticon MEGAbolite
and NatDiverse databases of over 20 000 NP and NP-like
structures against the X-ray crystal structure of the quaternary
APPBP1-UBA3-NEDD8-ATP complex (PDB: 1R4N) using the
docking software Molsoft. The 10 highest-scoring compounds
were purchased and tested in biological validation experiments,
and the biflavonoid 6,600 -biapigenin emerged as the top candi-
date, with micromolar potencies against NAE in enzyme and
cell-based assays. While 6,600 -biapigenin itself has not been found
in nature, it has been synthesized from 6,600 -biapigenin hexa-
aceate, in turn obtained from the naturally occurring succeda-
neaflavanone (6,600 -binarigenin). Interestingly, molecular
modeling analysis of the ligand–receptor interaction indicated
a binding mode very different to that of ATP or the nucleotide
mimic NAE inhibitor MLN4924, tentatively suggesting that
Fig. 2 Molecular model of a) virtual screening hit 6,600 -biapigenin, b) MLN4924 and c) ATP bound to the NAE heterodimer generated by virtual ligand
docking. Molecular modeling analysis revealed a putatively different binding mode of 6,600 -biapigenin compared to MLN4924 or ATP.67
This journal is ª The Royal Society of Chemistry 2011
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6,600 -biapigenin can be considered a new class of NAE inhibitor
(Fig. 2). Significantly, 6,600 -biapigenin was only the second NAE
inhibitor reported to date. While further kinetic or structural
evidence would be required to definitively establish the mecha-
nism of inhibition of NAE by 6,600 -biapigenin, this study high-
lights some of the chief advantages of molecular docking over
ligand-based pharmacophore screening. Firstly, for a relatively
new biomolecular target such as NAE, ligand-based pharmaco-
phore modeling is often impossible due to the fact that inhibitors
of the target have not yet been discovered. Secondly, since the
structures in the screening library are computed against the
pharmacophoric model of known inhibitors, pharmacophore
screening is inherently unable to identify bioactive compounds
with a novel mode of binding, and such compounds would be the
first to fall out of the virtual screening campaign due to their
incongruous binding mode. Besides its use in virtual screening,
molecular docking can also be used to explore the mode and
mechanism of enzyme inhibition by natural products. For
example, Monti et al. have used Molsoft to analyze the binding
of the marine natural products including scalaradial69 and
petrosaspongiolide M70 to phospholipase A2, a target for
inflammation.
Natural products targeting the receptor–ligand interaction
In 2003, Liu et al. screened the China Natural Products Database
of 50 000 compounds against a structural model of the potassium
ion channel using Dock.71 The 3D model of eukaryotic Shaker
K+ channel was constructed based on homology with the crystal
structure of prokaryotic Streptomyces Kcsa K+ channel (PDB
entry: 1BL8). The search area for the molecular docking was
restricted to the extracellular pore binding site formed by the
tetramer. Attention was focused on potential ligands that inter-
acted with the residues surrounding the entry of the ‘‘ion-selec-
tive filter’’ of the channel so that the compounds could inhibit K+
channel function. To reduce the size of the initial hit list, the top
200 compounds were reoptimized using the Sybyl molecular
mechanics force field, yielding a final collection of 14 compounds
of which 4 were procured for biological testing. All 4 compounds
inhibited the K+ channel in the electrophysiological assay by
whole-cell voltage-clamp recording in dissociated hippocampal
rat neurons, with 20–1000-fold higher potency than tetraethyl-
ammonium, a well-known K+ channel blocker.
Chem. Sci., 2011, 2, 1656–1665 | 1661

Brinton and co-workers (2005) docked an in-house database of
25 000 plant-based NP and NP derivatives against the X-ray co-
crystal structure of estrogen receptor-b (ERb) with the isoflavone
genistein (PDB entry: 1QKM) using the docking software Gold.72
ERb is postulated to play key roles in maintaining estrogen-
inducible neuronal morphological plasticity, brain development,
and cognition. On the other hand, ERa is more predominantly
expressed in the breast and the endometrium. Therefore, ER
agonists for the treatment of neurodegenerative diseases should
display high selectivity for ERb over ERa to avoid unwanted
profilerative effects in other tissues or organs. The top 500 scoring
molecules from the molecular docking were filtered by visual
inspection and 100 compounds were manually selected for further
analysis by the docking program Affinity, to refine the binding
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/C1SC00152C
modes predicted by Gold. The final result was that 31 compounds
were selected that possessed the critical hydrogen bond with
His475 of ERb as well as necessary hydrophilic and hydrophobic
characteristics matching that of endogenous 17b-estradiol or
genistein. Of the 12 tested compounds, 5 molecules displayed
significant selectivity for ERb over ERa (3 of these exhibited over
100-fold selectivity). However, the neurobiological effects of these
compounds will require further in vivo investigations as the
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agonistic or antagonistic activities of weak estrogenic binders are
known to be dependent on a number of factors, such as the
concentration of the drug, the concentrations of endogenous
estrogens, as well as the level of transcriptional coactivators or
corepressers present in the particular tissue.73
Peroxisome proliferator-activated receptor-gamma (PPAR-g)
plays a critical role in lipid and glucose homeostasis, and inhib-
itors of PPAR-g, such as the synthetic thiazolidinediones, are
potential therapeutic agents for the treatment of type II dia-
betes.74 Notable side effects associated with such compounds,
including fluid retention, weight gain, and cardiac hypertropy
have recently stimulated Hibbs and co-workers (2008) to
discover new NP scaffolds as PPAR-g agonists.75 The authors
docked an in-house natural product database of 200 compounds
against two structurally distinct X-ray co-crystal structures of
PPAR-g using rigid-receptor docking with Glide. One PPAR-g
structure was co-crystallized with the thiazolidinedione rosigli-
tazone (PDB entry: 1FM6), while the other incorporated the
tyrosine-based analogue farglitazar (PDB entry: 1FM9), one of
the earliest anti-diabetic lead compounds. Termed ‘‘multiple
rigid-receptor docking’’, the authors proposed that the use of two
structurally distinct induced-fit conformations of the protein
would admit a wider range of ligands, particularly if the hits
achieved ligand–receptor interactions comparable to one or both
of the known inhibitors. The 20 best-scoring compounds for each
of the two conformations were pooled together, and duplicates
(those compounds scoring highly for both conformations) were
removed, to achieve a hit list of 29 unique candidates, that were
observed to fall into three structural classes: the flavonoids, the
gingeroids, and the ginkolides. However, visual inspection of the
binding poses revealed that only the flavonoids were able to
engage key protein residues in the transcriptional activation
funtion 2 domain (AF-2 helix) characteristic of PPAR-g
agonists, and all of the flavonoids achieved much better docking
scores with the farglitazar-induced receptor rather than the
rosiglitazone-induced receptor. Biological validation using
a cellular transcriptional factor assay confirmed this analysis as
1662 | Chem. Sci., 2011, 2, 1656–1665
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only 6 out of the 29 compounds showed biological activity, and
all 6 were from the flavonoid family, with activities approxi-
mately 3.6–21-fold times less potent than rosiglitazone. Consid-
ering the considerable flexibility exhibited by the PPAR-g ligand
binding domain, the authors used an induced-fit docking (IFD)
protocol to further analyze the binding mode of the flavonoids.
Receptor flexibility was modeled using the protein structure
prediction and refinement package Prime in combination with
Glide. Significantly, induced-fit docking of the flavonoids into
the inferior model (the rosiglitazone-induced receptor) resulted
in significant structural rearrangements in the protein side chains
of the ligand-binding domain, reproducing the superior, lower-
energy binding pose observed in the farglitazar model. This study
showed that induced-fit docking can be used to reveal a more
plausible binding mode than by rigid-docking alone, allowing for
even significant rearrangements of the protein scaffold in order
to better accommodate the docked ligand. However, the protein
side-chain prediction and geometry minimization calculations
are more computationally demanding compared to those in
rigid-receptor docking. Furthermore, as with all molecular
models, the induced-fit binding pose would still have to be veri-
fied with experimental techniques such as NMR or X-ray
crystallography.
In 2008, the group of Ngai and Archer reported the discovery
of high-affinity agonists of an olfactory G protein-coupled
receptor (GPCR) using both receptor-based and ligand-based
virtual screening.76 The GPCR responds preferentially to long
chain amino acids such as lysine and arginine, as well as to other
amino acids with lower affinity. This relatively non-specific
binding profile is characteristic of odorant receptors, which
allows the olfactory system to recognize a diversity of chemical
structures exceeding the actual number of olfactory receptors in
the human genome. The authors wished to identify novel high-
affinity agonists of the V2R vomeronasal receptor-like goldfish
receptor 5.24 for use as molecular probes to study receptor
structure and olfactory function. Interestingly, while a database
comprising over 1.6 million commercially available compounds
was used for the molecular docking and pharmacophore-
screening, the most active hit compounds were all naturally
occurring, linear amino acid derivatives, and these were found to
be more potent agonists than the natural amino acids. For
example, diaminopimelic acid is a component of Gram-negative
bacterial cell walls and is used as an intermediate bacterial
biosynthetic pathways for lysine and peptidoglycans. Further-
more, the hit rate for the common virtual hits that scored highly
in both the molecular docking and pharmacophore screening was
extremely high (16/36 ¼ 44%), with activity defined as >40%
maximum receptor activity at 20 mM of the compound. Four of
these compounds were active as olfactory receptor agonists in
vivo as measured by electrophysiological recordings of goldfish
olfactory epithelium. Furthermore, molecular modeling was
performed to analyse specific features of the amino acid ligands
that may confer agonist or antagonist activities, based on the
structural analysis of the receptor’s ligand binding domain.
Compared to enzyme–substrate or ligand–receptor interac-
tions, targeting the protein–protein interface is usually regarded
as more difficult due to the relatively large and featureless nature
of most protein–protein binding pockets, that lack clearly-
defined binding crevices or mechanism-based contacts.77 In 2010,
This journal is ª The Royal Society of Chemistry 2011

our research group wondered if the enormous structural diversity
of NPs could be harnessed to discover novel small molecule
inhibitors of tumor necrosis factor-a (TNF-a).78 TNF-a is
a multifunctional cytokine that acts as a central biological
mediator for critical immune responses, including inflammation,
infection, and apoptotic cell death. Dysregulation of TNF-a has
been implicated in cases of tumorigenesis, diabetes, and espe-
cially in autoinflammatory diseases such as rheumatoid arthritis,
psoriatic arthritis and Crohn’s disease.79 Using the software
Molsoft, over 20 000 NP and NP-like structures from the Ana-
lyticon MEGAbolite and NatDiverse databases were docked
against the X-ray co-crystal structure of TNF-a dimer with
SPD304 (PDB code: 2AZ5), the strongest TNF-a small molecule
inhibitor reported to date. The top 16 compounds from the
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/C1SC00152C
virtual screening were obtained and tested in vitro, and two NP-
like compounds, a pyrazole-linked quinuclidine and an indolo
[2,3-a]quinolizidine, emerged as the top candidates. Despite the
mostly hydrophobic, formless nature of the TNF-a binding
pocket, both compounds occupied a similar region in the binding
site compared to SPD304 (Fig. 3). Furthermore, both
compounds were observed to be large enough to contact both
subunits of the TNF-a dimer simultaneously, thus occupying
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and blocking the binding site for the third TNF-a subunit and
preventing the formation of the biologically active TNF-a trimer
complex. Molecular modeling analysis indicated the absence of
extensive hydrogen bonding or salt bridge formation which was
consistent with our knowledge of the TNF-a binding pocket and
with the reported binding mode SPD304. The indoloquinolizi-
dine (IC50 ¼ ca. 10 mM) was found to be more potent against
TNF-a in the receptor binding assay compared to SPD304
(IC50 ¼ 22 mM by a comparable ELISA), while the quinuclidine
Fig. 3 Low-energy binding conformations of a) screening hit quinuclidine, b) screening hit indolo[2,3-a]quinolizidine, c) SPD304 bound to TNF-
a dimer and d) superimposition of quinuclidine (orange), indolo[2,3-a]quinolizidine (yellow) and SPD304 (blue) generated by virtual ligand docking.78
This journal is ª The Royal Society of Chemistry 2011
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(IC50 ¼ ca. 5 mM) had comparable activity to SPD304 (IC50 ¼ ca.
3 mM) against cellular TNF-a induced NF-kB luciferase activity.
These compounds also represented only the third and fourth
examples of TNF-a inhibition by a small molecule inhibitor at
that time. This study demonstrates that despite the seemingly
intractable nature of the protein–protein interface, molecular
modeling can be an effective tool for harnessing the structural
diversity of NPs towards identifying novel inhibitors of the
protein–protein interaction.
Natural products targeting DNA
DNA-targeting NPs are some of the most well-known anti-
cancer drugs. For example, mitomycin is a potent DNA cross-
linker from Streptomyces caespitosus or Streptomyces lavendulae
that finds use as a chemotherapeutic against gastrointestinal,
breast, and bladder cancers,80 while the actinomycins, also from
Streptomyces, are chemotherapeutics and antibiotics that bind
DNA at the transcription initiation complex and inhibiting RNA
polymerase.81 Recently, non-canonical secondary structures of
DNA such as the G-quadruplex have emerged as attractive
targets for therapeutic intervention due to their putative associ-
ation with oncogene promoter sequences such as c-myc, bcl-2,
VEGF, KRAS, and c-kit.82–84 While the NP telomestatin is the
most potent G-quadruplex ligand known to date,85 the majority
of G-quadruplex binding ligands reported in the literature have
been synthetic in origin. In 2010, our research group applied
high-throughput structure-based virtual screening methods to
identify natural product ligands of the c-myc G-quadruplex, as
potential chemotherapeutic agents.86 In order to develop a high-
throughput screening platform for G-quadruplex binding
Chem. Sci., 2011, 2, 1656–1665 | 1663

ligands, a model of the intramolecular c-myc G-quadruplex
1 : 2 : 1 loop isomer was constructed using the X-ray crystal
structure of the closely related intramolecular human telomeric
G-quadruplex DNA (PDB entry: 1KF1), since neither the NMR
nor X-ray crystallographic information for the predominant c-
myc 1 : 2 : 1 loop isomer was available. Using Molsoft, we
docked the Analyticon MEGAbolite and NatDiverse databases
of over 20 000 NP and NP-like structures against our the model
of the c-myc G-quadruplex, and the top 5 scoring compounds
were tested in an in vitro assay. The naphthopyrone pigment
fonsecin B, isolated from the fungus Aspergillus fonsecaeus,
emerged as the top candidate. Fonsecin B was found to inhibit
Taq-mediated extension through stabilization of the G-quad-
ruplex secondary structure (IC50 ¼ ca. 20 mM), with potency
Published on 20 June 2011 on http://pubs.rsc.org | doi:10.1039/C1SC00152C
comparable to the well-known G-quadruplex binder TMPyP4.
The molecular docking analysis revealed that fonsecin B was
stacked against the end of the G-quadruplex at the 30 -terminus,
with potential electrostatic interactions with a potassium ion in
the central channel (Fig. 4). Importantly, this study represented
the first large scale high-throughput virtual screening of a NP
database against the c-myc G-quadruplex. To our knowledge, no
other study has been published that utilizes high-throughput
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molecular docking to discover novel NP or NP-like nucleic acid-
binding compounds. With a growing awareness of the important
regulatory roles played by non-canonical DNA or RNA motifs
in human biology, as well as the ready availability of such nucleic
acid structures in the PDB, we believe that this is an area that
definitely deserves further attention in the literature.
Conclusions
Computational technologies are a practical solution to the
horrific experimental costs associated with high-throughput
screening of large compound libraries. A variety of modeling
techniques are available for today’s medicinal chemists for the
rapid and efficient discovery of lead molecules against biomo-
lecular targets. Meanwhile, natural products are re-emerging as
a valuable source of bioactive scaffolds that display remarkable
chemical diversity in structure and function. The use of virtual
screening technologies ameliorates many of the problems asso-
ciated with the incompatability of natural products with high-
throughput screening. The combination of virtual screening and
natural products allows the medicinal chemist to harness the
extraordinary potential of natural products in an efficient and
inexpensive manner. Molecular docking, while regarded as more
complex and computationally demanding compared to
Fig. 4 Hypothetical molecular model of virtual screening hit fonsecin B
with the c-myc G-quadruplex.86
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pharmacophore modeling, has the potential to accurately predict
binding affinities of screening hits as well as potentially reveal
lead structures with novel modes of binding. As scoring algo-
rithms become more refined, together with the continuous
improvement in computer processing power and capabilities, we
believe that molecular docking has great promise in virtual lead
discovery. In this review, we have highlighted some recent
examples of the use of molecular docking for the identification of
bioactive compounds from natural product databases. These
works demonstrate that molecular modeling can be used to
reveal natural products as highly potent and selective enzyme
inhibitors, blockers of protein-protein interactions, as well as
ligands targeting non-canonical DNA structures. Given that
most of these studies were published within the last three years,
we are confident that this powerful combination of molecular
docking and natural compounds will continue to thrive as an
active area of research in the coming years.
This work is supported by the Hong Kong Baptist University
(FRG2/09-10/070 and FRG2/10-11/008), Centre for Cancer and
Inflammation Research, School of Chinese Medicine (CCIR-
SCM, HKBU) and The Hong Kong Anti-Cancer Society
(HKACS) Research Grant.
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