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Virtual screening of natural products database

Article  in  Mini Reviews in Medicinal Chemistry · July 2020

DOI: 10.2174/1389557520666200730161549

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Virtual Screening of Natural Products Database

José Alixandre de Sousa Luisa,b, Renata Priscila Costa Barrosb, Natália Ferreira de Sousab, Eugene
Muratovc, Luciana Scottib and Marcus Tullius Scottib*

a
Education and Health Center, Federal University of Campina Grande, Cuité, Paraíba, Brazil; bPostgraduate Program
in Natural Products and Synthetic Bioactive, Federal University of Paraíba, João Pessoa, Paraíba, Brazil; c University
of North Carolina, USA

Abstract: Constant research with natural products has generated, over time, a large number of compounds with potential
to be evaluated in several biological tests and subsequently have been cataloged in databases that allow other researchers
perform virtual screenings on activity in various biological systems. This considerably reduces the time for the
development of new drugs. This review describes the main databases of Natural Products available for searching for
bioactive compounds. It also describes the main features of Virtual Screening strategies for identification of molecules
with potential to be used as new drugs. In adittion, a search was made in the Web of Science database, using as search
term "Virtual screening of natural products databases" from 2003 to 2018. The search criterion resulted in 230 articles,
which had their abstracts evaluated as to the pertinence to the criteria required for this work, which are: a) be a research
article; b) performing a virtual screening from databases of natural products or containing natural products; c) works that
identified drug candidate molecules. Based on these criteria, the bibliographic review work on the topic was excluded.
After this analysis, 104 works were selected for this review. Were selected relevant papers describing the obtaining of
potential drug candidates that were distributed in 15 classes, of which the anticancer, antibacterial and anti-inflammatory
hits were the most abundant. There are also described works showing efforts to search for new molecules against various
other diseases in distinct biological systems. In this way, this work shows an overview of several methodologies and we
hope they can help and inspire the development of new research to improve people's quality of life.
Keywords: Natural Products, Natural Products Databases, Virtual Screening, Ligand-Based, Structure-Based, Drug
candidates.

1. INTRODUCTION
From the beginnings of humanity, nature is used as a potential source for new drugs, but in a rudimentary and empirical
way. From the twentieth century onwards with the scientific development of the areas of ethnopharmacology, chemistry and
techniques of extraction, purification, identification and biological testing of molecules from natural products, it became
possible to study the raw materials to obtain candidate molecules be used therapeutically. So much so that the Food and Drug
Administration (FDA) reports that of the 1221 new small molecules approved in the period 1981 to 2014, more than 50 % come
_______________________________________________
*Address correspondence to this author at the Postgradute Program in Natural Products and Synthetic Bioactive, Federal University of Paraíba, João Pessoa,
Paraíba, Brazil, CEP 58.051-900; Tel.: +55 83 99869-0415; e-mail: mtscotti@gmail.com, jalixluis@hotmail.com
directly or indirectly from natural metabolites[1, 2]. However, this process has for some time faced some financial and
technical barriers. To speed up, natural product databases and computational tools have been used to optimize the search of new
drugs.
Constant research with natural products (whether with extracts or isolated molecules) has generated, over time, a large
number of compounds with potential to be evaluated in several biological tests and subsequently have been cataloged in
databases that allow other researchers perform virtual screenings on activity in various biological systems. This considerably
reduces the time for the development of new drugs[3, 4]. Currently, there are several accessible databases for a systematic
search of molecules from natural products. From now on we will show the databases that we consider the most important, then
we will make an explanation about the main strategies of Virtual Screening and to conclude we will show a series of examples
showing the application of these approaches.

2. DATABASES OF NATURAL PRODUCTS

When a researcher on Natural Products (NPs), for the purpose of obtaining new drugs, plans his work, he must keep in mind
a possible applicability in a biological target. There are databases that catalog thousands of molecules that have been tested in
certain biological systems with effect or not. They help to check if a particular molecule has already been tested and avoid
duplicate work. These databases can be searched with minimal information, such as chemical structure and biological data, but
the stage of dereplication, which is the process known as rapid characterization of known compounds, requires more
information, such as biogeographic and taxonomic information, compound (new or not) in other individuals of the same
species, genus, subfamily and family. This information can help reduce the number of failures in structural identification by
dereplication. This strategy has become a powerful tool for the discovery of lead compounds and has been adopted by almost
every major pharmaceutical company in the world as it provides a starting point for understanding the chemical factors that
interfere with biological activity, physicochemical properties responsible for a given biological response[4-6].
ChemSpider, PubChem and ChEMBL are large structure-based databases, but they do not allow differentiation of synthetic
origin molecules from those of natural origin and this can leave the researcher confused[7-10]. Due to these limitations, a
number of databases specialized in both natural and commercial products have emerged. A group of German researchers has
prepared a review on the major databases of natural products existing around the world that are useful in helping to develop
new drugs. Characteristics of some of the databases we consider most important will be described below.
The Dictionary of Natural Products (DNP) (http://dnp.chemnetbase.com) is one of the most comprehensive banks. It is a
commercial database that provides names and synonyms, physical chemical properties, spectroscopic data and molecular
structures, as well as biological source and use; this database is available both online and in the form of a CD room. There are
Marinlit for marine natural products, and Antibase for microorganisms and higher fungi materials. However, none of these
provide structural collections in a format that can be rapidly integrated into a software package such as ACD/Structure
Elucidator[11].
Reaxys is a database containing over 220k natural products collected from various journals and contains information on
chemical structures, reactions, properties, sources and bioactivity data. NPs can be accessed via the Web and the results can be
downloaded in SD or SMILES format[11].
Super Natural II is a database that gathers information from 16 supplier banks and 5 available databases (KEGG, MetaCyc,
UNPD, HMDB and ZINC). This database provides information such as chemical structures, physico-chemical properties and
predicted toxicity. The site also allows research on the possible mechanism of action[11-14].
Universal Natural Products Database (UNPD) is the largest free and full database access database. It has more than 229 k
compounds. This database provides 3 D structures with their stereochemical definitions in 3 D coordinates as well as molecular
descriptors that can be downloaded as SD files to individual compounds or as an SD or CSV file containing the complete
database[11, 14].
There are databases focused on Traditional Chinese Medicine (TCM), among them TCM Database @ Taiwan with more
than 60k compounds derived from herbs, minerals, animals and other products compiled from TCM texts and dictionaries. The
Traditional Chinese Medicine Integrated Database (TCMID) is a database that promotes an interface between TCM and modern
Western Medicine, another bank that has this characteristic is the Chemical Database of Traditional Chinese Medicine (Chem-
TCM). The Herbal Ingredients' Targets database (HIT) relates ingredients derived from herbs to their biological activities,
whereas The Herbal Ingredients in vivo Metabolism database (HIM) focuses on characteristics about ADMET, particularly
about metabolism[11, 15-20].
The main databases focused on African natural products are AfroDb, AfroCancer (The African Anticancer Natural Products
Library), AfroMalariaDB (The African Antimalarial Natural Products Library), SANCDB (The South African natural
compound database) and (The Northern African Natural Products Database)[11, 21-24].
Other important regional databases are The Natural Products database of The State University of Feira De Santana (UEFS)
in Brazil contains structures of around 500 NPs collected from the literature. This data set is included in ZINC and does not
have its own Web site. Another is the NuBBE Database, focused on NPs and derivatives from plants and microrganisms native
to Brazil, the NuBBE database is freely accessible online database[11].
 In addition to the databases of natural products with a focus on metabolomics studies with relation, species-metabolite,
KNApSAcK Family, TIPdb-3D and AsterDB are examples of databases where it is possible to search for chemical structure by
species and other associated information[11]. Nevertheless, some data is lacking for exact dereplication since information such
as exact mass and geographic data have been shown to be very important for this type of study. Recently, SistematX[25] was
created as database, and it can be used for chemosystematics studies, dereplication and botanical correlation. Figure 1
summarizes the main databases of natural products.

3. VIRTUAL SCREENING STRATEGIES

The main objective of the virtual screening of compounds is to reduce the number of tests performed on animals and,
consequently, reduce the time spent on the development of new drugs. There are two major approaches that are commonly
used: Ligand Based (LB) when the biological target for the molecule under study is unknown and the Structure Based (SB)
when the biological target for the molecule is known and has its structure deposited in databanks. Each of these approaches can
be worked through several strategies that use Machine learning, computational pattern recognition or statistical modeling
algorithms to generate quantitative correlations between molecular structures and chemical properties or biological activities.
The LB strategy explores similarities between known molecules that are responsible for biological activity, whereas the SB
observes chemical interactions between the molecules and a known biological target.

3.1. LB methods
Linear Free Energy Relationships (LFER) are based on the interpretation of molecular descriptors that represent
characteristics of the molecules under study. There are descriptors that allow the analysis of 2-D and 3-D molecules depending
on the representation model used. These descriptors can be classified into families that differ in computational interpretability,
level of detail and molecular characteristics that they capture. The main families are physicochemical, electronic and steric
descriptors and the correct use of these descriptors allows the realization of Quantitative Structure Activity Relationship
(QSAR) in the development of new drugs[26-28].
Topological Indices use descriptors that are based on the linear sequence of (1-D) atoms, the most used are those
recommended by the Lipinski Rules and provide important information about the oral bioavailability of drugs. There are also 2-
D models in QSAR studies that relate number of atoms and bonds, degree of branching and number of electrons[28-32].
Substructural Descriptors, these approaches use binary fingerprints based on 2-D structures that represent the presence or
absence of sub-structural fragments. As examples we have MACSS key descriptors, Tripos's Sybyl®, Extended Connectivity
Fingerprints (ECFP) that allow to verify local or global characteristics of the molecules and are very useful in VS. There are
also Pharmacophores that are 3-D descriptors that represent specific geometric arrangements of combinations of atoms of
different classes that allow to capture characteristics essential to the biological activity of the drugs. For the success of this
methodology is essential to choose appropriate descriptors to the model, otherwise it may provide inappropriate results[28, 33,
34].
Field Based Models are methods where 3-D descriptors are constructed by encoding conformational information by
aligning molecules in an interaction field. Comparative Molecular Field Analysis (CoMFA®) is a technique where molecules
are aligned in a 3-D space generating a superposition that maximizes the steric effect and electrostatic overlap or uses a
pharmacophoric model. The values of the steric and electrostatic fields at each point of the Grid are used to construct a 3-D
QSAR equation using a set of molecules with activities known as training set. By using this method, it is possible to generate 3-
D representations of invariant molecular fragments by topology using deterministic rules that specify absolute configuration,
conformation and orientation that are called topomers. These topomers have been employed to search for similarity in large
virtual databases to generate CoMFA alignments and to aid the drug development process[28, 35-37].
Local Surface Area Descriptors are commonly used in QSAR/QSPR models, because they quantify the surface areas of
polar atoms such as oxygen and nitrogen in atomic fragments in molecules or areas of molecular surfaces accessible to solvents,
this is important to incorporate electrostatic and desolvation effects in 3-D QSAR models to predict a number of biological
properties, such as ability to bypass the blood-brain barrier, intestinal absorption and oral bioavailability, and to quantify the
effects of solvents on protein folding and stability and the effects of desolvation at protein-protein and protein-ligand bonds[38-
46].
Shape Descriptors, this methodology is based on the Rapid Overlay of Chemical Structures (ROCS), that is, on the idea
that molecules have similar shape if their volumes have significant overlap and any incompatibility of volume is a measure of
dissimilarity. In ROCS, the molecular form is represented as a continuous function constructed from Gaussian functions
centered on atoms. The form of a query molecule is used as scaffold for other molecules with similar 3-D forms. GRIND
descriptors represent an independent class of alignment that encode field-level distributions of molecular interaction at key
points around a molecule in the form of correlograms, rather than capturing molecular information of shape or surface. There
are other methods, dependent and independent of alignment, that can be used for the purpose of analyzing molecular surfaces
such as: Zauhar's Shape Signature, Property Encoded Surface Translator (PEST), Ultrafast shape recognition (USR) and Chiral
Shape Recognition (CSR)[28, 47-50].
 Data Fusion is the process of combining multisensor data from different sources so that the resulting information or model
is better than when sources are used individually. The data fusion processes are classified into three levels: data level fusion,
feature level fusion and decision level fusion. Data level fusion combines several sources of raw data to produce new raw data
that is expected to be more informative and synthetic than the inputs. For molecular modeling this is equivalent to combining
different sets of descriptors. Several applications along these lines have already been discussed above. Feature level fusion
combines various features, as for example in the combination of several latent variable sets extracted from principal component
analysis, partial-least squares analysis, or independent component analysis[51-53]. Decision fusion combines decisions from
several individual models with either the same or different descriptor sets. Consensus models are examples of the latter, where
individual model predictions based on the same or different descriptor sets are combined into a single meta-learning model. In a
recent application, kernel partialleast squares (K-PLS) models with data fusion have shown a significant boost in performance
compared to traditional K-PLS models in predicting the binding affinity for the human serum albumin. Several consensus
scoring approaches combining highly diverse descriptor sets such as structural keys, property-based fingerprints, shape scores
and 3-D pharmacophores, have been investigated and shown to give better and more consistent rankings of active
molecules[28, 54-58].
Non-Linear Models comprise the Artificial Neural Networks (ANNs) that are the most widespread representatives of this
methodology applied to the process of drug development. These models belong to the class of selforganizing algorithms in
which the neural network learns the relationship between descriptors and biologic activity through iterative prediction and
improvement cycles. A major drawback of neural networks is the fact that they are sensitive to overtraining, resulting in
excellent performance within the training set but reduced ability to assess novel compounds. Therefore, care is taken to always
measure ANN performance on “independent” datasets not used for model generation. O Support Vector Machines (SVM) is a
kernel-based supervised learning method that was introduced by Vapnik and Lerner, it is based on statistical learning theory
and the Vapnik-Chervonenkis dimension and seek to minimize the sum of the training error and the model complexity (known
as the generalization error) instead of minimizing the training error itself, leading to more robust models with better predictive
ability on molecules not included in the training set. SVM can be used for classification as well as for regression models[28, 59,
60].
Models Validation are required not only to characterize the predictive capacity of the models, but also the Applicability
Domain of them[61, 62]. The use of these techniques is important to solve the "Kubinyi paradox" where it says models that
perform best, retrospectively, are often the worst prospectively. Thus, methods to characterize not only the predictive ability but
also the domain of applicability of models are occupying the attention of researchers. The domain of applicability of a QSAR
model is the physicochemical, structural or biological space, the information in which has been used to train the model, and it is
within this space that the model is applicable to make predictions for new compounds. The applicability domain of a QSAR
model is described in terms of the parameters that are descriptors of the model. Ideally, the QSAR should only be used to make
predictions within that domain by interpolation, and not by extrapolation. Compounds which are highly dissimilar from all
compounds of the training set (in the space of selected descriptors) can not be predicted with any degree of confidence.
Statistical methods to estimate the model applicability domains include range-based (either descriptor range or principal
components range may be used), distance-based, geometric and probability-density distribution-based methods. Thus, for a
robust model to be constructed, it is essential to use appropriate descriptors, modeling methods with high external validation
capability, and models within their demonstrated applicability domains. Sukumar and Das[28] list some good practices in
Cheminformatics so that predictive errors are minimized: a) There should be a plausible (not necessarily a known or a well-
understood) mechanism or connection between the descriptors and response. Otherwise we might just as well be doing
numerology; b) Robustness: you cannot keep tweaking parameters until you find one that works just right for a particular
problem or data set and then apply it to another. A generalizable model should be applicable across a broad range of parameter
space; c) It is important to know the domain of applicability of the model and stay within it; d) Likewise, it is important to
know the error bars on the experimental data: there is no point expending a lot of effort modeling the noise in the data; e) The
minimum requirement for developing a predictive model or hypothesis is the “No cheating” principle, i.e. no looking at the
“answer” or the responses of the prediction set during model building; f) Divide the data set into training, validation and test
sets; g) Validate the training set models using an external validation set; h) Of course, if a data set contains too much noise, no
QSAR/QSPR technique can extract a meaningful signal. One should not look too hard for something that may not be there,
because one is then liable to be modeling the noise in the data; i) Consider the use of “filters” to scale and then remove
correlated, invariant and “noise” descriptors from the data, and to remove outliers from consideration; j) Modeling is meant to
assist human intelligence – not to replace it. So, it is important to try to understand the chemistry of the problem at hand. In this
context, however, it is worth re-emphasizing the difference between predictive and retrospective QSAR. Descriptors selected
for their ease of interpretation are unlikely to yield optimal predictive models. Conversely, descriptor selection methods
designed to generate highly predictive models are often not suitable for mechanistic analysis[63, 64].

3.2. SB methods
Structure-based (SB) methods for the development of new drugs are complementary to LB and depend on the ability to
determine and analyze 3-D structures of biological molecules. This method is used when we know the structures of the
biological targets for the drugs (proteins and nucleic acids). This knowledge may be derived from crystallographic studies or
obtained by homology from known structures. The main objective of this strategy is to verify if a molecule has the capacity to
interact with a specific target and to trigger a biological response, as well as to analyze the interactions responsible for
triggering this response. The correct interpretation of these interactions is useful for production of new molecules that fit
optimally with the active sites and produce a more effective biological response[17, 65, 66].
The crucial point of this strategy is the structural knowledge of target macromolecules for the drugs. In the last decades, the
development of X-ray crystallography and NMR spectroscopy has been able to completely identify the structures of proteins
and nucleic acids that are related to diseases. After their complete structural identification these molecules are deposited in
Databases for consultations and studies. The main databases for this purpose are: Protein Data Bank (PDB) and PDBbind.
These databases have extensive collections of experimentally measured affinity data for high-value protein-ligand complexes
for studies on the molecular recognition of interactions between molecules in biological systems[17, 66].
Thus, several research groups and pharmaceutical companies have developed methodologies that allow for virtual
screenings that allow the rapid identification of potential binders for biological targets of interest. The following will show
some usual protocols for this purpose.
The first step in a SB study is the Preparation of a Target Structure and when a protein is deposited, for example in the
PDB, it is a good starting point for a docking study because the success of this approach depends on the quality of the structural
information on the target, as well as the molecules to which they are anchored. The process then starts by analyzing active wells
for the binding of molecules and this is done by analyzing the cocrystalline structures of complex target-ligands or using in
silico methods to identify new binding sites[67, 68]. Another approach is Comparative Modeling which is performed when
there is no experimental structural data, then computational methods are used to predict the 3-D structures of target proteins
based on the principle that proteins with similar sequences have similar structures. Homology modeling is a specific type of
comparative modeling in which the template and target proteins share the same evolutionary origin. Comparative modeling
involves the following steps: (1) identification of related proteins to serve as template structures, (2) sequence alignment of the
target and template proteins, (3) copying coordinates for confidently aligned regions, (4) constructing missing atom coordinates
of target structure, and (5) model refinement and evaluation[69-72]. Another step of the process is the binding site detection
and characterization, considering that the protein-binding interaction is a prime factor for drug activity. Binding sites for
small molecules are known from the characterization of the cocrystalline structure of a complexed protein to a ligand.
Computational methods like POCKET, SURFNET, Q-SITEFINDER, etc are often used for binding site identification.
Computational methods for identifying and characterizing binding sites can be divided into three general classes: (1) geometric
algorithms to find shape concave invaginations in the target, (2) methods based on energetic consideration, and (3) methods
considering dynamics of protein structures. There are methods for representing small molecules and target protein for docking
simulations, the main ones are atomic, surface and grid representations. Atomic representation of the surface of the target is
usually used when scoring and ranking is based on potential energy functions. Surface methods represent the topography of
molecules using geometric features. The surface is represented as a network of smooth convex, concave, and saddle shape
surfaces. These features are generated by mapping part of van der Waals surface of atoms that is accessible to probe a sphere.
Docking is then guided by a complementary alignment of ligand and binding site surfaces[66, 73].
In the docking studies, a series of algorithms are used to characterize the protein-ligand complexes. The methods can be
classified as rigid-body docking que consider only static geometric/physiochemical complementarities between ligand and
target and ignore flexibility and induced-fit binding models and flexible docking applications depending on the degree to which
they consider ligand and protein flexibility during the docking process. More advanced algorithms consider several possible
conformations of ligand or receptor or both at the same time according to the conformational selection paradigm. Rigid docking
simulations are generally preferred when time is critical, i.e., when a large number of compounds are to be docked during an
initial virtual HTS. However, flexible docking methods are still needed for refinement and optimization of poses obtained from
an initial rigid docking procedure. With the evolution of computational resources and efficiency, flexible docking methods are
becoming more commonplace. Some of the most popular approaches include systematic enumeration of conformations,
molecular dynamic simulations,Monte Carlo search algorithms with Metropolis criterion (MCM), and genetic algorithms[66,
74-79].
The docking experiments generate hundreds or thousands of poses and are necessary Scoring functions for evaluation
Protein-Ligand Complexes. More complex scoring functions attempt to predict target-ligand binding affinities for hit-to-lead
and lead-to-drug optimization. Scoring functions can be grouped into four types: (1) force-field or molecular mechanics-based
scoring functions, (2) empirical scoring functions, (3) knowledge-based scoring functions, and (4) consensus scoring
functions[80-84].
All these techniques aim to perform binding site characterization and this depends on the understanding of
physiochemical interactions between molecules. Optimization of lead molecules into high-affinity compounds that are worth
studying in vivo often requires optimization of its binding affinity along with pharmacological properties. This process of
optimization requires a deep understanding of the molecular interactions between ligand and target and the optimization process
can be accelerated by the use of computational methods like molecular docking, molecular dynamics simulation, and quantum-
mechanical simulations[66, 85-87].
In this approach, models are also developed that evaluate steric and electronic factors necessary for optimal target-ligand
interaction, for example the Pharmacophore Model. Most common properties that are used to define pharmacophores are
hydrogen bond acceptors, hydrogen bond donors, basic groups, acidic groups, partial charge, aliphatic hydrophobic moieties,
and aromatic hydrophobic moieties. Pharmacophore features have been used extensively in drug discovery for virtual
screening, de novo design, and lead optimization. A pharmacophore model of the target binding site can be used to virtually
screen a compound library for putative hits. Apart from querying data base for active compounds, pharmacophore models can
also be used by de novo design algorithms to guide the design of new compounds. Figure 1 summarizes the main strategies
used for virtual screening of natural products[66, 88-90].

4. EXAMPLES OF VIRTUAL SCREENING OF NATURAL PRODUCTS

For the construction of this topic, a search was made in the Web of Science database, using as search term "Virtual
screening of natural products databases" from 2003 to 2018. The search criterion resulted in 230 articles, which had their
abstracts evaluated as to the pertinence to the criteria required for this work, which are: a) be a research article; b) performing a
virtual screening from databases of natural products or containing natural products; c) works that identified drug candidate
molecules. Based on these criteria, the bibliographic review work on the topic was excluded. After this analysis, 104 works
were selected for this review, distributed chronologically as shown in Figure 2.

 
Fig. (1). Summary of key Natural Product Databases and Virtual Screening strategies

 
Fig. (2). Review workflow
4.1. Obtaining candidates for potassium channel blockers
Liu and colleagues[91] conducted a Structure Based (SB) study through a search in the China Natural Products Database for
molecules that act as potassium channel blockers in a three-dimensional model of eukaryotic cell channels, taking into account
that they are attractive for the development of drugs. In this channel model, the extracellular binding site and the pore are
formed by tetramers and was used as a target for a Virtual Screening (VS) by the use of the DOCK 4.0 program and later, to
reduce the false-positive, complexes of the 200 best ranked compounds were optimized in Sybyl 6.7. They focused on potential
ligands that interact with the residues surrounding the ion-selective filter of K+ channels, so that the selected compounds could
bind to the filter. The compounds were ranked according to the relative binding energy, favorable shape complementarity, and
potential to form hydrogen bonds with the K+ channel. Then, four candidate compounds were screened for VS to be
investigated by Whole Cell voltage-clamp recording in dissociated hippocampal neurons and it was found that compound 1
markedly depressed the delayed rectifier K+ current (IK) and fast transient K+ current (IA), whereas compounds 2, 3 and 4
exerted a more potent and selective effect on IK. The intracellular application of the 4 compounds had no effect on the K +
currents. Figure 3 shows the chemical structures of compounds 1-4 as well as the general model of their interaction with the K+
channel by using the LIGPLOT program.

 
Fig. (3). Chemical structures of the compounds 1-4 (Adapted from Liu and colleagues[91])

4.2. Obtaining candidates for the treatment of Alzheimer's disease

Alzheimer's disease (AD) is a disease whose etiology remains unknown, but it is known that neuropathological events
associated with memory loss are associated with the presence of numerous plaques and a cholinergic deficiency due to
degeneration or atrophy of neurons in the basal forebrain. Thus, inhibition of acetylcholinesterase (AChE) is a promising
strategy for the treatment of this disease and others, such as senile dementia, ataxia, myasthenia gravis and Parkinson's disease.
AChE inhibitory drugs increase the effectiveness of cholinergic transmission by the metabolic inhibition of acetylcholine[92].
Brains from AD patients possess a large amount of amyloid β-peptide (Aβ) plaques and there is evidence that AChE could play
a key role in accelerating the deposition of these plaques[93]. To select potential AChE inhibitors from natural sources,
Rollinger and co-workers[94] constructed a structure-based pharmacophore model using an in silico filtering experiment for the
discovery of drug candidates from a 3 D multiconformational database with more than 110,000 natural products. The three-
dimensional structure of the Galanthamine (GNT)-AChE complex was used as the starting point for the generation of the
pharmacophore model in the study. The model exhibits four essential features: one H bond-donor function (HBD) pointing
from the GNT (5) hydroxyl group to GLU-199, one H-bond-acceptor function (HBA) located at GNT’s methoxy oxygen atom,
and two hydrophobic features (HY) positioned on the aromatic ring and on the cyclohexene moiety of the ligand. Exclusion
volume spheres are located in this model on the aromatic moieties of the surrounding amino acids Phe290, Phe330, Tyr121, and
Trp84. To further restrict the 3 D search space, a molecular shape constraint generated from the structure of GNT aligned with
the pharmacophore model was added. From the computational study, scopoletin (SCT) (6) and its scopolin glucoside (SCN) (7)
were selected as potential inhibitors of AchE (Fig. 4). These molecules were then isolated by various chromatographic
techniques from the Scopolia carniolica Jaqc plant and were tested in an enzyme assay using Ellman's reagent. They showed
moderate, but significant, dose-dependent and long-lasting inhibitory activities. In the in vivo experiments (icv application of 2
µmol) 6 and 7 increased the extracellular acetylcholine (ACh) concentration in rat brain to about 170 % and 300 % compared to
basal release, respectively. At the same concentration, the positive control galanthamine increased the ACh concentration to
about the same level as 6. These are the first in vivo results indicating an effect of coumarins on brain ACh.
 In another study, Schuster and co-workers[95] conducted a pharmacophore-based virtual screening study of natural product
databases. After a first VS they selected morphinan and isoquinolinic compounds to be tested in vitro as possible inhibitors of
AChE and compounds with activity in the micromolar range were found. Active and inactive compounds were used to
construct an LB pharmacophore model with the aim of serving as a useful tool for the selection of compounds to have their
cholinergic activities evaluated in future synthesis projects. Among the virtual Hits tested, the selection of actives was
significantly greater than in a random selection of test compounds (Fig. 5) and the most active compounds were tested
biochemically to verify their activities on opioid receptors μ, δ and κ.

 
Fig. (4). Structures of compounds 6 and 7 (From Rollinger and co-workers[94]).

 
Fig. (5). A) Best AChE inhibitors by virtual hits (n = 14) from the in-house database derived from a flexible search using the
ligand-based model. B) Best AChE inhibitors by random compounds not included in their virtual hitlist (n = 50) (From
Schuster and co-workers[95])
 By analyzing drugs approved by the FDA for the treatment of Alzheimer's Disease (AD), it has been observed that the
mechanism of action occurs through interaction with the main catalytic site of AChE, as well as with the Peripheral Anionic
Site (PAS) that is responsible by triggering the aggregation of beta-amyloid peptide. Lakshmi, Kannan and Boopathy[96]
constructed a Pharmacophore model using PHASE, based on a set of fifteen best known AChE inhibitors. All these models on
validation were further restricted to the best seven. These were transferred to PHASE database screening platform for screening
89,425 molecules deposited at the “ZINC natural product database”. Novel lead molecules retrieved were subsequently
subjected to molecular docking and ADME profiling. A set of 12 compounds were identified with high pharmacophore fit
values and good predicted biological activity scores. These compounds not only showed higher affinity for catalytic residues,
but also for Trp86 and Trp286, which are important, at PAS of AChE. The knowledge gained from this study, could lead to the
discovery of potential AChE inhibitors that are highly specific for AD treatment as they are bivalent lead molecules endowed
with dual binding ability for both catalytic site and PAS of AChE (Figure 6).

 
Fig. (6). Lead molecules retrieved from the ZINC natural product database as potent AChE inhibitors. These are derived by
pharmacophore screening and docking combined virtual screening. The ZINC ID and their corresponding structure are given
(From Lakshmi, Kannan and Boopathy[96]
 Natural marine products can help increase the quality of life in patients with neurological diseases. Lorenzo et al[97] studied
a large number of marine products that act against Alzheimer’s disease through varying pathways. According to structure- and
ligand-based analyses, caulerpin, an alkaloid primarily isolated from the genus Caulerpa, possesses activity against monoamine
oxidase B. To predict the activity of caulerpin, they employed Volsurf descriptors and the machine learning Random Forest
algorithm in parallel with a structure-based methodology that included molecular docking. Using caulerpin as a lead compound,
a database containing 108 analogs was evaluated, and nine were selected as active. The structures selected as active exhibited
polar and non-polar substitutions on the caulerpin skeleton, which were relevant for their activity. Dragon consensus drug-like
scoring was applied to identify the active analogs that might serve as good drug candidates, and the entire group presented
satisfactory performance. These results indicate the possibility of using these analogs (Figure 7) as potential leads against
Alzheimer’sdisease.

Fig. (7). Caulerpin anologs most likely to be MAO B inhibitors

A promising target for the development of new drugs against AD is the beta-site APP cleaving enzyme1 (BACE1), as it
catalyzes the rate determining step in the generation of amyloid fibrils (Aβ) peptide. Aggregation and deposition of Aβ is the
most popular hypothesis for the cause of AD. Aβ are neurotoxic species and generate oxidative stress in brain that alters the
cortico-cortical connectivity. This leads to cessation of the cerebral cortex and eventual death of brain cells. Active site of
BACE1 contains catalytic aspartic (Asp) dyad and flap. Asp dyad cleaves the substrate amyloid precursor protein (APP) with
the help of flap. In the search for innovative inhibitors of BACE1, Kumar and colleagues[98] screen the natural database
InterBioScreen, followed by 3D QSAR pharmacophore modelling, mapping, in-silico ADME/T predictions. Further, molecular
dynamics of selected inhibitors were performed to observe the dynamic structure of protein after ligand binding. All
conformations and the residues of binding region were stable but the flap adopted a closed conformation after binding with the
ligand. Bond oligosaccharide interacted with the flap as well as catalytic dyad via hydrogen bound throughout the simulation.
This led to stabilize the flap in closed conformation and restricted the entry of substrate. Carbohydrate drugs have been earlier
used in the treatment of AD because of their low toxicity, high efficiency, good biocompatibility and easy permeability through
the blood brain barrier. Their finding will be helpful in identify the potential leads to design novel.

4.3. Obtaining candidates for human mood disorders and neurodegenerative diseases drugs
In this field of research, 5-hydroxytryptamine (5-HT) receptors are important targets for the development of new drugs. In
this class of receptors the 5-HT1A subtype appears as the most promising because it binds to the endogenous neurotransmitter
serotonin (5-HT), and is the most abundant in the Central Nervous System (CNS) being distributed in the cerebral cortex,
hippocampus, septum, amygdala and raphe nucleus in high densities, while it is also located at the basal ganglia and thalamus
in low amounts. It had been among the most important molecular targets that were actively being pursued for drug discovery
efforts in psychoactive treatment. Because of its dense concentration on cortical and hippocampal pyramidal neurons, 5-HT1A
receptor had been actively studied in recent years for novel strategies to treat the cognitive deficits in schizophrenia4. In fact,
atypical antipsychotic drugs modestly enhance cognition and several drugs in this class were found to show 5-HT1A partial
agonist activity (e.g. aripiprazole, clozapine, olanzapine, ziprasidone and quetiapine). Aiming to obtain new ligands from the 5-
HT1A receptor, Luo and colleagues[99] developed binary quantitative structure-activity relationship (QSAR) models for 5-
HT1A binding using data retrieved from the WOMBAT database and the k-Nearest Neighbor (kNN) machine learning method.
A rigorous QSAR modeling and screening workflow had been followed, with extensive internal and external validation
processes. The models’ classification accuracies to discriminate 5-HT1A binders from the non-binders were as high as 96% for
the external validation. These models were employed further to mine two major natural products screening libraries, i.e.
TimTec Natural Product Library (NPL) and Natural Derivatives Library (NDL). In the end five screening hits were tested by
radioligand binding assays with a success rate of 40%, and two new compounds (Figure 8) were confirmed to be binders at the
μM concentration against the human 5-HT1A receptor. The combined application of rigorous QSAR modeling and model-
based virtual screening presents a powerful means for profiling natural products compounds with important biomedical
activities.

 
Fig. (8). Compounds confirmed as binding of 5-HT 1A receptors

4.4. Obtaining candidates for anti-inflammatory drugs

The inflammatory response is a complex process involving metabolic pathways of arachidonic acid. An important enzyme
in this process is Cyclooxygenase (COX), which has two widely studied isoforms, COX-1 and COX-2, with functions that
determine the activation of the inflammation process. Phospholipases, especially cytosolic phospholipase A2, liberate the
phospholipid-bound arachidonate, and then the cyclooxygenase-complex transforms the liberated arachidonate by a sequential
introduction of two moles of oxygen (lipoxygenase reaction), followed by a COX reaction. This transformation results in a 15-
hydroperoxide prostaglandin (PGG2) that is reduced to the corresponding hydroxy product (PGH2). Then, a battery of enzymes
is leading to different inflammation mediators such as prostaglandins. COX-1 is a constitutively expressed gene, whereas COX-
2 is mainly induced by various inflammatory stimuli. Conceptually, COX-2 selective inhibitors should be expected to retain
antiinflammatory efficacy by inhibition of the prostaglandins, while reducing or eliminating the gastric, renal, and haemostatic
side effects commonly associated with nonsteroidal antiinflammatory drugs (NSAIDs) use. However, this simple idea of
focusing on the selective inhibition of COX-2 to profit exclusively from the desired effects is only valid with caveats. The
family of prostaglandins induced by COX-2 may replace that by COX-1 in some situations and vice versa. Both COX
isoenzymes contribute to mucosal defense, and the inhibition of the two isoforms contributes to the pathogenesis of NSAID-
induced gastric damage[100-102].
To solve the collateral problems caused by the use of current NSAIDs, there is a constant search for more selective drugs. In
order to discover new, more selective COX-1 and COX-2 inhibitors, Rollinger and co-workers[103] explored the efficiency of
using a biased 3 D database, including secondary metabolites obtained from anti-inflammatory medicinal plants as a source for
a VS. To that end, pharmacophore models of COX-1 and COX-2 were generated with structure-based as well as common
feature-based modeling, resulting in three COX hypotheses. Four different multiconfomational 3 D databases in molecular
weight between 300 and 700 were applied to the screening in order to compare and obtain the obtained hit rates. Two of them
were created in-house (DIOS, NPD). The database DIOS consists of 2752 compounds from phytochemical reports of anti-
inflammatory medicinal plants described by the ethnopharmacological source ‘De material medica’ of Pedanius Dioscorides,
whereas NPD contains almost 80,000 compounds gathered arbitrarily from natural sources. In addition, two available
multiconformational 3 D libraries comprising marketed and development drug substances Derwent World Drug Index (WDI)
and National Cancer Institute Database (NCI), mainly originating from synthesis, were used for comparison. As a test of the
pharmacophore models’ capability in natural sources, the models were used to search for known COX inhibitory natural
products. Depending on the hypothesis used, DWI and NCI library searches produced hit rates in the range of 6.6 % to 13.7 %.
A slight increase of the number of molecules assessed for binding was achieved with the database of natural products (NPD).
Using the biased 3 D database DIOS, however, the average increase of efficiency reached 77 % to 133 % compared to the hit
rates resulting from WDI and NCI. The statistical benefit of a combination of an ethnopharmacological approach with the
potential of computer aided drug discovery by in silico screening was demonstrated exemplified on the applied targets COX-1
and COX-2 (Figure 9) and allowed the selection of new hits with possible anti-inflammatory activity (Figure 10).
  
Fig. (9). Flurbiprofen (38) fitted on COX-1 pharmacophore model (Adapted from Rollinger et al[103])

Fig. (10). Structures of new hits with possible anti-inflammatory activity (Adapted from Rollinger et al[103])
Another useful target for the search for anti-inflammatory drugs is Microsomal prostaglandin E synthase-1 (mPGES-1).
PGE2 is an important product of the COX pathway and has been considered as a mediator of inflammation, pain, fever and
cancer; it is also known to regulate physiological functions in the gastrointestinal tract, in the kidney and in the immune and
nervous systems. Three major isoforms of PGE2 synthases have been identified: cytosolic PG E synthase (cPGES), microsomal
PGES 1 (mPGES-1) and mPGES-2. Both cPGES and mPGES-2 are constitutively expressed in various cells and tissues, while
mPGES-1 is localised to microsomal compartment of the cell. Recently, mPGES-1 has attracted much attention as a potential
drug target for inflammation and pain, tumorigenesis, arthritis and atherosclerosis, stroke, cancer and tissue repair. mPGES-1 is
induced by pro-inflammatory agents and is functionally coupled to COX-2 in various models of inflammation. Previous studies
using mPGES-1 knockout mice have shown that mPGES-1 contributes to the inflammatory production of PGE2. Hence,
mPGES-1 is a potential drug target for inflammation-related diseases. Chen et al have investigated mPGES-1 pharmacophore
features by Catalyst HypoGen[104], and as a continuation of the project, they applied 3 D quantitative structure–activity
relationship (3D-QSAR) analysis and molecular docking to search for novel inhibitors for mPGES-1. They performed virtual
screening using our traditional Chinese medicine and natural products database (http://tcm.cmu.edu.tw/) and constructed
comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) using a
training set of 30 experimentally tested mPGES-1 inhibitors. The CoMFA and CoMSIA models derived were statistically
significant with cross-validated coefficient values of 0.808 for CoMFA and 0.829 for CoMSIA and non-cross-validated
coefficient values of 0.976 for CoMFA and 0.980 for CoMSIA. Docking and de novo evolution design gave three top
derivatives, 2-O-caffeoyl tartaric acid-Evo_2 (57), glucogallin-Evo_1 (58) and 3-O-feruloylquinic acid-Evo_7 (59) that have
higher binding affinities than the control, glutathione. These three derivatives have interactions with Arg70, Arg73, Arg110,
Arg126 and Arg38, which all are mPGES-1 key active site residues. In addition, these derivatives fit well into the CoMFA and
CoMSIA models, with hydrophobic, hydrophilic and electropositive substructures mapped onto corresponding contour plots.
Hence, they suggested that these three de novo compounds could be a starting basis for new mPGES-1 inhibitors[105] (Figures
11).

 
Fig. 11. (a) Binding conformation of 2-O-Caffeoyl tartaric acid Evo_2 (57) and (b) Glucogallin-Evo_1 (58) and (c) 3-
OFeruloylquinic acid-Evo_7 (59) at the mPGES-1 binding site and common features observed among the top derivatives. Red
circle 1 indicates electropositive characteristics; red circle 2 indicates steric favour region. Blue circle 1 indicates hydrophobic
features; blue circle 2 indicates hydrophilic features. The red circles are features observed from CoMFA ligand mapping, while
the blue circles are obtained from CoMSIA ligand mapping (Adapted from Li et al[105])
Sala and colleagues[106] developed a protocol for the identification of inhibitors of the human inhibitor nuclear-factor κВ
kinase 2 (hIKK-2). This target is a serine-threonine protein kinase belonging to the IKK complex and is the primary component
responsible for activating the nuclear-factor kB transcription factor (NF- κВ) in response to inflammatory stimuli. The
importance of the NF-κВ pathway in regulating the expression of the genes controlling cellular immune and inflammatory
responses has motivated research groups in both academia and the pharmaceutical industry to devote increasing efforts toward
developing ATP-competitive inhibitors for hIKK-2, which could be of therapeutic use, e.g., in patients affected by chronic
inflammatory diseases. The hIKK-2 subunit is a polypeptide of 756 residues with a kinase domain in its 15–300 sequence
segment. At present, there are only two entries in the Protein Data Bank (http://www.pdb.org; PDB) for hIKK-2 (PDB codes
3BRT and 3BRV), and in both cases, they correspond to the NEMO-binding region located at the C-terminal end of the protein
(therefore, no experimental structure for the kinase domain of hIKK-2 is known). Moreover, obtaining a homology model for
this kinase domain is not trivial because the more similar protein in the PDB (i.e., the catalytic domain of human ZIP kinase;
PDB code 1YRP) has a pairwise sequence alignment with hIKK-2 of only 217 residues long, with a low percentage of
sequence identity (i.e., 32 %). With this knowledge Sala and colleagues[106]: (1) developed a homology model for the hIKK-2
kinase domain which could stand the test of your validation criteria; (2) docked ATP-competitive molecules known to be potent
and specific inhibitors of hIKK-2 with this model; (3) identified which of the resulting poses were knowledge-based coherent
by analyzing whether they satisfied the experimentally known generic binding features of ATP-competitive inhibitors of
kinases; (4) used the knowledge-based coherent poses to derive a structure-based common pharmacophore containing the key
intermolecular interactions between hIKK-2 and its inhibitors; (5) obtained exclusion volumes from our homology model and
added them to the pharmacophore; (6) validated the selectivity of the resulting pharmacophore and of the VS process using a
large database of kinase decoys and ATP-competitive inhibitors for hIKK-2 that were not used during the pharmacophore
building; (7) used the previously validated structure-based pharmacophore and VS protocol to find ATP-competitive inhibitors
for hIKK-2 in a database of NPs[107]; and, finally, (8) proved the reliability of the prediction by testing the inhibitory effect of
some selected hits on hIKK-2 in vitro. They demonstrated that your virtual-screening protocol was successful in identifying
lead compounds for developing new inhibitors for hIKK-2, a target of great interest in medicinal chemistry. Additionally, all
the tools developed during the study (i.e., the homology model for the hIKK-2 kinase domain and the pharmacophore) will be
made available to interested readers upon request.
In another study, Sala and colleagues[108] produced a stricter version of your protocol and applied to an in-house database
of 29,779 natural products annotated with their natural source. The search identified 274 molecules (isolated from 453 different
natural extracts) predicted to inhibit hIKK-2. An exhaustive bibliographic search revealed that anti-inflammatory activity has
been previously described for: (a) 36 out of these 453 extracts; and (b) 17 out of 30 virtual screening hits present in these 36
extracts. Only one of the remaining 13 hit molecules in these extracts shows chemical similarity with known synthetic hIKK-2
inhibitors. Therefore, it is plausible that a significant portion of the remaining 12 hit molecules are lead-hopping candidates for
the development of new hIKK-2 inhibitors. Additionally, they predicted that there are 414 other extracts with undescribed anti-
inflammatory activity that contain at least one out of 274 VS hits. Consequently, your work opens the door to the discovery of
new anti-inflammatory extracts of natural origin that could be of use, for example, in the design of functional foods aimed at
preventing diseases that are the result of chronic inflammation.
In this constant search for new anti-inflammatory drugs, another promising target is Lanthionine synthetase component C-
like protein 2 (LANCL2). Prokaryotic LanC is a part of a multimeric membrane associated lanthionine synthetase complex
involved in the modification and transport of peptides[109]. LanC itself is a zinc containing enzyme that acts in concert with
specific dehydratases to facilitate intramolecular conjugation of cysteine to serine or threonine residues, yielding macrocyclic
thioether analogs of cysteine known as lanthionines. The first member of the eukaryotic lanthionine synthetase component C-
like (LANCL) protein family, LANCL1, was isolated from human erythrocyte membranes[110]. A related protein, LANCL2,
was subsequently identified in human brain and testis[111]. LANCL2 is most highly expressed in testis, and its exogenous
introduction has been shown to cause increased cellular sensitivity to the anticancer drug, adriamycin, by suppressing the
expression of MultiDrug-Resistance 1 and its cognate protein, P-glycoprotein. On the other hand, overexpressed LANCL2
interacted with the actin cytoskeleton, implying that LANCL2 may also have a role in cytoskeletal reorganization and cellular
movement. in vitro results suggesting that LANCL2 is required for abscisic acid (ABA) binding to the membrane of human
granulocytes and for transduction of the ABA signal into cell-specific functional responses in granulocytes[112]. ABA is an
isoprenoid phytohormone that plays important roles in plant responses to environmental stresses and host responses. In
addition, ABA has received recent attention due its peroxisome proliferator-activated receptor (PPAR) γ-activating and anti-
inflammatory properties, which make it a target for development of potent anti-inflammatory and insulin sensitizing
therapeutics[113]. Lu and co-workers[114] performed homology modeling to construct a three-dimensional structure of
LANCL2 using the crystal structure of lanthionine synthetase component C-like protein 1 (LANCL1) as a template using
SWISS-MODEL Workspace. Using this model, structure-based virtual screening was performed using compounds from NCI
(National Cancer Institute) Diversity Set II, ChemBridge, ZINC natural products, and FDA approved drugs databases. Several
potential ligands were identified using molecular docking. In order to validate the anti-inflammatory efficacy of the top ranked
compound (NSC61610) in the NCI Diversity Set II, a series of in vitro and pre-clinical efficacy studies were performed using a
mouse model of dextran sodium sulfate (DSS)-induced colitis. Your findings showed that the lead compound, NSC61610,
activated peroxisome proliferator-activated receptor gamma in a LANCL2- and adenylate cyclase/cAMP dependent manner in
vitro and ameliorated experimental colitis by down-modulating colonic inflammatory gene expression and favoring regulatory
T cell responses. Figure 12 illustrates results obtained in silico.

 
Fig. (12). A) Structure of NSC61610 (60) (From Lu et al[114]).
 Among inflammatory diseases, ulcerative colitis (UC) is of great importance because it is a chronic intestinal disease
mediated by the immune system and relapsing. Interleukin (IL) -6, a pro-inflammatory cytokine, plays a key role in the
uncontrolled inflammatory process, which is a main characteristic of UC. Galvez-Llompart et al[115] built a quantitative
structure–activity relationship model based on molecular topology (MT) to predict the IL-6 mediated anti-UC activity. After an
external validation of the model, a virtual screening of the MicroSource Pure Natural Products Collection and Sigma-Aldrich
databases was carried out looking for potential new active compounds. From the entire set of compounds labeled as active by
the model, four of them, namely alizarin-3-methylimino-N,N-diacetic acid (AMA), Calcein, (+)-dibenzyl-l-tartrate (DLT), and
Ro 41-0960, were tested in vitro by determination of IL-6 production in two cell lines (RAW 264.7 and Caco-2). The results
demonstrate that three of them were able to significantly reduce IL-6 levels in both cell lines and particularly one, namely Ro
41-0960. These results confirm MT’s efficacy as a tool for the selection of compounds potentially active in UC.
Aswad and colleagues[116] developed an interesting study to index natural products for less expensive preventive or
curative anti-inflammatory therapeutic drugs. They used a set of 441 anti-inflammatory drugs representing the active domain
and 2892 natural products representing the inactive domain was used to construct a predictive model for bioactivity-indexing
purposes. The model for indexing the natural products for potential anti-inflammatory activity was constructed using the
iterative stochastic elimination algorithm (ISE). ISE was capable of differentiating between active and inactive anti-
inflammatory molecules. By applying the prediction model to a mix set of (active/inactive) substances, we managed to capture
38% of the anti-inflammatory drugs in the top 1% of the screened set of chemicals, yielding enrichment factor of 38. Ten
natural products that scored highly as potential anti-inflammatory drug candidates are disclosed (Figure 13). Searching the
PubMed revealed that only three molecules (Moupinamide, Capsaicin, and Hypaphorine) out of the ten were tested and
reported as anti-inflammatory. The other seven phytochemicals await evaluation for their anti-inflammatory activity in wet lab.
The proposed anti-inflammatory model can be utilized for the virtual screening of large chemical databases and for indexing
natural products for potential anti-inflammatory activity.
OH
O N O

O O N

trichostachine (61) Pipernonaline (62) O

OH
O O
H3CO H3C OCH3
N N
H H
CH3
HO OH
Moupinamide (63) Capsaicin (64)

NH2 OCH3
H
N N HO O

N N N
O H
NH
H3C CH3
Triacanthin (65) Piperolactan (66) N-2-Phenylethylcinnamamide (67)
O H
O N
O CH3
N HN H3C CH
H O 3
O CH3 N CH
O 3
4,5-Dihydropiperlongumine (68) Noroxyhydrastinine (69)
O
O
Hypaphorine (70)  

Fig. (13). Natural products that were scored highly as potential anti-inflammatory drug candidates by ISE based anti-
inflammatory indexing model[116]
Psoriasis is a chronic immune-mediated inflammatory skin disorder which generally results into overgrowth of skin tissue.
This disease is associated with both the genetic and environmental causes of factors depending upon the particular conditions of
the patient. Heat shock proteins (HSPs) have been witnessed as a potential drug target for inhibition of psoriatic cell
differentiation. The expression level of HSP is increased when the cells get exposed to elevated temperature, oxidative stress
and nutritional deficiencies and thus plays major role in psoriatic progression pathway. Immunoreactivity intensity distribution
index scores for HSP70 expression is significantly higher in psoriatic patients compared to normal. Based on this knowledge,
Mishra et al[117] performed a virtual screening against leadlike, drug-like and some natural product of ZINC database. The
screened ligands were further introduced to ADMET prediction and simulations to see the drug proficiency and likeness
property. The molecular dynamic of system was found stable during simulation trajectory and not much of significant changes
occurred in the conformation of the protein–ligand complex. Thus, present study in all probability might prove useful for future
design of new derivatives with higher potency and specificity. The Figure 14 shows the six most promising compounds
obtained in the study as potential new drugs.

4.4. Obtaining candidates for anti-lipemic drugs

The farnesoid X receptor (FXR) belonging to the metabolic subfamily of nuclear receptors is a ligand induced
transcriptional activator. Its central function is the physiological maintenance of bile acid homeostasis including the regulation
of glucose and lipid metabolism[118]. It is expressed in liver, intestine, kidney, adrenal glands, and also in the vasculature
where FXR might play a role in the pathogenesis of cardiovascular diseases. FXR has been found to be a key target for bile
acids (BAs). Representing endogenous signaling molecules, BAs negatively regulate their own synthesis by repressing the
transcription of cholesterol 7α-hydroxylase (CYP7A1), a cytochrome P450 enzyme essential for the synthesis of cholesterol. In
publications the relevance of FXR as BA activated receptor was illuminated with regard to the treatment of atherosclerosis and
its counter-regulatory role in immunity and inflammation. Beneficial effects of FXR agonists are reported for the modulation of
lipids and glucose as well as hepatobiliary and gastrointestinal diseases[119]. On the one hand, FXR holds a regulative function
in many endogenous pathways and the features of its active site are well-characterized. These essential facts contribute to the
attractiveness of this nuclear receptor as novel drugable target to find innovative agents showing favorable effects in the
prevention and treatment of, for example, the metabolic syndrome, dyslipidemia, atherosclerosis, and type 2 diabetes. For the
identification of FXR ligands from natural sources, Grienke and co-workers[120] selected a virtual screening approach. Based
on the protein data bank (PDB)[121] crystal structure entry 1osh[122] comprising a y-shaped hydrophobic ligand within the
binding site of the nuclear receptor FXR, a structure-based pharmacophore model (1osh-1) was created and validated[123]. The
generated model consists of five hydrophobic features, one hydrogen bond acceptor with His294, and 27 exclusion volume
spheres. Virtual screening of your in-house Chinese Herbal Medicine (CHM) database, comprising 10,216 compounds, with the
1osh-1 model resulted in a list of 572 virtual hits (VHs) ranked according to their computationally derived best fit values. The
analysis of the VHs comprising various compound classes involved several parameters, for example, already known FXR-
related effects, commercial availability of the natural starting material or the pure compound, and accessibility of the selected
VHs from natural sources. Considering these aspects, a set of representatives has been selected for pharmacological evaluation
of the predicted FXR inducing potential taking into account diverse structure classes such as triterpenes, flavonoids,
furanocoumarins, quinolone derivatives, carotenoids, and fatty acid derivatives. The study revealed mainly lanostane-type
triterpenes of the TCM fungus Ganoderma lucidum Karst. as putative FXR ligands. To verify the prediction of the in silico
approach, two Ganoderma fruit body extracts and compounds isolated thereof were pharmacologically investigated.
Pronounced FXR-inducing effects were observed for the extracts at a concentration of 100 µg/mL. Intriguingly, five lanostanes
out of 25 secondary metabolites from G. lucidum, that is, ergosterol peroxide (77), lucidumol A (78), ganoderic acid TR (79),
ganodermanontriol (80), and ganoderiol F (81), dose-dependently induced FXR in the low micromolar range in a reporter gene
assay[120] (Figure 15). In another study, Diao et al[124] carried out a structure-based virtual screening strategy using an in-
house natural product database (NPD) containing over 4,000 compounds and subsequent hit-based similarity searching method.
Six FXR antagonists were identified by method and two of the most potent compounds 82 and 83 could antagonize the CDCA-
induced SRC-1 recruitment to FXR-LBD with the IC50 values of 1.29 μM and 1.79 μM, respectively (Figure 16). A docking
study further showed the mode of interaction of compound 82 with the active site of FXR.

 
Fig. (14). Top six compounds ranked as potential new drugs against psoriasis[117].
  
Fig. (15). Structures of lanostanes with Pronounced FXR-inducing effects

 
Fig. (16). Chemical structures of FXR antagonists[124]
Statins belong to a class of molecules of great commercial importance in the category of anti-lipemic drugs. They are
inhibitors of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, a key enzyme in cholesterol
metabolism. Na busca por novos inibidores da HMG-CoA reductase que sejam mais seletivos e menos tóxicos, Lin et al[125]
realizaram um virtual screening para a obtenção de potential hHMGR inhibitors from natural product to discover hypolipidemic
drug candidates. They used the 3 D structure 1HWK from the PDB (Protein Data Bank) database of hHMGR as the target to
screen for the strongly bound compounds from the traditional Chinese medicine database. Many interesting molecules
including polyphenolic compounds, polisubstituted heterocyclics, and linear lipophilic alcohols were identified and their
ADMET (absorption, distribution, metabolism, excretion, toxicity) properties were predicted. Finally, four compounds were
obtained for the in vitro validation experiments. The results indicated that curcumin and salvianolic acid C (Figure 17) can
effectively inhibit hHMGR, with IC50 values of 4.3 μM and 8 μM, respectively.

 
Fig. (17). Compounds obtained as hHMGR inhibitors
4.5. Obtaining candidates for antidiabetic drugs
Type 2 diabetes mellitus (T2DM) is considered to be the ‘‘epidemic of the 21st century’’ and, consequently, the
development of new therapies is one of the main challenges in drug discovery[126]. The inhibition of human dipeptidyl
peptidase-IV (DPP-IV) is a treatment option for T2DM. This enzyme belongs to the serine protease family and selectively
removes N-terminal dipeptides from substrates containing proline or alanine as the second residue[127]. The most important
substrates of DPP-IV are incretins, such as glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide
(GIP). DPP-IV inhibitors are of considerable interest to the pharmaceutical industry, and intense research activities in this area
have resulted in the launch of sitagliptin, saxagliptin, alogliptin, linagliptin and vildagliptin to the market[128]. The DPP-IV
binding site is highly druggable in the sense that tight and specific binding to the enzyme can be achieved with small molecules
with drug-like physicochemical properties. The different interaction motifs used by these DPP-IV ligands include Ser630 (that
together with Asp708 and His740 form the enzyme catalytic triad), the hydrophobic S1 pocket (formed by Tyr631, Val656,
Trp659, Tyr662, Tyr666 and Val711), the hydrophobic S2 pocket (formed by Arg125, Phe357, Arg358, Tyr547, Pro550 and
Asn710) and the N-terminal recognition region (formed by Glu205, Glu206 and Tyr662). Based on the analysis of the DPP-IV
crystal structures and interpretation of the structure-activity relationship (SAR) data, both the lipophilic S1 pocket and the
Glu205/Glu206 dyad can be considered as crucial molecular anchors for DPP-IV inhibition[129, 130]. Due to the importance of
this target, Guasch and co-workers[131, 132] developed two works where they predicted that 446 out of the 89,165 molecules
present in the natural products subset of the ZINC database would inhibit DPP-IV with good ADMET properties. Notably,
when these 446 molecules were merged with 2,342 known DPP-IV inhibitors and the resulting set was classified into 50
clusters according to chemical similarity, there were 12 clusters that contained only natural products for which no DPP-IV
inhibitory activity has been previously reported. Nine molecules from 7 of these 12 clusters were then selected for in vitro
activity testing and 7 out of the 9 molecules were shown to inhibit DPP-IV (where the remaining two molecules could not be
solubilized, preventing the evaluation of their DPP-IV inhibitory activity). Then, the hit with the highest activity was used as a
lead compound in the prediction of more potent derivatives (Figure 18).

 
Fig. (18). A) Schematic overview of the VS workflow and the procedure used for selecting the VS hits that were tested for
DPP-IV inhibitory activity. For the VS, the number of compounds that passed each step and the programs used are showed. For
the selection of VS hits for bioactivity testing, the numbers show either how many VS hits are scaffold-hopping candidates for
DPP-IV inhibition (Fingerprint similarity analysis step) or how many molecules were experimentally tested for bioactivity
(Biological test step; B) Chemical structures and ZINC codes for the 9 molecules selected for experimentally testing whether
these compounds exhibited DPP-IV inhibitory activity. The insolubility of C4 and C6 prevented these compounds from being
assayed for DPP-IV inhibitory activity. Positions in the C5 structure that will be replaced by substituents to identify derivatives
with higher binding affinity on the DPP-IV binding site are indicated with a grey background and annotated with the label of
the corresponding site in the common structure-based pharmacophore (Adapted from Guasch and co-workers[131]).
 Peroxisome Proliferator-Activated Receptor (PPAR) are fatty acid activated transcription factors that belong to the
thyroid/retinoid nuclear receptor family. The α, δ, and γ subtypes of PPAR found in mammals, coordinate pathways involved in
glucose and lipid homeostasis. PPARs are implicated in the pathology of various disease states including type II diabetes,
obesity, dyslipidemia, atherosclerosis, neoplastic diseases and tumors, inflammatory conditions, and neurodegenerative
diseases[133]. PPARγ is predominately expressed in adipose tissue and its significant role in lipid metabolism, adipogenesis,
glucose homeostasis, and insulin sensitization. Natural ligands of PPARγ are fatty acids as well as eicosanoids. Among a large
variety of synthetic ligands are thiazolidinediones (TZDs) and some nonsteroidal anti-inflammatory drugs. TZDs have been
used in clinical practice to treat type II diabetes for many years and have been shown to lower blood glucose levels and improve
insulin sensitivity and PPARγ was identified as their target and that its activation was shown to be responsible for their
therapeutic benefits. However, despite their excellent potencies, administration of TZDs has been associated with severe side
effects such as fluid retention, weight gain, cardiac hypertrophy, and hepatotoxicity[134-136]. Petersen and co-workers[137]
have combined in silico and in vitro approaches for the identification of natural compounds that act as PPARc agonists. They
have used pharmacophore based Virtual Screening (VS) for the initial identification of target compounds and subsequently
verified one of the hits using bioassay-guided chromatographic fractionation. The model was used for the virtual screening of
the Chinese Natural Product Database (CNPD). From the resulting hits, they selected methyl oleanonate (Figure 19), a
compound found, among others, in Pistacia lentiscus var. Chia oleoresin (Chios mastic gum). The acid of methyl oleanonate,
oleanonic acid, was identified as a PPARγ agonist through bioassay-guided chromatographic fractionations of Chios mastic
gum fractions, whereas some other sub-fractions exhibited also biological activity towards PPARγ. The results from the work
are two-fold: on the one hand they demonstrate that the pharmacophore model developed is able to select novel ligand scaffolds
that act as PPARγ agonists; while at the same time it manifests that natural products are highly relevant for use in virtual
screening-based drug discovery.

 
Fig. (19). Structure of oleanonic acid (from Petersen and co-workers[137]).
One approach that may be used for production of antidiabetic drugs is the design of inhibitors of the Acetyl-CoA
carboxylases (ACCs) enzymes that have crucial roles in obesity-induced type 2 diabetes. These enzymes are responsible for
biotin-dependent carboxylation of acetyl-CoA to malonyl-CoA, which is an important intermediate in controlling the rate of
fatty acid metabolism. Inhibition of ACC blocks the de novo fatty acid synthesis and enhances β-oxidation of fatty acid which
could be utilised for the treatment of metabolic disorders, such as obesity and type 2 diabetes. In humans, there are two
isoforms of ACC (1 and 2) with distinct subcellular localization. Acetyl-CoA carboxylase1 (ACC1), a 265-kDa cytosolic
protein, is principally expressed in lipogenic tissues, while Acetyl-CoA carboxylase2 (ACC2), a 280-kDa protein, is
predominantly expressed in oxidative tissues, such as liver, skeletal muscles and heart. Non-selective inhibition of ACC has
been shown to be more effective in fat oxidation than selective inhibition of ACC2. Bhadauriya and colleagues[138] have
identified three potential new ACC inhibitors with different scaffolds, through pharmacophore based virtual screening and
molecular docking approach. For that, they used a pharmacophore modeling approach, where the best HypoGen
pharmacophore model for ACC2 inhibitors (Hypo1_ACC2) consisted of one hydrogen bond acceptor, one hydrophobic
aliphatic and one hydrophobic aromatic feature, whereas the best pharmacophore (Hypo1_ACC1) forACC1 consisted of one
additional hydrogen-bond donor (HBD) features. These hypotheses have been validated, as well as the test set, decoy set and
Cat-Scramble methodology. In this way, the three potential new inhibitors mentioned (ZINC33086616, ZINC19924472 and
ZINC33086598) were identified from screening in Specs, NCI, Chem-Div and Natural product databases using filters as
estimated IC50 value, quantitative estimation of drug-likeness and molecular docking analysis.
Vuorinen et al[139] na busca por novos agentes antidiabéticos estudaram a goma da oleorresina da Pistacia lentiscus var.
chia, também chamada de Mastic gum, pois havia relatos que essa planta possuía essa atividade. Então eles realizaram um
Pharmacophore-based virtual screening para filtrar o natural product database DIOS na intenção de obter potenciais inibidores
da enzima 11β-hydroxysteroid dehydrogenase 1. A inibição dessa enzima está relacionada ao combate de distúrbios
metabólicos, incluindo diabetes. The hit list analysis was especially focused on the triterpenoids present in Pistacia species.
Multiple triterpenoids, such as masticadienonic acid and isomasticadienonic acid, main constituents of mastic gum, were
identified. Indeed, masticadienonic acid and isomasticadienonic acid (Figure 20) selectively inhibited 11β-hydroxysteroid
dehydrogenase 1 over 11β-hydroxysteroid dehydrogenase 2 at low micromolar concentrations. These findings suggest that
inhibition of 11β-hydroxysteroid dehydrogenase 1 contributes to the antidiabetic activity of mastic gum.
  
Fig. (20). Structures of isomasticadienonic acid and masticadienonic acid
Fatty acids are a class of straight-chain hydrocarbons with a carboxyl tail group. They are metabolized to yield large
quantities of adenosine triphosphate (ATP), thus acting as a preferred fuel for the human metabolism. Abnormally elevated
fatty acid levels in circulation have been reported to be involved in the pathogenesis of chronic metabolic disorders, including
type 2 diabetes, obesity, and atherosclerosis. Fatty acid trafficking in cells requires fatty acid binding proteins (FABPs), a
family of approximately 15 kDa lipid chaperones. Both endogenous and exogenous fatty acids can reversibly bind to FABPs
with high affinity, and the fatty acid-bound FABPs are shuttled in the cytoplasm for the lipid metabolic process or storage. The
inhibition of fatty acid binding protein 4 (FABP4) by using small molecules could potentially provide therapeutic opportunities
for metabolic disorders treatment. Wang and co-workers[140] performed a virtual screening and molecular docking on the
herbal molecules database. They reported flavonols as an ideal scaffold for FABP4 inhibitors development. Among the popular
flavonols examined, they identified hyperoside (98) (figure 21) as a promising FABP4 inhibitor. Identical to the well-known
FABP4 inhibitor BMS309403, hyperoside induced lipid accumulation and upregulated peroxisome proliferator-activated
receptor γ (PPARγ) protein expression during the adipocyte differentiation process. Furthermore, both PPARγ antagonist and
FABP4 overexpression attenuated hyperoside-induced adipogenesis, indicating that hyperoside promoted adipogenesis in
adipocytes via the FABP4/PPARγ pathway.They anticipate hyperoside to be a promising, novel FABP4 inhibitor for
antidiabetic drug development.

 
Fig. (21). A) Structure of Hyperoside (98); B) An ideal scaffold of flavonol for novel FABP4 inhibitors development is
summarized[140]
 Another important target for the development of antidiabetic drugs is Protein tyrosine phosphatase non-receptor type 1
(PTPN1) also known as protein tyrosine phosphatase 1B (PTP1B). The protein tyrosine phosphatases are enzymes that catalyze
protein tyrosine dephosphorylation in regulation of insulin action by dephosphorylation of activated auto phosphorylated
insulin receptor and downstream substrate proteins. Bibi and Sakata[141] proposed a pipeline for the development of effective
and plant-derived anti-diabetic drugs that may be safer and better tolerated. PTPN1 inhibitors possessing antidiabetic activity
less than 10 μM were used as a training set. A common feature pharmacophore model was generated. Pharmacophore-based
screening of plant-derived compounds of the ZINC database was conducted using ZINCpharmer. Screened hits were assessed
to evaluate their drug-likeness, pharmacokinetics, detailed binding behavior, and aggregator possibility based on their
physiochemical properties and chemical similarity with reported aggregators. Through virtual screening and in silico
pharmacology protocol isosilybin (ZINC30731533) (Figure 22) was identified as a lead compound with optimal properties.
This compound can be recommended for laboratory tests and further analyses to confirm its activity as PTPN1 inhibitor.

 
Fig. (22). Structure of isosilybin (ZINC30731533) (99) selected as potential PTPN1 inhibitor
Zeidan and colleagues[142] used a set of 97 approved anti-diabetic drugs, representing the active domain, and a set of 2892
natural products, representing the inactive domain to construct predictive models in silico and to index anti-diabetic bioactivity.
Their recently-developed approach of ‘iterative stochastic elimination’ was utilized. They describes a highly discriminative and
robust model, with an area under the curve above 0.96. Using the indexing model and a mix ratio of 1:1000 (active/inactive),
65% of the anti-diabetic drugs in the sample were captured in the top 1% of the screened compounds, compared to 1% in the
random model. Some of the natural products that scored highly as potential anti-diabetic drug candidates are disclosed. One of
those natural products is caffeine, which is noted in the scientific literature as having the capability to decrease blood glucose
levels. The other nine phytochemicals await evaluation in a wet lab for their anti-diabetic activity (Figure 23). The indexing
model proposed herein is useful for the virtual screening of large chemical databases and for the construction of anti-diabetes
focused libraries.

 
Fig. (23). Some of the natural products that scored highly as potential anti-diabetic drug candidates according to ISE-based,
anti-diabetic activity indexing model[142]
 The G protein-coupled bile acid receptor 1 (GPBAR1), also commonly named M-BAR or Takeda G-protein-coupled
receptor 5 (TGR5), is a rhodopsin-like G protein-coupled receptor (GPCR) expressed in various tissues and has been
recognized as a promising new target for the treatment of diverse diseases, including obesity, type 2 diabetes, fatty liver disease
and atherosclerosis. The identification of novel and potent GPBAR1 agonists is highly relevant, as these diseases are on the rise
and pharmacological unmet therapeutic needs are pervasive. Thus, Kirchweger and colleagues[143] developed a proficient
workflow for the in silico prediction of GPBAR1 activating compounds, primarily from natural sources. A protocol was set up,
starting with a comprehensive collection of structural information of known ligands. This information was used to generate
ligand-based pharmacophore models in LigandScout 4.08 Advanced. After theoretical validation, the two most promising
models, namely BAMS22 and TTM8, were employed as queries for the virtual screening of natural product and synthetic small
molecule databases. Virtual hits were progressed to shape matching experiments and physicochemical clustering. Out of 33
diverse virtual hits subjected to experimental testing using a reporter gene-based assay, two natural products, farnesiferol B and
microlobidene (Figure 24), were confirmed as GPBAR1 activators reaching more than 50% receptor activation at 20 μM with
EC50s of 13.53 μM and 13.88 μM, respectively. This activity is comparable to that of the endogenous ligand lithocholic acid.

 
Fig. (24). Structures of farnesiferol B (110) and microlobidene(111)[143]

4.6. Obtaining candidates for cytochrome P450 inhibitors drugs

The human cytochrome P450 2D6 (CYP2D6) enzyme is part of phase-I metabolism in which xenobiotics are oxidized to
increase their excretion from the body. Xenobiotics are chemicals that are foreign to the human body; examples include
synthetic drugs, environmental chemicals, pesticides, herbicides, preservatives, flavourings and natural products, some of
which are omnipresent in food and beverages. Therefore, it is an important target for drug-drug interaction (DDI) studies. High-
throughput screening (HTS) assays are commonly used tools to examine DDI, but show certain drawbacks with regard to their
applicability to natural products. Hochleitner and colleagues[144] proposed an in silico – in vitro workflow for the reliable
identification of natural products with CYP2D6 inhibitory potential. In order to identify candidates from natural product-based
databases that share similar structural features with established inhibitors, a pharmacophore model was applied. The virtual hits
were tested for the inhibition of recombinant human CYP2D6 in a bioluminescence-based assay. By controlling for unspecific
interferences of the test compounds with the detection reaction, the number of false positives were reduced. The success rate of
the reported workflow was 76%, as most of the candidates identified in the in silico approach were able to inhibit CYP2D6
activity. The presented combined virtual - in vitro workflow may help to screen natural compound libraries fast and reliably for
new CYP2D6 inhibitors. Moreover, detailed information on CYP2D6 inhibition is essential when aiming to combine herbal
preparations and synthetic drugs at the same time safely. The Figure 25 shows the compounds classified as potential inhibitors
of CYP2D6.
Cytochrome P450 3A4 (CYP3A4) is an important member of the CYP family. Potential drug–drug interactions (DDIs)
caused by CYP3A4 inhibitors could lead to increasing risk of side-effects/toxicity or decreasing effectiveness. The evaluation
of CYP3A4 inhibitory activity is time-consuming, labor-intensive, and costly, and it is necessary to establish virtual screening
models for predicting CYP3A4 inhibitors. Pang et al[145] performed a study where four classifier algorithms, including
support vector machine (SVM), naive Bayesian (NB), recursive partitioning (RP), and K-nearest neighbor (KNN), were applied
to discriminate CYP3A4 inhibitors from the non-inhibitors. Correlation analysis and stepwise linear regression methods were
used for descriptor selection and optimization. The performance of classifiers was measured by 5-fold cross-validation, Y-
scrambling and test set validation. Finally, the optimal NB model with Matthews correlation coefficients of 0.894 for the test
set was developed to screen FDA-approved drugs and natural products database. As a result, 90 compounds from FDA-
approved drug databases were predicted as inhibitors, and 46% of them were identified as known CYP3A4 inhibitors. Six
natural products were selected for further bioactivity assay and molecular docking (Figure 26). Two of them with good docking
score also exerted significant CYP3A4 inhibitory activities with IC50 values of 0.052 and 1.120 mM, respectively. This study
proved the feasibility of a new method for predicting CYP3A4 inhibitory activity and preventing the occurrence of DDIs at
early stage in drug development.

 
Fig. (25). Structures of the compounds classified as potential inhibitors of CYP2D6[144].

Isoimperatorin Bergaptin Bisdemethoxycurcumin

Azulene
Pterostilbene Ellipticine
 
Fig. (26). Structures of CYP3A4 inhibitors[145]
4.7. Obtaining candidates for antiviral drugs
Around the world there are number of diseases caused by viruses that affect a large part of the world population. Some of
these diseases are very serious and can lead to death. Severe Acute Respiratory Syndrome (SARS) is an epidemic viral disease
spread in several countries. To illustrate the severity of the disease, in China between March and May 2003, 5327 people were
infected, of which 349 died. This disease is caused by a coronavirus that has a target elucidated for obtaining potential
inhibitors which is the SARS coronavirus (SARS-CoV) protease[146-148]. Liu and Zhou[149] performed a VS to find potent
SARS-CoV protease inhibitors using two natural product databases, the marine natural products database (MNPD) and the
traditional Chinese medicines database (TCMD). Before the procedure, the databases were filtered by Lipinski’s ROF and Xu’s
extension rules. The results were analyzed by statistic methods to eliminate the bias in target-based database screening toward
higher molecular weight compounds for enhancing the hit rate. Eighteen lead compounds were recommended by the screening
procedure. They were useful for experimental scientists in prioritizing drug candidates and studying the interaction mechanism.
The interaction and binding mechanism were elucidated by the complex structure of SARS–M4367 (131). The similarity of the
protein binding mode between screened compounds and Wu-2, AG7088, which were reported as possible molecules of SARS
inhibitors, showed certain values of the research for experimental scientists in prioritizing drug candidates. The results show
that high-affinity drugs for the SARS protein may have the characteristic of direct interaction with the functional residues,
His41 and Cys145, which act as a crucial role in the regulation of the SARS life cycle (Figure 27)[149].

 
Fig. (27). Structure of SARS–M4367 (131) (From Liu and Zhou[149])
Rollinger and co-workers[150] performed a SBVS to obtain human Rhinovirus inhibitors (HRV). This virus belongs to the
picornavirus family and comprises more than 100 different serotypes and are etiological agents of the common cold that,
despite being a simple and self-limiting disease, causes socioeconomic impact due to the fact that it causes work and school
absences[151]. In the study, they aimed to find antiviral compounds from nature, focusing on the HRV coat protein. To do so,
they used an in-house 3 D database containing 9,676 plant metabolites. They selected sesquiterpene coumarins from the gum
resin asafetida as promising natural products. Chromatographic separation steps resulted in the isolation of microlobidene
(134), farnesiferol C (135), farnesiferol B (136), and kellerin (137). Determination of the inhibition of the HRVinduced
cytopathic effect for serotypes 1A, 2, 14, and 16 revealed a dose-dependent and selective antirhinoviral activity against serotype
2 for asafetida (IC50 = 11.0 μg/mL) and its virtually predicted constituents 2 (IC50 = 2.5 μM) and 3 (IC50 = 2.6 μM). In study,
entries from the Protein Data Bank (PDB) 1QJU and 1C8M, both complexes of HRV serotype 16 with highly active inhibitors,
served as starting points for model generation. The model was designed to combine features characteristic for ligand binding in
the hydrophobic pocket within the HRV capsid. Four hydrophobic features (H) represent the predominately lipophilic character
of the pocket realized in all known inhibitor structures. A hydrogen bond acceptor (HBA) shows another typical interaction, a
direct or more often water-mediated hydrogen bond to Leu100. The features are arranged in a long stretched out fashion
modeling the “tunnel”-shape of the pocket. For this closed and sterically, well defined, binding site, the application of a shape
was useful in providing spatial limitation of the ligand size. The 3 D coordinates for feature setting and the shape were derived
from the PDB complex 1QJU and its bound inhibitor WIN 61209. For enhanced restrictivity, another interaction possibility
seen in the most well-known HRV coat protein inhibitor pleconaril was realized within the model. A customized hydrogen
bond acceptor containing fluorine (HBAF) to Tyr144, which is located in the very inner part of the pocket, was added using
crystal structure information from PDB entry 1C8M. The resulting five-feature hypothesis is shown in Figure 28. For natural
compound database screening, the HBAF feature could have been replaced by a regular HBA feature, but we preferred to keep
the original model as described in the previous study unchanged. They also performed a Docking study using LigandFit to
generate a Pharmacophore model and the results are shown in Figure 22. With the aid of the BLAST program 2.2.17 an
alignment of the serotypes was also performed to verify the conformations of the hydrophobic cannons of the HRV-2 and
HRV-16 serotypes and the results are shown in Figure 29.

 
Fig. (28). Highly Active HRV Coat Protein Inhibitors WIN61209 (132) and Pleconaril (133). (from Rollinger and co-
workers[150])
 This study exemplarily confirms the applicability and potency of the combined theoretical (in silico) and empirical
(ethnopharmacological) efforts for the rational search for naturally derived drug leads, enabling a fast and efficient
identification of potential new inhibitors of the HRV-2 coat protein[150].

 
Fig. (29). Chemical Structures of Sesquiterpene Coumarin Ethers Microlobidene (134), Farnesiferol C (135), Farnesiferol B
(136), Kellerin (137). (From Rollinger et al[150]).
Among the viral diseases, some remain a serious public health problem, including those caused by the virus Influenza like
H5N1 and H1N1 that have caused several deaths around the world recently. These strains of this virus have three viral-coat
proteins: hemagglutinin (HA), neuraminidase (NA), and M2. HA and NA are glycoproteins that recognize terminal sialic-acid
(SA) residues on host-cell surface receptors, and M2 is a proton channel critical for virus assembly and replication. Upon
attachment via SA binding, HA mediates viral entry into the cell[152]. Following viral replication, NA facilitates the liberation
of new virions from the cellular surface by cleaving the α(2−6)- or α(2−3)-ketosidic linkages that connect terminal SA
residues to cell-surface glycoproteins. NA is conserved in all wild-type influenza viruses, and its inhibition halts viral
propagation by interfering with effective shedding. Consequently, it is an attractive target for anti-influenza drug design[2,
153]. Ikram et al[154] performed a virtual screening approach for identifying plants with anti H5N1 neuraminidase activity.
They reported computational and experimental efforts to identify influenza neuraminidase inhibitors from among the 3000
natural compounds in the Malaysian-Plants Natural-Product (NADI) database. These 3000 compounds were first docked into
the neuraminidase active site. The five plants with the largest number of top predicted ligands were selected for experimental
evaluation (Table 1). Twelve specific compounds isolated from these five plants were shown to inhibit neuraminidase,
including two compounds with IC50 values less than 92 μM. Furthermore, four of the 12 isolated compounds (Table 2) had also
been identified in the top 100 compounds from the virtual screen. Together, these results suggest an effective new approach for
identifying bioactive plant species that will further the identification of new pharmacologically active compounds from diverse
natural-product resources.
Table 1. Percent NA Inhibition of Plant Fractions Selected Based on Virtual Screening

% inhibition of HSNI NA at 250 µg/mL

Extract Fraction

Plant EXT F1 F2 F3 F4 F5 F6

G. mangostana 82,95 ±0,21 83,73 ± 0,28 78,25 ± 0,05 84,49 ± 0,19 80,15 ± 0,27

M. charantia 72,61 ± 0,34 58,03 ± 0,9 34,12 ± 0,10 70,70 ± 0,47 72,43 ± 0,13 46,93 ± 0,17 66,08 ± 0,20

T. divaricate 62,79 ± 0,20 35,95 ± 0,19 20,06 ± 0,05 33,47 ± 0,11 28,00 ± 0,23 20,95 ± 0,09 11,90 ±0,21

B. javanica 55,80 ±0,03 49,64 ± 0,44 58,38 ± 0,12 57,49 ± 0,03

E. longifolia 29,62 ± 0,36 9,14 ± 0,22 8,00 ± 0,36 23,02 ± 0,30 56,47 ± 0,17

Another strain of the Influenza virus that caused many deaths was H7N9. It emerged in China in 2013 and caused many
deaths in Asia, mainly because it had mechanisms of resistance to all drugs available for treatment at the time. In an attempt to
solve the problem, Tran and colleagues[155] performed virtual screening targeting the Neuraminidase (NA) protein against
natural compounds of traditional Chinese medicine database (TCM) and ZINC natural products. Compounds expressed high
binding affinity to the target protein was then evaluated for molecular properties to determine drug-like molecules. 4
compounds showed their binding energy less than -11 Kcal/mol were selected for molecular dynamics (MD) simulation to
capture intermolecular interactions of ligand-protein complexes. The molecular mechanics/Poisson-Boltzmann surface area
(MM/PBSA) method was utilized to estimate binding free energy of the complex. In term of stability, NA-compound 7181
achieved stable conformation after 20ns and 27ns for ligand and protein root mean square deviation, respectively. In term of
binding free energy, compound 7181 (152) gave the negative value of -30.031 (KJ/mol) indicating the compound obtained a
favorable state in the active site of the protein (Figure 30).
Table 2. AutoDock Score, IC50 Values, and Percent Inhibition of Compounds Isolated From Five Plantsa

Compound AutoDock score IC50 (µM) % inhibition

(kcal/mol) (at 250 µg/ml)

-8,87 91,95 ± 0,09 93,08 ± 0,04

(at 609 µM)

α – mangostin* (138)

-8,85 95,49 ± 0,08 90,13 ± 0,02

(at 609 µM)

Rubraxantone*(139)

-8,85 95,49 ± 0,08 90,13 ± 0,02

(at 603 µM)

Garcinone C*(140)

-11,07 126,64 ± 0,13 90,13 ± 0,02

(at 603 µM)

Gartanin*(141)

-8,99 275,45 ± 0,03 60,65 ± 0,29

(at 529 µM)

Daucosterol(142)

-11,49 ≥250 15,58 ± 0,29

Momordicin 1(143)
 -10,21 ≥250 21,42 ± 0,50
(at 550 µM)

Kuguacin J(144)

-10,49 ≥250 20,95 ± 0,09

(at 800 µM)

Voaphylline(145)

-10,89 ≥250 20,84 ± 0,67

(at 612 µM)

Eurycomanone(146)

-9,83 ≥250 21 ± 02,18

(at 609 µM)

Eurycomanol(147)

-9,92 ≥250 2,90 ± 0,34

(at 631 µM)

13α,21 - epoxyeurycomanone(148)

-10,45 ≥250 34,50 ± 0,27

(at 589 µM)

13 α,21 - epoxyeurycomanone(149)

34,82 ± 0,05 94,19 ± 0,02

(at 858 µM)

DANA(150)
 4,80 E-03 100
(at 879 µM)

Oseltamivir(151)
a
Compounds marked with asterisks were subjected to additional testing to rule out nonspecific inhibition by self aggregation

 
Fig. (30). Structure of compound 7181 (152).

4.8. Obtaining candidates for anticancer drugs

Cancer is a generic term for a vast number of diseases that are characterized by a lack of control in the regulation of the cell
cycle. Thus, there are large number of strategies and targets for the prospection of new agents suitable for use as anticancer
drugs.
One such target is the Androgen Receptor (AR) that is related to prostate cancer. The molecules that act in this pathway can
act by blocking the synthesis of androgens (testosterone (T) and dihydrotestosterone (DHT)) or inhibiting the binding of these
hormones to AR, both strategies produce interference in the androgenic effects responsible for the stimulation of the
development of prostate cancer. Purushottamachar et al[156] developed the first Phamacofore model (common feature based
model or Catalyst HipHop algorithm) for a rationale identification of androgen receptor down-regulating agents (ARDAs). This
was accomplished by generating a 3 D pharmacophore model based on a training set of five well-known ARDAs (Figure 31).
The model containing one hydrophobic group, one aromatic group, and two hydrogen bond acceptors identified 48 and 93
compounds from the NCI (59,652 compounds) and Maybridge (238,819 compounds) databases, respectively. The study
resulted in the identification of six small molecules that were experimentally confirmed as ARDAs (EC50 values 17.5 – 212
μM) and five compounds also exhibited significant human prostate cancer LNCaP proliferation inhibitory activities. These new
scaffolds can be used as leads for rational design of potent ARDAs and hence anti-cancer agents.

 
Fig. (31). Chemical structures of five known androgen receptor down-regulating agents (ARDAs) used to generate the
pharmacophore model
Another useful target for the development of anticancer drugs is the stem cell factor receptor (c-Kit), a member of the
platelet-growth factor receptor (PDGFR), which plays an important role in the processes of differentiation, migration and cell
metabolism growth[157, 158]. The c-Kit kinase functional abnormity is thought to associate with mastocytosis and several
highly malignant human cancers such as gastrointestinal stromal tumours. Given its importance, the kinase activity is tightly
regulated by several mechanisms in normal cells. All of these indicate its potential as a therapeutic target[159]. Based on this
knowledge, Jiang and co-workers[160] investigated a 3 D pharmacophore model of c-Kit inhibitors by using HypoGen
algorithms implemented in the Catalyst software package. By the model used, the best hypothesis (correlation coefficient
0.989) consisted of a hydrogen bond acceptor, two hydrogen bonding donors and a hydrophobic region. From there, the VS of
the China Natural Products Database was conducted to search for potential lead compounds. Hit compounds were subjected to
a filter by using Lipinski's rules and a Docking study to refine and retrieve hits as well as to reduce the rate of false positives.
Then, 28 compounds were purchased or synthesized for in vitro assays against the A549, MCF-7, HepG2 and PC-3 tumor cell
lines in which c-Kit is overexpressed. Two compounds show very low micromolar inhibition potency against PC-3 and HepG2
cell lines respectively. And they were selected for further modification and testing (Figure 32).

 
Fig. (32). Structures of two potential lead compounds obtained in this study (Adapted from Jiang and co-workers[160]).
Another route is the production of DNA methyltransferase inhibitors (DNMTs). These enzymes promote epigenetic changes
by DNA methylation in carbon 5 of cytosine residues. Three DNMTs have been identified in the human genome, DNMT1,
DNMT3a and DNMT3b. DNMT1 is responsible for replicating the pattern of DNA methylation during replication and is
essential for mammalian development. These enzymes are key regulators of gene transcription and their roles in carcinogenesis
have been a topic of considerable interest over the last few years. Therefore, specific inhibition of DNA methylation is an
attractive and novel approach for cancer therapy[161-163]. However, all DNMT inhibitors currently in clinical use are
nonselective cytosine analogs with significant cytotoxic side-effects. Medina-Franco and colleagues[164] performed a
systematic VS from a large collection of natural products as inhibitors of DNMT1. They started using public databases with
more than 89,000 compounds and selected a lead-like subset with approximately 14,000 molecules, making a comparison of
their properties with those of a collection of approved drugs and observed the biological relevance of the subset characteristics
of natural products. In addition, a scaffold analysis of the lead-like subset revealed that, other than benzene, the most frequent
scaffolds in this set are benzopyran-2-one (chromen-2-one or coumarin), indole, and 2-phenyl-1,4-benzopyrone or flavone
(Figure 33). Virtual screening of the lead-like natural product set with a validated homology model of the catalytic domain of
DNMT1 was conducted following a multistep docking-based approach using Glide, Gold, and Autodock. A total of 58
consensus hits with high and similar or better docking scores than the corresponding scores for RG108 were identified. The
consensus hit ZINC04028795 or NCI408488 has reported DNMT1 inhibitory activity[165]. Another hit, compound
ZINC00488396, is a structural isomer of RG108 (Figure 34). The consensus hits are characterized by side chains containing
carboxylic groups which permit hydrogen bonds with amino residues that have an important role in the mechanism of DNA
methylation. The molecular properties of the virtual screening hits are compliant with the biological relevant property space
covered by drugs. A major perspective of this work is the experimental validation of the consensus hits. In addition, the
multistep docking-based strategy used in this work can be implemented to screen other larger in-house or commercial natural
products databases such as the CRC Dictionary of Natural Products or the database implemented in the Drug Discovery Portal.
On a more general level, the results also reinforce the notion that compounds with DNA demethylating activity can be found in
natural sources, including dietary products. The results suggest that natural products are a rich source for demethylating agents
that need to be rigorously characterized in biochemical and cellular assays[164].
The production of stabilizing ligands of c-myc G-quadruplex DNA has been studied, since they inhibit Taq polymerase-
mediated DNA extension through the stabilization of the G-quadruplex secondary structure. This strategy is useful for
production of new anticancer drugs. Most of the drugs that act in this way described in the literature are of synthetic origin
(cationic porphyrins, quindoline derivatives, platinum complexes, isoalloxazines and triarylpyridine compounds) and these
compounds present high toxicity[166-169], but the most potent of the ligands is of natural origin, the telomestatin[170].
Encouraged by the high molecular diversity of natural products, and their potentially, lower toxicity profiles compared to
synthetic compounds, Lee et al[171] developed high-throughput, ligand docking-based virtual screening methods to identify
small molecule leads from natural product databases. In order to develop a high-throughput screening platform for G-
quadruplex binding ligands, a model of the intramolecular G-quadruplex loop isomer of NHE III1 was constructed using the X-
ray crystal structure of the intramolecular human telomeric G-quadruplex DNA (PDB code: 1KF1). This NHE III1 G-
quadruplex isomer model has been previously employed to design quindoline compounds that stabilize c-myc G-quadruplex
DNA. The aforementioned model was utilized in conjunction with in silico hit-to-lead optimization to develop new platinum
(II) Schiff-base complexes with superior c-myc regulatory properties, presumably through stabilization of the NHE III1 G-
quadruplex structure. Using this approach, over 20,000 compounds in a natural product database obtained from AnalytiCon
Discovery GmbH were screened in silico. The continuously flexible ligands were docked to a grid representation of the
receptor and assigned a score reflecting the quality of the complex according to the ICM method [ICM-Pro 3.6-1d molecular
docking software (Molsoft)]. The 5 highest scoring compounds (Figure 28) were tested in a preliminary polymerase stop assay
to assess their ability to stabilize the c-myc G-quadruplex, and fonsecin B emerged as the top candidate and was demonstrated
to inhibit Taq-mediated extension through stabilization of the G-quadruplex secondary structure (IC50 =ca. 20 µM). Fonsecin B
(181) is a naphthopyrone pigment originally isolated from the fungus Aspergillus fonsecaeus in 1974 (Figure 35)[171].

 
Fig. (33). Chemoinformatic analysis of 14,053 compounds in the lead-like set of natural products with Most frequent molecular
scaffolds (cyclic systems). Chemotype identifier, frequency and percentage are shown. The chemotype identifier of scaffolds
also frequent in the non-filtered natural product database is in bold.. (From Medina-Franco et al[164])

 
Fig. (34). Discovery of novel candidate DNMT1 inhibitors by multistep docking-based virtual screening approach. A)
Schematic outline of the screening cascade used in this work. B) Chemical structures of 12 selected consensus hits with
potential DNMT1 inhibitory activity (From Medina-Franco et al[164]).
  
Fig. (35). Chemical structure of Fonsecin B
In this research field, the ubiquitinylation of the unwanted proteins is achieved by the coordinated efforts of the E1
activation, E2 conjugation and E3 ligase enzymes. Ubiquitin-like protein (Ubl) conjugation pathways, including NEDD8 and
SUMO, have also been identified as key players in the protein degradation process[172]. Similar to the ubiquitin pathway,
NEDD8 is first activated by the E1-like NEDD8-activating enzyme (NAE) and is then transferred to Ubc12, the NEDD8-
conjuating enzyme. Finally, the NEDD8 is conjugated to cullin proteins to activate the cullin-RING ubiquitin E3 ligase (CRLs)
activity[173]. Therefore, NAE could be considered as a more selective upstream target, controlling the specific degradation of
proteins regulated by CRLs, such as the cancer related substrates p-IκBα and c-myc. The NAE inhibitor MLN4924[174, 175],
currently in Phase 1 clinical has been shown to be effective against both solid and haematological human cancers. Other work
has shown that the natural product-like 6,6’’-biapigenin dose-dependently inhibited NAE activity in cancer cells and suppressed
the growth of a human epithelial colorectal adenocarcinoma cell line[176]. Zhong and co-workers[177] reported herein the
identification of the dipeptide-conjugated deoxyvasicinone derivative (182) (Figure 36) as an inhibitor of NAE by virtual
screening of over 90,000 compounds from the ZINC database of natural products. Molecular modelling results suggested that
182 may be a non-covalent competitive inhibitor of NAE by blocking the ATP-binding domain. Compound 1 was able to
inhibit NAE activity in both cell-free and cell-based assay with potencies in the micromolar range and selectivity over
analogous E1 enzymes UAE and SAE. They envisage that the identification and molecular docking analysis of this bioactive
scaffold as an NAE inhibitor would provide the scientific community with useful information in order to generate more potent
analogues.

 
Fig. (36). Chemical structures of small molecule NAE inhibitors 182, 6,6’’-biapigenin (183) and MLN4924 (184). (From
Zhong and co-workers[177]).
Nicotinamide phosphoribosyltransferase (NAMPT), an enzyme taking part in main NAD biosynthetic pathway, is an
attractive target for anticancer therapy. Yi and colleagues[178] developed a study where they generated 3 D-QSAR models and
performed virtual screening techniques. First, they produced a Phamacophore model through the Catalyst / HypoGen algorithm.
Hypo 1 was validated through test set prediction and Fischer’s randomization methodologies. Then they screened some public
compound libraries (Asinex, Ibscreen and Natural products database) using Hypo1 for a 3 D query. The screened hits were
further refined by Lipinski’s rule of five, ADMET properties as well as molecular docking studies. Finally, six molecules
(Figure 37) with diverse scaffolds exhibited the right pharmacophore features and good binding modes between the receptor
and ligands and were selected as possible candidates against NAMPT for further study.
Protein tyrosine kinases (PTKs) play important roles in the processes of proliferation, differentiation and progression of cell
cycle, as well as in angiogenesis and inhibition of the apoptosis process[179]. Thus, an abnormal functioning of these enzymes
can lead to the development of several types of cancers. The human epidermal receptor (HER) and Epidermal growth factor
receptor (EGFR, ErbB) family are the most studied representatives of this class of targets and it has been observed that the
exaggerated expression, amplification and mutation of EGFR and other ErbB family receptors are directly related to the
malignancy of some cells and thus are of great interest as targets for the discovery of anticancer drugs. There are various reports
that EGFR is a cause of mostly non-small cell lung cancer (NSCLC), while ErbB2 is implicated in breast cancer[180, 181].
Sawatdichaikul and co-workers[182] performed a VS with 29,960 compounds from the Chemiebase medicinal compound
database (http://chemiebase.ku.ac.th), contains a collection of Thai natural compounds, were virtually screened against the
EGFR-TK using AutoDock 4.0, GOLD and GLIDE (XP). The results revealed eight potential hits: CAS nos. 104096-45-9,
112649-21-5, 113866-89-0, 142608-98-8, 142608-99-9, 144761-33-1, 155233-17-3 and 80510-05-0. These compounds have
been reported to show anticancer activities in the literature. With the help of SiMMap and MOE interaction analysis, the
protein–ligand interaction patterns between the functional groups of these compounds and the binding pocket residues were
analyzed. Hydrogen bonding and hydrophobic forces are the main components of the interactions of these hits, similar to those
observed for the known inhibitors erlotinib, gefitinib and AEE. The physicochemical filter indicates that compounds CAS nos.
104096-45-9 and 144761-33-1 are likely to be potential leads in the drug discovery process (Table 3 and Table 4).

 
Fig. (37). Structures of six potential inhibitors of NAMPT.
Table 3. Chemical name(s) and plant source(s)

Common name(s) of plant

CAS no. (Compound) Structure name Scientific name(s) of plant sources(s)
source(s)

104096-45-9 (191) N/A Diospyros mollis Griff, Diospyros ehretioides wall.ex G.Don Ebony tree

112649-21-5 (192) Garcinone E Garcinia mangostana L. Mangosteen

113866-89-0 (193) AC 5-1 Annona squamosa L., Citrus hystrix DC. Custard apple and makrut lime

142608-98-8 (194) Exiguaflavanone A Artemisia indica Wild, Sophora exigua Legume

142608-99-9 (195) Exiguaflavanone B Artemisia indica Wild, Sophora exígua Legume

144761-33-1 (196) 1,6-Heptadiene-3,5- Curcuma aromatica Salisb, Curcuma xanthorrhiza Wild turmeric
dione

155233-17-3 (197) Caloxanthone B Calophyllum inophyllum Ballnut

80510-05-0 (198) Euchrestaflavanone A; Azadirachta indica A.Juss Neem

Lespedezaflavanone B
N/A not available

 
Fig. (38). structures of the two final hit molecules, ZINC08234189 (209) and ZINC03871891(210)[183]
Table 4. Score of each docking method and total electrostatic energy values.

CAS no. ADT4 GOLD

Structures name ΔGa ADT Oechemscore KCSscore ΔGa GOLD XPscore

(Compound) (kcal/mol) (kcal/mol) (kcal/mol (kcal/mol

(199) 183321-74-6 Erlotinib -7.71 -7.86 28.65 -7.42 -8.32

(200) 104096-45-9
N/A* -8.93 -9.62 32.81 -8.23 -9.22

(201)
112649-21-5
Garcinone E -10.07 -10.24 34.18 -8.41 -9.13

(202)
113866-89-0
AC 5-1 -8.27 -9.97 34.18 -9.25 -8.85

(203)
142608-98-8
Exiguaflavone A -8.05 -10.18 31.20 -7.60 -9.43

(204)
142608-99-9
Exiguaflavone B -8.38 -10.38 33.43 -8.23 -9.53

(205)
144761-33-1
1,6-Heptadiene-3,5-dione -8.24 -8.30 33.02 -9.05 -8.48

(206)
155233-17-3
Caloxanthone B -10.24 -9.60 35.07 -8.48 -8.57

(207)
80510-05-0 Euchrestaflavone A; -9.24 -8.85 35.07 -8.68 -8.61
(208) Lespedezaflavone
N/A* not available
a
Estimated binding energy from docking programs

In order to obtain inhibitors of c-Met receptor tyrosine kinase, Aliebrahimi and colleagues[183] performed a consensus
docking approach using Autodock Vina and Autodock 4.2 to virtual screen Naturally Occurring Plant-based Anti-cancer
Compound-Activity-Target (NPACT) database against active and inactive conformation of c-Met kinase domain structure. The
initial database had 1574 compounds and were subjected to Lipinski and Veber filters, which reduced the database to 738
compounds. Then, the compounds were submitted to docking and molecular dynamics (MD) procedures providing 98
candidates who after ranking allowed the discovery of two hits (ZINC08234189 and ZINC03871891) (Figure 38). Using a 20
ns MD simulation, the stability of each complex was evaluated. Their results showed that both protein and ligand backbone of
ZINC08234189 achieved stability after 5 ns. Nevertheless, ZINC03871891 experienced stable conformation in each case
during the entire simulation process. Given that hydrogen bond with residues of the hinge region (P1158, M1160) is a hallmark
of kinase domain inhibitors, their analysis showed that both hit molecules formed hydrogen bonds with key residues in the
active form. In summary, based on hydrogen bond analysis and MM-PBSA studies, they predicted that ZINC08234189 is a
plausible inhibitor for the active state of c-Met. In line with their docking results, ZINC03871891 showed more effectiveness
toward active c-Met kinase domain than the inactive form due to higher binding energy. The results disclosed here can be
useful to overcome Met-addicted cancers.
Another important representative of this class is the ErbB3, this receptor plays an important role in mediating cellular
growth and differentiation. Recent research works identified that CD74-NRG1 fusions lead to overexpression of the EGF-like
domain of NRG1, and thus activate ERBB3 and PI3K-AKT signaling pathways. The fusion was detected in lung
adenocarcinomas, and served as an important oncogenic factor for ErbB3 driven cancers. Guo et al[184] applied a sequential
virtual screening directed to this receptor using databases of natural products (~ 150,000 compounds) and Chinese traditional
medicine compounds (~ 30,000) and led to identification of a group of small molecular compounds potentially capable of
blocking ErbB3. Six small molecular compounds were selected for in vitro analysis (Table 5). Five of these molecules
significantly inhibited the growth of A549 cells. Among them, compound VS1 is the most promising one with IC50 values of
269.75 μM, comparing to the positive control of nimustine hydrochloride with IC50 values of 264.14 μM. With good specificity
and predicted ADMET results, their results support the feasibility by using a pharmacophore of the compound VS1 for
designing and optimization of ErbB3 inhibitors.
Table 5. Structures and bioassay data of the compounds.

A549 Cell linea MRC-5 Cell lineb

Compound Structure
IC50 (µ M) IC50 (µ M)

VS1 269,75 305,14

(211)

VS2 1162,71 737,52

(212)

VS3 150,48 119,66

(213)

VS4 612,34 369,37

(214)

VS5 540,63 466,30

(215)

VS6 541,54 330,92

(216)

Nimustine Hydrochloride 264,14 436,94

(217)

a
A549 cells are human lung adenocarcinoma epithelial cells.
b
MRC-5 cells are human lung normal cells.
 In this field of protein kinases other important representatives are polo-like kinase 1 (PLK1), cyclin-dependent kinase 1 and
2 (CDK1) and (CDK2). Shi and colleagues[185] developed a high-throughput virtual screening study aimed at finding new
inhibitors for these enzymes. They computationally constructed the appropriate global human protein–protein interaction
network with data from online databases, and then modified it into a kinase-related apoptotic protein–protein interaction
network. Subsequently, they identified several kinases as potential drug targets according to their differential expression
observed by microarray analyses. Then, they predicted relevant microRNAs, which could target the abovementioned kinases.
Ultimately, they virtually screened a number of small molecule natural products from Traditional Chinese Medicine
(TCM)@Taiwan database and identified a number of compounds that are able to target polo-like kinase 1, cyclin-dependent
kinase 1 and cyclin-dependent kinase 2 in HeLa cervical carcinoma cells. Figure 39 shows the most promising molecules
obtained for each target.

 
Fig. (39). Best small molecule compounds targeting PLK1, PLK1 PBD, CDK1 and CDK2 in HeLa cells (CDK1, cyclin-
dependent kinase 1; CDK2, cyclin-dependent kinase 2; PLK1, polo-like kinase 1; PLK1 PBD, polo-like kinase 1 polo-box
domain)[186].
Janus kinases (JAKs) play an important role in intracellular signal transduction via cytokine and growth factor receptors. In
mammals, the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway is the principle signaling
mechanism for cell proliferation, differentiation, cell migration, and apoptosis. The JAK2V617F mutation not only results in
constitutive protein tyrosine kinase (PTK) activity, but also confers factor-independent growth and changes in metabolic
activity to various hematopoietic cell lines. Consequently, JAK2 represents an attractive target for the development of anti-
cancer therapeutics. Thus, Zhong et al[187] carried out a structure-based high-throughput virtual screening from a natural
product database containing over 20,000 compounds. Molecular docking was performed by using the ICM-Pro 3.6-1d program.
Emodic acid (1) and 6-chloroemodic acid (2) were identified as useful scaffolds for the future development of novel JAK2
inhibitors. Low-energy binding conformations of 222 and 223 in the JAK2 PTK domain were generated by virtual ligand
docking and were found to overlap considerably with the binding pose of CMP6(224), a known JAK2 inhibitor (Figure 40).
Compounds 222 and 223 displayed low micromolar efficacies against JAK2 enzyme activity and JAK2 autophosphorylation in
human erythroleukemia cells, and inhibited STAT3 DNA binding activity in a human hepatocarcinoma cell line (HepG2).

 
Fig. (40). Structure of compounds 222, 223 and CMP6(224)
 Cancer cells have been reported to acquire at least ten different pro-survival capabilities to dictate malignant growth among
which the MAPK signal plays a significant role in sustained angiogenesis, limitless replicative potential and evasion of
apoptosis, offering hopes that inhibition of this signaling could have an influence on down-regulation of pro-survival pathways.
Accumulating data demonstrates that Raf serine/threonine kinase is the most characterized downstream effector of the central
signal transduction mediator Ras. A most prevalent B-Rafv600e mutant in Raf kinase family exhibits elevated kinase activity and
results in constitutive activation of the MAPK pathway, thus making it a promising drug target for cancer therapy. Wang et
al[188] built a three-dimensional kinase domain structure of B-Rafv600e by Swiss-Model server (http://swissmodel.expasy.org/),
using the structure of 3SKC as template. They also built a screening library containing 6818 small molecular natural products
from TCM@Taiwan subset in the ZINC database. Virtual screening was applied to identify its potential inhibitors.
Subsequently the 25 ns molecular dynamic (MD) simulations, ZINC38541768, ZINC38541767, and ZINC12496469 (Figure
41) were identified as B-Rafv600e potential inhibitors in a DFG-in conformation. Furthermore, according to the molecular
mechanics/generalized born surface area (MM/GBSA) method, these three small molecules exhibit similar and good binding
affinity toward B-Rafv600e (−38.76 kcal mol−1, −42.60 kcal mol−1, and −39.04 kcal mol−1). At the same time, several critical
residues, such as I463, V471 in the P-loop, and DFG motif residue D594 within the A-loop, are also well clarified. All these
results may not only indicate some future applications of inhibitors targeting B-Rafv600e, but also benefit B-Rafv600e harboring
cancer patients.

 
Fig. (41). Structures of compounds selected as potential B-Rafv600e inhibitors
Chronic myeloid leukemia (CML) is a myeloproliferative disease that arises due to the reciprocal chromosomal
translocation of abl gene from chromosome 9 to bcr gene on chromosome 22. The resulting bcr-abl fusion gene and the short
chromosome generated thereby is called ‘Philadelphia chromosome. The fusion gene product - Bcr-Abl protein - exhibits
constitutive tyrosine kinase activity and phosphorylates and activates downstream signalling pathways such as Ras and MAP
kinase, PI3 Kinase, Crkl, Myc, Jak-Stat involved in cell proliferation and survival. In addition, Bcr-Abl inhibits DNA repair
thus leading to progressive blast phase of CML. The constitutive activity of Abl kinase critically promotes CML pathogenesis.
Even though targeting the fusion gene product Bcr-Abl protein is a successful strategy, development of drug resistance and
drug intolerance are currently the limitations for Bcr-Abl targeted CML therapy. Parcha et al[189] developed natural Bcr-Abl
inhibitors. They performed virtual screening (VS) of ZINC natural compound database by docking with Abl kinase using Glide
software. Two natural inhibitors ZINC08764498 (228) and ZINC12891610 (229) were selected by considering their high Glide
docking score and critical interaction with the hinge region residue Met-318 of Abl kinase. The reactivity of the two molecules
was assessed computationally by Density Functional Theory calculations. Further, the conformational transition, hydrogen
bond interactions and the binding energies were investigated during 10 ns molecular dynamics simulation of the Abl-hit
complex. When tested in vitro, hit1compared to hit2 (Figure 42) showed selective inhibition of cell proliferation and induction
of apoptosis in Bcr-Abl positive K-562 leukemia cells. Their results demonstrate that ZINC08764498, a coumarin derivative
identified through VS, is a potential natural inhibitor for the treatment of CML.
Other hormone receptors that have important role as targets for anticancer drugs are Estrogen Receptors (ERs). There are
two subtypes of this receptor, ER-α (is distributed in breast, uterus and bone) and ER-β (is expressed in prostate, lungs, central
nervous system and cardiovascular system). It has been shown that ERs receptors are responsible for several diseases regulated
by hormones, among them, mainly breast cancer, where some modulators of these receptors are used clinically. However, some
clinical observations suggest that using mimetic substances from these steroids increases the risk in patients of developing
breast and prostate cancer. Thus, there is interest in the development of Selective Estrogen Receptor Modulators (SERMs) that
may exert a specific effect on particular tissue, ensuring the possibility to selectively exert agonistic or antagonistic estrogen-
like action in various tissues. One such example is Raloxifene, a marketed ER modulator has estrogenic actions on bone and
anti-estrogenic actions on the uterus and breast; thus, it can be used to treat osteoporosis while decreasing the risk of uterus and
breast cancer[190, 191]. Cao et al[192] built an in-house natural product database (NPD) for drug screening. This NPD
contained over 4,000 natural products isolated from 100 medicinal plants by their group. Therefore, they adopted structure-
based virtual screening for the in-house NPD against both ERα and ERβ. All the natural products in their database were
virtually docked to ligand binding domain (LBD) of both ERα (PDB: 3ERT14) and ERβ (PDB: 1NDE15). The crystal
structures in this study were retrieved from Protein Data Bank (PDB) and prepared by ‘Protein Preparation Wizard’ workflow
in Maestro 9.0 graphical user interface of the Schrödinger program suite. All the molecular docking simulation was performed
with default parameters by using Glide 5.5 in Schrödinger program suite and ranked by GlideScore. In order to rationalize SAR
and understand the types of modulation, molecular docking simulation was applied to obtain the putative binding poses. Major
difference between agonists and antagonists was due to the conformation of C-terminal helix, helix 12 (H12). When the
hydrophobic LBD pocket was bound with agonists, H12 orientated vertically to H11. Conversely, the binding with antagonists
will make H12 occupy the region where co-activator protein is expected to bind, which led to the prevention from dimerization
and transcription, and thus, exerted antagonistic effect. Likewise, our discovered ER agonists conformed to the rules that ER
agonists had small and straight shape, mimicking endogenous steroid E2 and occupying the binding pocket. For compound 233,
the di-phenol ring stretched into a hydrophobic cavity and interacted with residues Leu 343, Phe 356, Leu 301 and Leu 339
through hydrophobic Van der Waals contacts. Di-phenol fragment of 233 formed two pairs of essential hydrogen bonds with
Arg 346 and Glu 305, which mimicked the hydroxyl group attached to the A-ring of E2, forming the water-mediated hydrogen
bond network with the amide group of Arg 346 and the carboxyl group of Glu 305. The presence of those binding features
played an important role in maintaining activity of ER modulators, which could also illustrate why their discovered agonists
kept these interactions. Similar to another interaction between the D-ring of E2 and the receptor, another phenolic group
attached to monophenolic ring of 233 could also form hydrogen bond with His 475, which could contribute to the potency of
these agonists. Thus, eleven compounds were identified as estrogen receptor modulators (Figure 43). Among them, three
compounds were confirmed as ER agonists and eight compounds were confirmed as ER antagonists by yeast two-hybrid (Y2H)
assay, with EC50 values ranging from several micromolar to 100 micromolar. In this study, a novel series of cycloartane
triterpenoids isolated from Schisandra glaucescens Diels was found to have ER antagonistic effect, the most potent antagonist
of which exhibited activity with EC50 value of 2.55 and 4.68 µM for ERα and ERβ, respectively. Moreover, the types of
modulation and subtype selectivity were also investigated through molecular docking simulation. Pang et al[193] also
developed Ligand-based machine learning methods and structure-based molecular docking for the identification of ERα
antagonists from an in-house natural product library. 162 compounds were predicted as ER antagonists and were further
evaluated by molecular docking. According to docking score, they selected eight representative compounds (Figure 44) for both
ERα competitor assay and luciferase reporter gene assay. Genistein, daidzein, phloretin, ellagic acid, ursolic acid, (−)-
epigallocatechin-3-gallate, kaempferol, and naringenin exhibited different levels for antagonistic activity against ERα.

 
Fig. (42). Structures of the compounds selected that showed selective inhibition of cell proliferation and induction of apoptosis
in Bcr-Abl positive K-562 leukemia cells[189]
  
Fig. (43). Structures of natural estrogen receptor modulators discovered (Adapted from Cao et al[192]).

 
Fig. (44). The investigation of the binding modes in ERα of 6 different skeleton structures. They were genistein (243),
naringenin (244), EGCG (245), phloretin (246), ellagic acid (247), and ursolic acid (248), which belong to isoflavone, flavone,
catechin, dihydrochalcone, polyphenol, and triterpenoid (From Pang et al[193]).
Mutations in Protein Tyrosine Phosphatase (PTP) PTPN11 (SHP2) that cause hyperactivation of the SHP2 catalytic activity
are identified in patients with Noonan Syndrome and in various types of leukemias, including Juvenile Myelomonocytic
Leukemia (JMML), Myelodysplastic Syndrome, B cell Acute lymphoblastic leukemia / lymphoma, and Acute Myeloid
Leukemia (AML). These mutations have also been found in adult AML as well as in some solid tumors, such as lung cancer,
colon cancer, melanoma, neuroblastoma, and hepatocellular carcinoma. SHP2 encoded by the PTPN11 gene is a non-PTP
receptor containing two N-terminal Src Homology-2 (SH2) domains, one PTP domain and one C-terminal tail. In the basal
state, the N-terminal SH2 domain (N-SH) blocks the catalytic site in the PTP domain until tyrosine-phosphorylated partners
bind to SHP2. PTPN11 mutations found in Noonan syndrome, leukemias, and tumors disrupt the self-inhibition between N-
SH2 and PTP domains, leading to gain-of-function by allowing constitutive access to the catalytic site of the enzyme. SHP2 is
widely expressed, and involved in multiple cell signaling processes, such as Ras-Erk, PI3K-Akt, Jak-Stat, and NF-κB pathways.
Thus, specific and potent SHP2 inhibitors with pharmacological properties are greatly needed for the research on SHP2
functions and the development of new drugs that ultimately serve the treatments for PTPN11-associated malignancies[194-
196]. Liu and co-workers[197] identified an inhibitor of Oncogenic Protein Tyrosine Phosphatase SHP2 (PTPN11). They
screened a database of natural products using CADD screening based on the 3 D structure of the SHP2 protein. A structural
alignment between SHP2 and the highly related SHP1 phosphatase was performed in order to search for a potential drug-
docking site in the SHP2 catalytic domain that would be specific for that protein. Not surprisingly, the catalytic pockets were
almost identical in SHP2 and SHP1, except four residues, i.e. Lys358, Arg362, Lys364, and Ser 365 in SHP2, which
correspond to Arg352, Lys356, Arg358, and Asn359 in SHP1. This led to the identification of a putative site in the entrance of
the SHP2 catalytic cleft that appeared to be different from that in SHP1 in both structure and amino acid composition. Ligands
bind to those amino acids which in the periphery of the active center through hydrophobic stabilization or hydrogen-bond (H-
bond) interactions could mask the catalytic signature residues (Cys459 and Ser460), and this represents a logical place for
active site-directed screening. So, In silico screening was carried out against a library of ∼10,000 small molecular weight
natural products targeting the peripheral site in the entrance of the SHP2 catalytic cleft, and the primary screening should meet
the following criteria: (1) The nearest distance from a compound must be within 5 Å of residues Cys459 or Ser460 in SHP2 and
beyond 5 Å of residues Cys453 or Ser454 in SHP1; (2) The interaction energy between a compound and one of the
hydrophobic residues in SHP2 must be more than 1KJ/mol. The obtained complex structures were refined using Embrace
method of MacroModel for secondary screening. After the primary screening with Glide Module and the secondary screening
with MacroModel, top 200 molecules with interaction energy less than -50 kJ/mol were selected for further analyses, such as
binding modes and diversity analysis. The further selection process was mainly based on the distributions of three indexes of
each compound including principle moment of inertia, dipole moment, and molecular weight. The indexes were normalized and
divided into ten parts, and each dot represented a compound in the grid system of three indexes. Compounds in the same lattice
were clustered into a group. One or two compounds were selected from each group. As a result, a total of 43 natural products
were selected and subsequently obtained from commercial sources and subjected to experimental testing. This effort led to the
identification of Cryptotanshinone (Figure 45) as an inhibitor of SHP2. Cryptotanshinone inhibited SHP2 with an IC50 of 22.50
μM. Fluorescence titration experiments confirmed that it directly bound to SHP2. Enzymatic kinetic analyses showed that
Cryptotanshinone was a mixed-type and irreversible inhibitor. This drug was further verified for its ability to block SHP2-
mediated cell signaling and cellular functions. Furthermore, mouse myeloid progenitors and patient leukemic cells with the
activating mutation E76K in PTPN11 were found to be sensitive to this inhibitor. Cryptotanshinone is used to treat
cardiovascular diseases in Asian countries, this drug has a potential to be used directly or to be further developed to treat
PTPN11-associated malignancies.

 
Fig. (45). Chemical structure of Cryptotanshinone
Among the enzymatic targets involved in the initiation and treatment of cancer is the Mitogen- and stress-activated kinase 1
(MSK1), that is a nuclear serine/threonine protein kinase that acts downstream of both extracellular signal-regulated kinases
and p38 mitogen-activated protein kinase in response to stress or mitogenic extracellular stimuli. Increasing evidence has
shown that MSK1 is closely associated with malignant transformation and cancer development. MSK1 should be an effective
target for cancer chemoprevention and chemotherapy. However, very few MSK1 inhibitors, especially natural compounds,
have been reported. Liu and colleagues[198] used virtual screening of a natural products database and the active conformation
of the C-terminal kinase domain of MSK1 (PDB ID 3KN) as the receptor structure to identify chrysin and its derivative,
compound (250), as inhibitors of MSK1. Compared with chrysin, compound (250) more strongly inhibited proliferation and 12-
O-tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic transformation of JB6 Pþ cells with lower cytotoxicity. Western
blot data demonstrated that compound 69407 suppressed phosphorylation of the MSK1 downstream effector histone H3 in
intact cells. Knocking down the expression of MSK1 effectively reduced the sensitivity of JB6 Pþ cells to compound 69407.
Moreover, topical treatment with compound 69407 before TPA application significantly reduced papilloma development in
terms of number and size in a two-stage mouse skin carcinogenesis model. The reduction in papilloma development was
accompanied by the inhibition of histone H3 phosphorylation at Ser10 in tumors extracted from mouse skin. The results
indicated that compound (250) exerts inhibitory effects on skin tumorigenesis by directly binding with MSK1 and attenuates
the MSK1/histone H3 signaling pathway, which makes it an ideal chemopreventive agent against skin cancer (Figure 46).

 
Fig. (46). A) Chemical structure of compound (250 (From Liu et al[198]).
Microtubules play an important role in the process of cell division. They are formed by α and β tubulins arranged non-
covalently in 13 longitudinally arranged protofilament structures in a head-tail formation to form a closed tube. α/β-tubulin duo
belongs to the broad class of GTP binding proteins and have a 40% sequence identity. Both the subunits show a slight structural
variation from the conventional GTPases, whereas the functional domains are well conserved. The N-terminal domain includes
a nucleotide binding site comprising of six parallel β-strands (S1-S6) altered with the same number of α-helices (H1-H6). The
intermediate domain has mixed β-strands (S7-S10) and three helices (H8-H10). This region is connected to the N-terminal
domain by the helix H7 and contains a hydrophobic taxol binding pocket. The C-terminal domain, which is a binding surface
for motor protein, is formed by two antiparallel helices (H11-H12) crossing over the two other domains. Apart from these
structural elements, the tubulin protein has three loops H1-S2, M and S loop. Both the monomeric subunits exhibit lateral and
longitudinal contacts along with head to tail protofilament formations. The major lateral interactions occur between the H1-S2
loop and helix H3 with the M-loop of the adjacent protofilaments. Tubulin comprises of three distinct drug binding domains
which includes taxol, vinca alkaloid and colchicine binding sites. In spite of their different binding sites, they are categorically
involved in arresting mitotic spindle formation. The drugs against β-tubulin are mainly derived from terrestrial plants and
marine resources[199-201]. Kumar, Gadewal and Mohammed[202] investigated the role of 517 compounds available in the
Seaweed Secondary Metabolites Database (SWMD), identified by virtual screening, against modeled human β-tubulin. For the
construction of the Homology Modeling, a query sequence of human β-tubulin was retrieved from the Swiss-Prot protein
database (accession number Q9BVA1.1). In the absence of the crystal structure of human β-tubulin, BLAST search was done
against Protein Data Bank. PDB ID: 1TUB obtained from the Protein Data Bank was taken up as a template for modelling of
the query sequence. Using the single template protocol of Modeller9v10, 30 structures were generated. Amongst these, the
model with the least DOPE score (Discrete optimized protein energy) was considered for energy minimization with 500
iterations using steepest descent in SwissPdb Viewer. The optimized structure was further validated using SAVES server
(http://nihserver.mbi.ucla.edu/SAVES/). In continuation, drug like compounds were derived from the SWMD database
(www.swmd.co.in) which comprises of 517 entries mostly from the red algae of the genus Laurencia (Ceramiales,
Rhodomelaceae). The MOL file (2 D structure) of each compound was prepared with Ligprep tool (Schrodinger 9.2) to
generate multiple conformers at physiological pH. The availability of different conformers in their native state and exploration
of their conformational search space by altering torsion angle values, led to the detection of 2382 conformers from 517
compounds. For docking studies, β-tubulin protein was prepared using ‘protein preparation wizard’ of Schrodinger 9.2 at
physiological pH. All the 2,382 generated conformers were docked at the taxol binding site of β-tubulin along with taxol,
eleutherobin and Sarcodictyin A. The binding site was generated using receptor-grid generation protocol of Glide by selecting
the centroid of taxol for grid file generation. A scaling factor of 1.0 was set to van der Waals (VDW) radii for the atoms of
residues that presumably interact with ligands and the partial atomic charge was set to less than 0.25. The initial screening of
2,382 conformers was done using Glide HTVS (High Throughput Virtual Screening) which filtered 1,955 conformers with
better spatial restraints. By keeping the cut off glide score above -4.0 kcal, 1955 conformers were brought down to 999
conformers. These selected conformers were subjected to Glide extra precision (XP) mode of docking which does extensive
sampling and provides reasonable binding poses. The pharmacokinetic properties of 999 conformers were assessed using
Qikprop to calculate the ADME properties of the compounds which includes physically significant descriptors and
pharmaceutically relevant properties. The Qikprop properties considered for analysis include glide score, glide energy
(kcal/mol), molecular weight (g/mol), molecular volume (Å), PSA, HB donors, HB acceptors, rotatable bonds, QP logP(o/w),
QP logS, QP PCaco, QP logHERG, QP PMDCK and % human oral absorption. All the conformers of lead compounds
(RL381, RL366, RL376 and RG012) when docked at the taxol binding site showed better interactions with the H1-S2 loop and
M-loop which are actively involved in lateral interactions of tubulins and also with the helix H7 which is the connecting link
between N-terminal and intermediate domain. Important residues involved in polar interaction by these lead compounds were
D224, H227, R276, R282 and R359 which closely mimicked the interaction of taxol with β-tubulin. They then attempted to
calibrate the available molecules based on their Lipinski rule, binding affinity and other descriptor based comparison to identify
potential lead molecules which could be used as drugs against human β-tubulin (Figure 47).
  
Fig. (47). Two dimensional structures of the four lead molecules: (251), (252), (253) and (254). (From Kumar, Gadewal and
Mohammed[202])
Mutations in β-tubulin structure give resistance to chemotherapeutic agents. Notably, mutations at R306C, F270 V, L217R,
L228F, A185T and A248V positions in β-tubulin give high resistance for paclitaxel binding. To discover novel inhibitors of β-
tubulin from natural sources, particularly alkaloids, Verma et al[203] employed a virtual screening approach to find potent lead
molecules from the Naturally-occurring Plant-based Anti-cancer Compound-activity Target (NPACT) database. Alkaloids have
great potential to be anti-cancer agents. Therefore, they have screened all alkaloids from a total of 1574 molecules from the
NPACT database for their study. Initially, Molinspiration and DataWarrior programs were utilised to calculate
pharmacokinetics and toxicity risks of the alkaloids, respectively. Subsequently, AutoDock algorithm was employed to
understand the binding efficiency of alkaloids against β-tubulin. The binding affinity of the docked complex was confirmed by
means of an intermolecular interaction study. Moreover, oral toxicity was predicted by using ProTox program. Further,
metabolising capacity of drugs was studied by using SmartCYP software. Additionally, scaffold analysis was done with the
help of scaffold trees and dendrograms, providing knowledge about the building blocks for parent-compound synthesis.
Overall, the results of the computational analysis indicate that isostrychnine (255) (Figure 48), obtained from Strychnosnux
vomica, satisfies pharmacokinetic and bioavailability properties, binds efficiently with β-tubulin. Thus, it could be a promising
lead for the treatment of paclitaxel resistant cancer types.

 
Fig. (48). Structure of isostrychnine (255).
Searching for new vinca domain tubulin inhibitors, Lone et al[204] performed a quantitative pharmacophore model based
on vinblastine analogues to identify the natural compounds possessing similar properties and to incorporate all the
complimentary features responsible for the activity. The constructed 3 D-QSAR model suggested that the aliphatic regions of
vinblastine scaffold should be devoid of HBD’s and must possess electron withdrawing groups in order to enhance the
biological activity. The R-group elaboration was linked to HBD and positive ionic centers. Receptor-ligand model revealed that
the central core having fused aromatic rings along with positively ionizable site is vital for enhancing the receptor ligand
interaction. However, the peptide-like features should be avoided for designing new derivatives. The stability of hit molecule
ZINC20758009 (256) (Figure 49) within the biological environment throughout the length of simulation run was confirmed via
MD simulations. In principle, the adopted virtual screening experiment is capable of scaffold hopping based on C20 substituted
vinblastine analogues. Therefore, the present approach can be employed for the identification of hit molecules rather than the
optimization of lead. In addition, the study also highlights the essential features accountable for enhancing the activity and
prioritizes the molecules that can inhibit the vinca binding site.

 
Fig. (49). 3 D view and 2 D interaction maps of the compound ZINC20758009 (256) at vinca binding site of tubulin[204]
In the constant search for new strategies to combat cancer, the mammalian target of rapamycin (mTOR), a member of the
phosphatidylinositol 3-kinase (PI3K) -related kinase family, is a promising target for the search for new drugs, functions,
regulate cell growth, proliferation and metabolism. The main ones are mTORC1 and mTORC2. The biological function of
mTORC1 is mainly regulated by inducing the phosphorylation of both eukaryotic translation initiation factor 4E-binding
protein 1 (4EBP1) and ribosomal S6 kinase 1 (S6K1), while mTORC2 mainly phosphorylates serine/threonine protein kinase
AKT at the serine residue S473. mTOR is a key node in PI3K/Akt/mTOR pathway and unusual mTOR signaling can promote
the development and progression of various cancers. Thus, developing clinical drugs based on mTOR kinase inhibition is of
great interest. Chen et al[205] proposed an in silico protocol to identify mTOR inhibitors from the ZINC natural product
database. A protocol flowchart is illustrated in Figure 34 and includes the following steps: (1) three dimensional quantitative
structure–activity relationship (3 D-QSAR) pharmacophore models were generated from known mTOR inhibitors and validated
by several methods, and then pharmacophore-based virtual screening was performed, (2) the Glide docking program was
employed to validate the compounds selected by the pharmacophore mapping procedure, (3) molecular mechanics/generalized
born surface area (MM-GBSA) analysis was utilized to refine the virtual hits, and (4) the compounds with the best MM-GBSA
scores and ideal-binding models were confirmed as the final hits. This protocol allows ligand- and structure-based virtual
results to be refined for the identification of novel mTOR inhibitors. The results showed that the model can predict the mTOR
inhibition activity of the tested compounds. Virtual screening was performed based on the best pharmacophore model, and the
results were then filtered using a molecular docking approach. In addition, molecular mechanics/generalized born surface area
analysis was used to refine the selected candidates. The top 20 natural products were selected as potential mTOR inhibitors, and
their structural scaffolds could serve as building blocks in designing druglike molecules for mTOR inhibition.
Signal transducer and activator of transcription (STAT) proteins are a family of transcription factors that mediate gene
expression in response to cytokines and growth factors. STAT3 regulates a variety of genes involved in cell proliferation,
differentiation, apoptosis, angiogenesis, metastasis, inflammation, and immunity. Dysregulated STAT3 activity has been
implicated in the development of a variety of solid and hematological tumors, including leukemia, lymphomas, and head and
neck cancer. Additionally, elevated STAT3 levels have been associated with poor prognosis of certain cancers[186, 206, 207].
Liu et al.[207] Carried out a Structure-based virtual screening for the identification of natural products as STAT3 dimerization
inhibitors. The virtual screening campaign furnished 14 hit compounds (259-272) (Figure 50) from a database of over 90,000
natural product and natural product-like compounds, from which compound C1 emerged as a top candidate. Compound 1
inhibited STAT3 DNA-binding activity in vitro and attenuated STAT3-directed transcription in cellulo with selectivity over
STAT1 and with comparable potency to the well-known STAT3 inhibitor S3I-201. Furthermore, compound C1 inhibited
STAT3 dimerization and decreased STAT3 phosphorylation in cells without affecting STAT1 dimerization and
phosphorylation. Compound C1 also exhibited selective anti-proliferative activity against cancer cells over normal cells in
vitro. Molecular docking analysis suggested that compound C1 might putatively function as an inhibitor of STAT3
dimerization by binding to the SH2 domain.
Another strategy for the production of anticancer drugs is the modulation of the production of Reactive oxygen species
(ROS). Catalase is a heme-enzyme and ubiquitous present in living organisms, the function of catalase is to destroys hydrogen
peroxide (H2O2) to two molecules of water and one molecule of oxygen, which is an important enzyme to against oxidative
damage in cell and tissues. ROS (Reactive Oxygen Species) were produced from aerobic organism, that including superoxide
anion, hydroxyl radical, and hydrogen peroxide. In normal metabolism, ROS are scavenged by antioxidant enzyme such as
superoxide dismutase, glutathione peroxidase and catalase. Hydrogen peroxides are linked to cellular damage. In some cases of
catalase deficiency, the increased levels of hydrogen peroxide and free radicals concentrations contribute to oxidative damage
in DNA, proteins, and cells and are implicated cell damage and lead to inflammation, aging and cancer. Huang and
colleagues[208] conducted a study to look for new natural products that prevent inactive catalase generation. The world largest
traditional Chinese medicine (TCM) database (http://tcm.cmu.edu.tw/) were employed to this study, which combined with
high-throughput virtual screening and molecular dynamics (MD) simulation to investigate potent nature compounds for keeping
catalase active. They found the two natural products, Hesperidin (273) and 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside
(THSG) (274) (Figure 51), the found ligands perform high binding affinity with catalase. The results of MD simulation show
that THSG is the most stable in trajectory analysis over all simulation times. Besides, THSG can affect the catalase structure
more compact during the process of MD simulation. In addition, the radical scavenging assay showing that THSG has more
potential antioxidant activity than Hesperidin. Therefore, They regard the nature TCM compound, THSG, could be used to
develop potential drugs that might have similar effect to keep catalase active and prevent the inactive form generation by
hydrogen peroxide.

 
Fig. (50). Structures of compounds (259-272) identified in the high-throughput virtual screening chosen for biological
validation

 
Fig. (51). The chemical scaffold of the TCM candidates: Hesperidin (273) and THSG (274)
DNA topoisomerases (Topo) catalyze breaking and rejoining of one (Topo I) or both the strands (Topo II) of duplex DNA.
These enzymes, thus maintain proper structural conditions of DNA molecule to release topological torsions generated in the
course of replication, transcription and other processes involving unwinding of DNA to access coded information.
Topoisomerases have been implicated in various types of cancers such as lung cancer, melanoma cancer, breast cancer and
prostate cancer. Therefore, anti-cancer drugs such as daunorubicin, doxorubicin (anthracyclines), etoposide and teniposide
(epipodophyllotoxins) targeted against Topo II have been developed. These drugs have been reported to cause accumulation of
cytotoxic non-reversible DNA double-strand breaks (cleavable complex). Looking to the roles of topoisomerases in various
types of cancers, Singh et al[209] performed a quantitative structure–activity relationship model, based on genetic function
approximation (GFA) method, was developed using 41 olivacine derivatives as inhibitors of topoisomerase IIb (Topo IIb). The
best predictive GFA model explained the biological activity of the training and test sets with correlation coefficient values (r2)
of 0.747 and 0.549, respectively, and a significant cross-validated correlation coefficient (q2) of 0.525. The model suggested
positive correlation between activity and descriptors, namely partition coefficient (ALogP), electrostatic energy, number of
hydrogen bond acceptors and number of rotatable bonds, while those of negative correlations with Jurs RPCG, Jurs TASA and
PHI. Furthermore, a common feature-based pharmacophore model for these inhibitors was also developed which comprised of
five features, namely one H-bond acceptor, two ring aromatic, one positive ionizable and one hydrophobic group. Screening of
a large database for natural compounds, using both these developed models, led to identification of the pyrrole derivative,
namely 1-(2-(dimethylamino)ethyl)-3-hydroxy-4-(4-methoxy-2-methylbenzoyl)-5-(3,4,5-trimethoxyphenyl)-1H-pyrrol-2(5H)-
one (compound ID: STOCK1N-31995) (275) with a predicted IC50 value of 0.3 x 10-6 µM as most potent inhibitor of Topo IIb.
The result of virtual screening was further validated using molecular dynamics (MD) simulation analysis. Thus, a 15 ns MD
simulation analysis revealed high stability and effective binding of 1-(2-(dimethylamino)ethyl)-3-hydroxy-4-(4-methoxy-2-
methylbenzoyl)-5-(3,4,5-trimethoxyphenyl)-1H-pyrrol-2(5H)-one (275) within the active site of Topo IIb (Figure 52).

 
Fig. (52). Structure of compound 1-(2-(dimethylamino)ethyl)-3-hydroxy-4-(4-methoxy-2-methylbenzoyl)-5-(3,4,5-
trimethoxyphenyl)-1H-pyrrol-2(5H)-one (275) with 2 D view of a showing the interacting residues of Topo IIb and DNA in the
binding of the best screened compound at the active site (From Singh et al[209])
Rho-associated, coiled-coil containing protein kinase (ROCK) is crucial for organizing the actin cytoskeleton and is
involved in a great variety of basic cellular functions, such as apoptosis, adhesion, migration, proliferation and contraction.
ROCK belongs to the Ras superfamily, which has been found to be mutated in about 30% of all human cancers. Aiming at the
selection of potential new anticancer agents, Chen et al[210] performed a combination of the multi-complex-based
pharmacophore (MCBP), molecular dynamics simulation and a hybrid protocol of a virtual screening method, comprised of
multipharmacophore-based virtual screening (PBVS) and ensemble docking-based virtual screening (DBVS) methods were
used for retrieving novel ROCK1 inhibitors from the natural products database embedded in the ZINC database. Ten hit
compounds were selected from the hit compounds, and five compounds were tested experimentally. The results showed that
three out (salvianolic acid B, 1-desgalloyleugeniin, plantamajoside) (Figure 53) of the five hit compounds can inhibit the
ROCK1 with a predicted dissociation constant lower than 10 μM. Therefore, they successfully identified several potent ROCK1
inhibitors with polyphenol scaffolds via a combination of structure- and ligand-based computational methods, and their studies
may provide novel lead compound of ROCK1 inhibitors as candidate drugs.

 
Fig. (53). Structures of the hit compounds as ROCK-1 inhibitors
Ntie-Kang and colleagues[211] developed and validated pharmacophore models for use in virtual screening protocols for
eight known anticancer drug targets, including tyrosine kinase, protein kinase B β, cyclin-dependent kinase, protein
farnesyltransferase, human protein kinase, glycogen synthase kinase, and indoleamine 2,3-dioxygenase 1. Pharmacophore
models were validated through receiver operating characteristic and Güner–Henry scoring methods, indicating that several of
the models generated could be useful for the identification of potential anticancer agents from natural product databases. The
validated pharmacophore models were used as three-dimensional search queries for virtual screening of the newly developed
AfroCancer database (~ 400 compounds from African medicinal plants), along with the Naturally Occurring Plant-based
Anticancer Compound-Activity-Target dataset (comprising ~ 1,500 published naturally occurring plant-based compounds from
around the world). Additionally, an in silico assessment of toxicity of the two datasets was carried out by the use of 88 toxicity
end points predicted by the Lhasa’s expert knowledge-based system (Derek), showing that only an insignificant proportion of
the promising anticancer agents would be likely showing high toxicity profiles. A diversity study of the two datasets, carried
out using the analysis of principal components from the most important physicochemical properties often used to access drug-
likeness of compound datasets, showed that the two datasets do not occupy the same chemical space (Figure 54).

 
Fig. (54). MCSS panel in (A) AfroCancer and (B) NPACT, featuring the most common substructures included in the databases.
Abbreviations: MCSS, most common substructure selection; NPACT, Naturally Occurring Plant-based Anticancer Compound-
Activity-Target (From Ntie-Kang et al[211]).
Chloride intracellular channel 1 (CLIC1), a newly discovered member of the highly evolutionarily conserved CLIC family
of chloride ion channel proteins, was first cloned because of its increased expression in activated macrophages. CLIC1 located
within the plasma membrane and other internal cell membranes are involved in diverse physiological processes, is involved in
the development of most aggressive human tumors, including gastric, colon, lung, liver, and glioblastoma cancers. It has
become an attractive new therapeutic target for several types of cancer. Wang and colleagues[212] developed a work to identify
natural products as potent CLIC1 inhibitors from Traditional Chinese Medicine (TCM) database using structure based virtual
screening and molecular dynamics (MD) simulation. First, structure-based docking was employed to screen the refined TCM
database and the top 500 TCM compounds were obtained and reranked by 𝑋-Score. Then, 30 potent hits were achieved from
the top 500 TCM compounds using cluster and ligand-protein interaction analysis. Finally, MD simulation was employed to
validate the stability of interactions between each hit and CLIC1 protein from docking simulation, and Molecular
Mechanics/Generalized Born Surface Area (MM-GBSA) analysis was used to refine the virtual hits. Six TCM compounds with
top MM-GBSA scores and ideal-bindingmodels were confirmed as the final hits. Their study provides information about the
interaction between TCM compounds and CLIC1 protein, which may be helpful for further experimental investigations. In
addition, the top 6 natural products structural scaffolds (Figure 55) could serve as building blocks in designing drug-like
molecules for CLIC1 inhibition.
A tumor-suppressor protein, p53 also known as the ‘‘guardian of the genome and policeman of the oncogenes’’ induces cell
cycle arrest or apoptosis. It is an integral part of many biological activities such as regulation of cell cycle, gene amplification,
programed cell death, chromosomal segregation, DNA repair, differentiation, DNA recombination and cellular senescence.
Eradication of mortalin–p53 interaction that leads to the inhibition of transcriptional activation or blocking of p53 from
functioning as a suppressor and induction of nuclear translocation of p53 can prove to be one of the useful approaches for
cancer management[213, 214]. Mortalin, a molecular chaperone belonging to the family of heat-shock proteins (Hsp70), was
initially discovered in the fibroblasts of mouse embryonic cells. It resides in multiple subcellular sites, interacts with multiple
binding partners and has important role in muscle activity, stabilization of mitochondrial protein, antigen processing, control of
cell proliferation, cell differentiation, cell fate determination, tumorigenesis and intracellular trafficking. Several studies have
demonstrated that mortalin gets upregulated in many cancers cases and contributes to tumorigenesis, poor prognosis, increased
tumor grade and drug resistance[215, 216]. Nagpal et al[217] used structure-based approach to target the p53-binding domain
of mortalin in order to prevent mortalin–p53 complex formation. They screened compounds from ZINC database against the
modeled mortalin protein using Glide virtual screening. The top two compounds, DTOM (ZINC 28639308) (285) and TTOM
(ZINC 38143676) (286) (Figure 56) with Glide score of -12.27 and -12.16, respectively, were identified with the potential to
abrogate mortalin–p53 interaction. Finally, molecular dynamics simulations were used to analyze the dynamic stability of the
ligand-bound complex and it was observed that residues Tyr196, Asn198, Val264 and Thr267 were involved in intermolecular
interactions in both the simulated ligand-bound complexes, and thus, these residues may have a paramount role in stabilizing
the binding of the ligands with the protein. Conclusion: These detailed insights can further facilitate the development of potent
inhibitors against mortalin–p53 complex.

Fig. (55). The molecular structure of the top 6 TCM compounds identified as building blocks in designing drug-like molecules
for CLIC1 inhibition[212]

 
Fig. (56). Structures of top two scoring ligands (A) DTOM (ZINC 28639308) (285) and (B) TTOM (ZINC 38143676
(286)[217].
Hexokinase 2 (HK2) enzyme plays a pivotal role in glycolytic pathway of cancer cells, which catalyzes the first step of
glycolysis by phosphorylating glucose to glucose-6-phosphate (G-6-P) and promotes tumor progression in mouse models. The
expression level of HK2 in cancer cells is higher than in normal cells, providing an interesting target for the development of
therapeutic strategies to preferentially kill cancer cells. Bao and colleagues[218] performed a structure-based virtual ligand
screening in a small in-house database of natural products predicted that a new steroid, (22E,24R)-6β-methoxyergosta-
7,9(11),22-triene-3β,5α-diol ((288) Figure 66) from Ganoderma sinense has high binding affinity to HK2 with significant
calculated binding free energy. Based on this prediction, compound (288), together with the other 12 steroid analogues (287,
289-299 Figure 57) from this plant were selected for further in vitro microscale thermophoresis (MST), enzyme inhibition, and
cell-based assays based on the HK2 target. And compound 2 was finally identified as an HK2 inhibitor. As the first natural
HK2 inhibitor, compound 2 can be considered as a potential drug candidate targeting at HK2 for cancer therapy.
Fig. (57). Structures of steroids 287-299[218].

Also studying Ganoderma sinense, Zheng and co-workers[219] conducted a study targeting the enzyme Isocitrate
dehydrogenase (IDH) which is a family of enzymes that catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate
using either NADP or NAD as a cofactor, which is one of the key reactions in tricarboxylic acid cycle. IDH mutations have
been associated with many cancers, including glioblastoma, sarcoma, acute myeloid leukemia, etc. Three natural steroids 300-
302 (Figure 58) from Ganoderma sinense, a unique and rare edible-medicinal fungi in China, were found as potential IDH1
inhibitors by virtual ligand screening method. Among the three compounds, 302 showed the highest binding affinity to IDH1
with significant calculated binding free energy. Enzymatic kinetics demonstrated that 302 inhibited mutant enzyme in a
noncompetitive manner. The half effective concentration of 3 for reducing the concentration of D-2HG in HT1080 cells was
35.97 μM. The levels of histone H3K9me3 methylation in HT1080 cells were reduced by treating with 302. Furthermore,
knockdown of mutant IDH1 in HT1080 cells decreased the anti-proliferative sensitivity to 3. In short, their findings highlight
that compound 302 may have clinical potential in tumor therapies as an effective inhibitor of mutant IDH1.

Fig. (58). Chemical structures of compounds 300–302 obteined from Ganoderma sinense[219].

Singh and Silakari[220] developed a protocol for the identification of potential kinases inhibitors from natural products that
could be used in the future for the treatment of lung cancer. A pharmacophore-based activity profiling protocol using parallel
pharmacophore-based virtual screening of ZINC. The study involves ligand-based pharmacophore modeling of various kinases,
including EGFR (T790 M), cMET, ErbB2, FGFR and ALK, which are well established targets of normal as well resistant lung
cancer. The generated pharmacophore models were then utilized for parallel and cross screening. The profiled molecules for
each target were then validated using molecular docking and molecular dynamic simulations. The results show that kinase
inhibitory activity profiling of some natural product molecules (Figure 59) was successfully achieved.
Taking into account that serine protease dipeptidyl peptidase-4 (DPP-4) inhibitors played a role in the management of
diabetes, obesity, and cancer, Kalhotra et al[221] carried out a study describing the development of field template, field-based
qualitative structure–activity relationship (SAR) model demonstrating DPP-4 inhibitors of natural origin, and the same model is
used to screen virtually focused food database composed of polyphenols as potential DPP-4 inhibitors. Compounds’ similarity
to field template, and novelty score “high and very high”, were used as primary criteria to identify novel DPP-4 inhibitors.
Molecular docking simulations were performed on the resulting natural compounds using FlexX algorithm. Finally, one natural
compound, chrysin (Figure 60), was chosen to be evaluated experimentally to demonstrate the applicability of constructed SAR
model. This study provides the molecular insights necessary in the discovery of new leads as DPP-4 inhibitors, to improve the
potency of existing DPP-4 natural inhibitors.

Fig. (59). Structures of ZINC00241889–(303), ZINC11866307––(304), ZINC38143676 (305), ZINC85643856 –(306),

ZINC85646292 (307), ZINC96221218 –(308), ZINC98364461 (309), ZINC98365505 –(310), (From Singh and
Silakari[220]).

Fig. (60). The three-dimensional view, representing the binding pose view of chrysin (311): at the active site of the DPP-(a);
and (b) a two-dimensional representation of interacting residues from the DPP-4 enzyme, involved at binding pose of
chrysin[221].
4.9. Obtaining candidates for antileishmanial drugs
Leishmaniasis is a complex of infectious diseases caused by parasites of the family Trypanossomatidae and genus
Leishmania. It affects around 12 million people around the world; there are reported cases in 98 countries spread across five
continents, mainly in poor countries, causing the disease to be classified as a neglected disease by the World Health
Organization. It is the second most parasitic disease with the highest mortality and an estimated 350 million people are at risk
of infection, being more prevalent in Brazil, Sudan, Ethiopia and India[222, 223]. In the search for new molecules more active
and with less toxic effects for the treatment of leishmaniasis, Costa and co-workers[224] developed a Ligand Based Virtual
Screening (LBVS) of natural products against Leishmania major promastigotes using as a starting point the alkaloid 1 and
coumarin 2 which are demonstrably active molecules. From there they searched the ZINC database (with approximately
800,000 compounds) and selected 40 compounds. For a detailed analysis of these 40 compounds, 14 had their antileishmanial
activities determined in vitro, confirming that these 14 molecules are characterized as lead candidates (Table 6).
Leishmaniasis shows wide spectra of infection ranging from self-healing localized cutaneous lesions caused by Leishmania
major to life threatening visceral infection caused by Leishmania donovani or Leishmania infantum. Available drugs against
leishmaniasis have limitations in terms of low efficacy, high cost of severe side effects. It was observed that these protozoan
parasites had developed resistance against these drugs. Pentavalent antimonial compounds which were used as the first line of
treatment are no more effective. In the case of visceral leishmaniasis drug resistance was observed in the Indian subcontinent, a
major endemic region of Leishmania donovani infection. Pathway involved in removal of oxidative stress in mammalian host
involves glutathione/glutathione reductase and thioredoxin/thioredoxin reductase systems. Leishmania parasite and other
trypanosomatid show an analogous but unique biochemical system, trypanothione metabolism, to fight oxidative stress and to
maintain redox balance. The trypanothione metabolism is based on trypanothione reductase (TR) and dithiol trypanothione. TR
is a unique NADPH dependent flavoenzyme that helps combat oxidative stress by keeping sufficient levels of reduced
trypanothione. Enzymes, glutathione reductase and trypanothione reductase show remarkable differences within active sites
which make them substrate specific. Moreover, differences at active site provide an opportunity to identify or synthesize
compounds which may specifically inhibit TR without interfering mammalian enzyme, glutathione reductase. In this way, with
the aim of identifying potential natural product inhibitors of trypanothione reductase which can be anti-leishmanial, Venkatesan
et al[225] performed in silico virtual screening of a in-house natural product data set containing 800 compounds with diverse
chemical entities. Leishmania infantum trypanothione reductase crystal structure (PDB ID: 2JK6)[226] was used in the virtual
screening process, docking studies were performed using AutoDock4[227] implementing Lamarckian genetic algorithm
considered as one of the best algorithm for small molecular conformational search to identify potential lead compounds. The
compounds were sorted based upon their binding energy and the top 50 ranked protein-inhibitor complexes were clustered
using AuPosSOM to ligand foot print the interactions. They reported a few alkaloids, sterols as potential trypanothione
reductase inhibitors for the first time; biological significance of the interactions were discussed. The footprinting of protein-
inhibitor interactions into clusters provided clues on various possible orientations that inhibitors can attain at the active site of
trypanothione reductase and the compounds were sorted based upon their binding energy and the top 50 ranked protein-
inhibitor complexes (Figure 61 ). The compounds reported in the study can be further assayed for their antileishmanial activity
and can be developed into drugs[225].

 
Fig. (61). Structures of Beta-Amyrin acetate (326), Lunarine (327), Ginkgetin (328) and Fucostanol (329). (From Venkatesan et
al[225]).
Table 6. Antileishmanial Properties of the Compounds Selected from the LBVS Studies

Cpd Structure LD50* (µM) Cpd Structure LD50* (µM)

(312) 100 (313) 104

(314) 125 (315) 200

(316) 120 (317) 42

(318) 8 (319) 190

(320) 155 (321) 52

(322) 25 (323) 1,5

(324) 3 (325) 0,1

 Pathogens of the genus Leishmania eliminate lipids and depend on lipid metabolism, especially for the host-pathogen
interaction, using the host lipids for energy and virulence. The rational approach is to target lipid metabolism of the pathogen
focusing lipid-catabolizing lipases. The LdLip3 lipase is considered as drug target as it is constitutively expressed in both
promastigote and amastigote forms. Since the LdLip3structure was not known, Parameswaran et al[228] modeled its three-
dimensional structure to implement structure-based drug discovery approach. Similarity-based virtual screening was carried out
to identify potential inhibitors utilizing NCI diversity set on ZINC database including natural products. Implementing
computational and experimental approaches, four anti-leishmanial agents were discovered. The non-availability of the
experimentally determined structure for lipases from pathogenic source hinders the use of structure-based drug discovery
approach for several deadly infectious diseases. Homology modeling of LdLip3 was carried out to address the same with the
aid of lipase from R. miehei in its inhibitor bound conformation (PDB id: 4TGL, resolution: 2.60 Å) to identify potential anti-
leishmanial agents. Sequence analysis revealed that LdLip3is highly conserved throughout the leishmanial species.
Conservation between LdLip3 and Rm-TGL is satisfactory in terms of fold and functional residues which is necessary for the
reliable LdLip3 model. The catalytic residues were conserved and correspond to Ser168, Asp232 and His288 in LdLip3. The
modeled structure confirmed that LdLip3 is a member of serine hydrolases with α/β fold (Fig. 2A). The 3 D model of LdLip3
(extends from 21 to 302 residues with 30 % identity and 44 % similarity toward Rm-TGL) was significantly conserved with
leishmanial homologs and Rm-TGL. LdLip3 has α/β hydrolase domain with five-stranded parallel β-sheet covered by two-
stranded anti-parallel β-sheet at the N-terminus. The nucleophile serine (Ser168) was found at the bend of the tight turn in the
‘nucleophilic elbow’ and other catalytic residues (Asp232 and His288) are positioned adjacent to each other within the active
site. Taken together, structural features of LdLip3 are similar to that of Rm-TGL and other triacylglycerol lipases. Apart from
conserved catalytic residues, strict conservation was observed between LdLip3 and Rm-TGL at the active site cavity (4 Å
around catalytic residues) namely His167, Gly170, Ala172, Gly202, Pro204, Val234, Pro235, Val283, Asp287 and Tyr291
which might play a role in the enzymatic activity. The residues of oxyanion hole were identified as Ser102 andVal169 which
are involved in substrate binding and tetrahedral intermediate stabilization. The energy minimized structure of LdLip3 model
has acceptable statistics of backbone dihedral angle distribution of amino acids in Ramachandran plot with 86.6 % in core, 12.2
% in additionally allowed and 0.4 % in generously allowed region which is comparable with Rm-TGL. Along with Asp51 and
Pro249, the catalytic nucleophile Ser168 was observed in disallowed region of Ramachandran plot, a typical feature found in
most of the energy minimized serine lipases/esterases. G-factor of LdLip3 was −0.19 which also indicates acceptable statistics
of distribution of phi and psi along with chi1, chi2 and chi3 angles (G-factor less than −0.5 is considered to be unusual). Errat
plot of LdLip3 showed structural quality of 81.319 with little steric hindrance between few amino acids. As expected, Verify-3
D also revealed that 79.86 % of the amino acids in the structure of LdLip3 have compatible 1 D-3 D score greater than 0.2. The
LdLip3 model has Z-score of −5.97 is in the range of native conformations of crystal structures with ProSA-web. The fold
quality of LdLip3from ProSA was comparable with experimentally determined Rm-TGL (Z-score: −7.01) indicating the
acceptability of the modeled LdLip3 structure. Molecular dynamics (MD) was carried out to assess stability of the energy-
minimized LdLip3 model to ensure its reliability for similarity-based virtual screening. Time evolution of RMSD of the
backbone Cα atoms of LdLip3 showed that it undergoes a significant change in the initial 5 ns of simulations and converged
with fluctuations less than 0.1 Å thus indicating stable conformation of LdLip3 model. MD simulations suggested that the
energy-minimized LdLip3 model is satisfactory for virtual screening process. Structural comparison of the energy minimized
LdLip3 model with Rm-TGL on Cα-backbone atoms (161 out of 219 aligned atoms) shows overall RMSD of 0.176 Å which
was carried out with PyMOL. Structural comparison also reveals that the catalytic site of LdLip3 (9 out of 12 Cα-backbone
atoms around 4 Å of catalytic residues with RMSD of 0.337 Å) is similar to Rm-TGL and residues Arg38, Ser102, Arg111,
Met285 and Arg286 in LdLip3 were exclusively found in the catalytic site. The leishmanial-specific residues at the catalytic
pocket have been exploited to screen potential leishmanial-specific lipase inhibitors. Similarity-based virtual screening was
carried out to identify potential inhibitors utilizing NCI diversity set on ZINC database including natural products.
Implementing computational and experimental approaches, four anti-leishmanial agents were discovered. The screened
molecules ZINC01821375, ZINC04008765, ZINC06117316 and ZINC12653571 had anti-leishmanial activity with IC50 (%
viable promastigotes vs. concentration) of 5.2 ± 1.8 µM, 13.1 ± 2.6 µM, 9.4 ± 2.6 µM and 17.3 ± 3.1 µM, respectively. The
molecules showed negligible toxicity toward mouse macrophages. Based on the contact footprinting analysis, new molecules
were designed with better predicted free energy of binding than discovered anti-leishmanial agents. Further validation for the
therapeutic utility of discovered molecules can be carried out by the research community to combat leishmaniasis[228].
Mattos Oliveira and colleagues[229] developed virtual screening by pharmacophore modeling and docking to identify one
potential inhibitor of the Farnesyl pyrophosphate synthase (FPPS) of Leishmania major. This enzyme acts in the early stages of
isoprenoid synthesis and is important for maintaining the integrity of the lipid bilayer of the parasite that causes the disease. A
total of 85,000 compounds from a natural products database (ZINC) was submitted for virtual hierarchical screening, and the
top ranked molecule in both methods was analyzed by intermolecular interaction profile and 20 ns molecular dynamics
simulations. The results showed a promising compound (Z84909) (Figure 62) from natural products that mimic the major
interactions present in the substrate/inhibitor.

4.10. Obtaining candidates for antibacterial drugs

Nowadays, a major public health problem is diseases caused by multiresistant bacteria, especially in nosocomial infections.
This is mainly caused by the development of microorganisms resistant to most of the available drugs in the therapy. In this
context, two species of clinical relevance are Staphylococcus aureus and Streptococcus epidermidis, due to the fact that they are
related to serious problems of hospital infections. Hospital infections account for 25 % of all deaths worldwide and 45 % of
deaths in those less developed countries. The cost of these hospitalizations, the use of antibiotics and bacterial resistance
(presented by some patients), represent, on average, US$ 1.3 billion per year in the United States[230]. Virtually all strains of
Staphylococcus aureus are resistant to penicillin and many are resistant to methicillin-related drugs (MRSA strains). Recently
cases of intermediate or complete resistance to vancomycin (VISA/VRSA strains) have been reported. Staphylococci are also a
common cause of infections related to bacterial biofilm formation on implanted devices. Infections may result in longer
hospitalization time, need surgery and can even cause death[231]. A joint strategy for antibiotic therapy to reduce resistance
phenomena is the use of substances that inhibit bacterial cell-cell communication. This is possible by the use of quorum sensing
inhibitor RNAIII-inhibiting peptide (RIP). Madanahally and co-workers[232] identified hamamelitannin (a natural product
found in the bark of witch hazel) (Figure 63) as a nonpeptide analog of RIP by virtual screening of a RIP-based pharmacophore
against a database of commercially available small-molecule compounds. To this end, they constructed a model from the three-
dimensional structure of the heptapeptide RIP (YSPWTNF-NH2) was constructed by homology to the crystal structure of the
residues 6-12 (YRPYTPS) of ribosomal protein L2 within the crystal structure of the 50 S ribosomal subunit from Deinococcus
radiodurans[233]. Program O was used for this purpose on an Octane workstation (Silicon Graphics Inc.)[234]. Screening for
small molecule nonpeptide analogs of RIP was carried out by a computer search with the ISIS software (Integrated Scientific
Information System) from Elsevier MDL against the Available Chemicals Database (ACD), a library of 300,000 commercially
available small molecule compounds. The model of RIP served as the basis for the search. First approach was to carry out
similarity searches with the RIP models against the ACD. As this search yielded only peptides it was abandoned. Next turned to
a search of the ACD based on a pharmacophore approach, in which queries were defined by a set of distance ranges between
aromatic rings (the midpoint of the Tyr, Phe and Trp rings was used) and hydrogen bond donors or acceptors, based on the RIP
model. Compounds with a molecular weight in excess of 1,000 Da and compounds deemed unsuitable for prophylaxis or
therapy, such as dyes and fluorescent compounds, were eliminated from the list of candidate compounds. The coordinates of
the top hits were converted from the internal MOL format to PDB format by program BABEL (OpenEye Scientific Software).
The structures of the top hits were superimposed on the RIP model and viewed either with program SwissPDBViewer on a PC
or with program O on a Silicon Graphics Octane workstation. With this medodology the Hamamelitannin was selected and in in
vitro tests it was observed that it has no effect on the growth of Staphylococcus, but like RIP, it does inhibit the quorum-sensing
regulator RNAIII. In a rat graft model hamamelitannin prevented device-associated infections in vivo, including infections
caused by methicillin resistant S. aureus and S. epidermidis (MRSA, MRSE) strains. These findings suggest that
hamamelitannin may be used as a suppressor to staphylococcal infections.

 
Fig. (62). Intermolecular interactions between the LmFPPS structure and Z84909. (From Mattos Oliveira and colleagues[229])

Fig. (63). A) Structure of hamamelitannin (2,5-di-O-galloyl-D-hamamelose) (331 (from Madanahally and co-workers[232])
 It has been observed in the therapy against bacterial infections the development of drug resistance. Multidrug resistant
bacteria resist a broad range of antimicrobials thereby reducing the treatment options and hence increasing the mortality.
Multidrug resistance is also a major difficulty in the treatment of the infectious diseases caused by some species, with efflux
pumps as one of the mechanisms of resistance. MexAB-OprM and AcrAB-TolC that belong to the Resistant Nodulation
Division (RND) family are the tripartite efflux pump assemblies, responsible for multidrug resistance in P. aeruginosa and E.
coli respectively. Aparna et al[235]. carried out a study in which a new criterion of excluding compounds with efflux substrate-
like features was used, thereby refining the selection process and enriching the inhibitor identification process. An in-house
database of phytochemicals was created and screened using high-throughput virtual screening against AcrB and MexB proteins
and filtered by matching with the common pharmacophore models. Phytochemical hits that matched with any one or more of
the efflux substrate models were excluded from the study. Hits that do not have features similar to the efflux substrate models
were docked using XP docking against the AcrB and MexB proteins. The best hits of the XP docking were validated by
checkerboard synergy assay and ethidium bromide accumulation assay for their efflux inhibition potency. Lanatoside C and
daidzein (Figure 64) were filtered based on the synergistic potential and validated for their efflux inhibition potency using
ethidium bromide accumulation study. These compounds exhibited the ability to increase the accumulation of ethidium
bromide inside the bacterial cell as evidenced by these increase in fluorescence in the presence of the compounds. With this
good correlation between in silico screening and positive efflux inhibitory activity in vitro, the two compounds, lanatoside C
and daidzein could be promising efflux pump inhibitors and effective to use in combination therapy against drug resistant
strains of P. aeruginosa and E. coli.

 
Fig. (64). Structures of lanatoside C (332) and daidzein (333)
Also in the attempt to solve the problem of resistance to antibiotics, Zang et al[236] described a new strategy that combats
multidrug resistance by using natural medicines to target the druggable enzymome (i.e., enzymatic proteome) of
Staphylococcus aureus. A pipeline of integrating in silico analysis and in vitro assay was purposed to identify antibacterial
agents from a large library of natural products with diverse structures, high drug-likeness, and relatively low flexibility, with
which a systematic interactome of 826 natural product candidates with 125 functionally essential S. aureus enzymes was
constructed via a high-throughput cross-docking approach. The obtained docking score matrix was then converted into an array
of synthetic scores; each corresponds to a natural product candidate. By systematically examining the docking results, a number
of highly promising candidates with potent antibacterial activity were suggested. Three natural products, i.e., radicicol,
jorumycin, and amygdalin, have been determined to possess strong broad-spectrum potency combating both the drug-resistant
and drug sensitive strains (MIC value < 10 µg/mL). In addition, some natural products such as tetrandrine, bilobalide, and
arbutin exhibited selective inhibition on different strains (Figure 65). Combined quantum mechanics/molecular mechanics
analysis revealed diverse non-bonded interactions across the complex interfaces of newly identified antibacterial agents with
their putative targets GyrB ATPase and tyrosyl-tRNA synthetase.

 
Fig. (65). Structures of selected natural products that showed good activity against resistant strains[236]
 Tuberculosis, one of the world’s most deadly infectious diseases, is caused by the bacterium called Mycobacterium
tuberculosis. Part of discovering new drugs to combat TB is by targeting novel enzymes. One of the new attractive drug targets
in M. tuberculosis is S-adenosyl-L-homocysteine hydrolase (SAHH), an enzyme in the activated methyl cycle. The activated
methyl cycle is responsible for the regeneration of S-adenosyl-methionine (SAM) from S-adenosyl-L-homocysteine (SAH).
Compounds that hamper the activated methyl cycle cause the accumulation of SAH and depletion of SAM. SAM, the end
product of the cycle, is a donor of active methyl groups in several essential cellular reactions and also a cofactor of certain
enzymes. Particular ratio of SAM to SAH in bacterial cell should be maintained for survival, and perturbation of this ratio level
leads to growth arrest[237, 238]. In order to detect potential agents against M. tuberculosis, Sampaco III and coworkers[239]
performed virtual screening of natural products from the Philippines and those in Ambinter database, molecular docking and
dynamics simulations on S-adenosyl-L-homocysteine hydrolase. At the end of the study, they reached the tautomer of the
compound test (Figure 66) as the most promising molecule, because it presented better binding energy (-307.64 kcal / mol) than
the substrate, SAH (-270,601 kcal / mol). Molecular dynamics simulations at body temperature indicated that the hit-SAHH
complex is more stable than the enzyme-substrate complex.

 
Fig. (66). Structure of the top hit compound as S-adenosyl-L-homocysteine hydrolase modulator
Isocitrate lyase (MtbICL) catalyzes the first step in the glyoxylate cycle, plays a pivotal role in the persistence of M.
tuberculosis, which acts as a potential target for an anti-tubercular drug. Shukla and co-workers[240] conducted a structure-
based virtual screening of natural compounds from the ZINC database (n = 1,67,748) against the MtbICL structure. The ligands
were docked against MtbICL in three sequential docking modes that resulted in 340 ligands having better docking score. These
compounds were evaluated for Lipinski and ADMET prediction, and 27 compounds were found to fit well with re-docking
studies. After refinement by molecular docking and drug-likeness analyses, three potential inhibitors (ZINC1306071 (341),
ZINC2111081 (342), and ZINC2134917 (343)) were identified. These three ligands and the reference compounds were further
subjected to molecular dynamics simulation and binding energy analyses to compare the dynamic structure of protein after
ligand binding and the stability of the MtbICL and bound complexes. The binding free energy analyses were calculated to
validate and capture the intermolecular interactions. The results suggested that the three compounds had a negative binding
energy with −96.462, −143.549, and −122.526 kJ mol−1 for compounds with IDs ZINC1306071, ZINC2111081, and
ZINC2134917, respectively (Figure 67). These lead compounds displayed substantial pharmacological and structural properties
to be drug candidates. They concluded that ZINC2111081 has a great potential to inhibit MtbICL and would add to the drug
discovery process against tuberculosis.

 
Fig. (67). Structures of three selected compounds as potential inhibitors of isocitrate lyase[240]
 Chlamydia is a Gram-negative pathogen which causes several diseases to mankind. Chlamydia trachomatis is the most
common sexually transmitted bacterial pathogen, with 100 million cases per year world wide. It also affects 400 million people
as the major cause of preventable blindness.4 In addition, genital C. trachomatis infection has been identifed as a cofactor that
is necessary for the transmission of human immunodeficiency virus (HIV). Macrophage infectivity potentiator (Mip) is the
virulence factor from Chlamydia trachomatis that is primarily responsible for causing sexually transmitted diseases (STDs) and
blindness. Mip possesses peptidyl-prolylcis/trans-isomerase (PPIase) activity that can be inhibited by FK506 or rapamycin.
Ramachandran et al[241] constructed the 3 D structure of the native and mutant Chlamydia trachomatis Mip based on the X-
ray structure of Legionella pneumophila Mip using homology modeling. The model was then validated and further used for
docking analysis. They developed a pharma cophore model based on the important features of rapamycin and FK506. The new
pharmacophore model was used to superimpose with the Universal Natural Product Database (UNPD) compounds and
resulting compounds with common chemical features were chosen. Furthermore, the lead molecules were docked into the
active site of the native and mutant Chlamydia trachomatis Mip. Docking analysis showed that the native Chlamydia
trachomatis Mip has the highest GOLD score of 85.75 in terms of compound-1 (UNPD131087) binding compared to the
D142L mutant that has a score of 83.21 and Y213A that has a score of 79.54. Similarly, compound-2 (UNPD175753) has a
GOLD score of 85.60 and D142L has a score of 82.47 and Y213A has a score of 78.86 which indicates that the binding affinity
is highly affected in both of the mutants due to changes in the conformation of the active site to which resistance has been
developed (Figure 68). Molecular dynamics simulation analysis infers changes in the binding pattern and structural
modification in the binding site by means of Rg, SASA, RMSD and RMSF. Hence, this study gives insight into the impact of
novel mutations on the activity of this protein, which can be attributed to the drug resistance.

 
Fig. (68). The chemical structure of the compounds with best binding affinity for C. trachomatis Mip
Buruli ulcer (BU) is a devastating chronic skin infection caused by Mycobacterium ulcerans. It is the third most common
mycobacterial disease after tuberculosis and leprosy. The disease is characterized by necrotizing skin lesions, which can cover
up to a third of the entire body surface area of the affected individual. It can also lead to irreversible deformity or long-term
functional disability when not properly treated. Due to the occurrence of resistance phenomena of this bacterium to the drugs
currently used in the treatment of this disease, there is a need to identify novel natural product-derived lead compounds, which
are potent and efficacious for the treatment of Buruli ulcer. Kwofie and colleagues[242] performed a study to predict natural
product-derived lead compounds with the potential to be developed further into potent drugs with better therapeutic efficacy
than the existing anti-buruli ulcer compounds. The three-dimensional (3 D) structure of Isocitrate lyase (ICL) of
Mycobacterium ulcerans was generated using homology modeling and was further scrutinized with molecular dynamics
simulations. A library consisting of 885 compounds retrieved from the AfroDb database was virtually screened against the
validated ICL model using AutoDock Vina. The molecular docking with the ICL model was validated by computing a Receiver
Operating Characteristic (ROC) curve with a reasonably good Area Under the Curve (AUC) value of 0.89375. Twenty hit
compounds, which docked firmly within the active site pocket of the ICL receptor, were assessed via in silico bioactivity and
pharmacological profiling. The three compounds, which emerged as potential novel leads, comprise ZINC38143792 (Euscaphic
acid), ZINC95485880, and ZINC95486305 (Figure 69) with reasonable binding energies (high affinity) of - 8.6, - 8.6, and - 8.8
kcal/mol, respectively. Euscaphic acid has been reported to show minimal inhibition against a drug-sensitive strain of M.
tuberculosis. The other two leads were both predicted to possess dermatological activity while one was antibacterial. The leads
have shown promising results pertaining to efficacy, toxicity, pharmacokinetic, and safety. These leads can be experimentally
characterized to assess their anti-mycobacterial activity and their scaffolds may serve as rich skeletons for developing anti-
buruli ulcer drugs.
  
Fig. (69). Structures of selected hits against Buruli Ulcer[242]
Bacteria coordinates their behavior through quorum sensing (QS), a mechanism that helps bacterial populations to enable
harmonious responses including biofilm formation and virulence factors expressions. Since, QS regulates the virulence arsenal
of many pathogenic bacteria, it seems to be a captivating drug target to combat bacterial infections. Drugs targeting the
virulence pathways could curb the bacterial pathogenesis and thereby prevents the disease development. Hence, interfering the
quorum sensing is an effective alternate strategy against various pathogens. Vinothkannan and colleagues[243] performed a
virtual screening from a natural products database, to find out potential CviR-mediated quorum sensing inhibitors (QSIs)
against Chromobacterium violaceum and in vitro biofilm and violacein inhibition assays have been performed. Biofilm
formation was investigated using confocal microscopy and gene expression studies were carried out using qRT-PCR. Further,
to study the biomolecular interaction of QSIs with purified CviR Protein (a LuxR homologue), microscale thermophoresis
(MST) analysis was performed. Results suggested that phytochemicals SPL, BN1, BN2, and C7X (Figure 70) have potential
GScore when compared to cognate ligand and reduced the biofilm formation and violacein production significantly. Especially,
100 μM of BN1 drastically reduced the biofilm formation about 82.61%. qRT-PCR studies revealed that cviI, cviR, vioB, vioC,
vioD genes were significantly down regulated by QSIs.MST analysis confirmed the molecular interactions between QSIs and
purified CviR protein which cohere with the docking results. Interestingly, they found that BN2 has better interaction with
CviR (Kd = 45.07 ±1.90 nm). Overall results suggested that QSIs can potentiallyinteract with CviR and inhibit the QS in a dose
dependent manner. Since, LuxR homologs present in more than 100 bacterial species, these QSIs may be developed as broad
spectrum anti-infective drugs in future.

 
Fig. (70). Structures selected as broad spectrum anti-infective drugs (From Vinothkannan and colleagues[243]).

4.11. Obtaining candidates for antibipolar drugs

Myo-inositol-1-phosphate (MIP) synthase is a key enzyme in the myo-inositol biosynthesis pathway. Disruption of the
inositol signaling pathway is associated with bipolar disorders. Azam et al[244] developed a study to obtain inhibitors of this
enzyme as potential drugs against bipolar disorders. Human MIP synthase structure was proposed for the first time in this
study. Two docking protocols were applied in order to have a comparative analysis and thus to generate a consensus. Structural
attributes of the active site mapped by the sequence to structure prediction was found to be involved in crucial interactions with
ligands yielding four potent inhibitors (ZINC03844661, ZINC03844222, ZINC00291743, (S)-2-penty-4-pentynoic acid
(Suvpd). The results of ligand docking illustrate that the amino acid residues Ser296, Gly297, Gln298, Thr299, Asp409,
Lys342, Lys346, Lys383, Lys455, Lys300 and Asn60 are pivotal for protein–ligand binding. These four novel inhibitors exhibit
much better scores and interactions as compared to the standard drug, indicating the probability of strong inhibition. Results
demonstrate the involvement of MIP synthase in the metabolic pathway of inositol biosynthesis and it is noteworthy that
hydrogen bond formation is the most significant interaction within the active site cleft. These chemical compounds could serve
as the lead for further cost-effective experimental screening. The inhibition of MIP synthase would help in reducing the risk of
severe psychiatric illness. This will also lead to a better understanding of catalytic and pharmacological properties of
aforementioned drug targets.

4.12. Obtaining candidates for antimalarial drugs

Malaria is one of the oldest parasitic diseases of mankind and is still reported today as one of the leading causes of mortality
in tropical regions. It has a strong influence on the economy, since the sick population has its work capacity significantly
reduced. It is an infectious disease, non-contagious, of chronic evolution, with episodic manifestations of acute character, that
affects millions of people in the tropical and subtropical zones of the world, mainly in Africa. Over time, several drugs have
been used in the treatment of malaria, but with problems of toxicity and emergence of resistant strains. For the production of
new agents several research groups have used strategies, among them the computational one to obtain new compounds for the
treatment of this disease.
In recent years, various new potential biochemical targets have been proposed for the antimalarial drug discovery. Among
them, the cysteine protease falcipain-2 (FP-2) has been considered as an attractive and most promising target for the
antimalarial agents discovery. Wuang et al.[245] performed a structured-based virtual screening using an in-house natural
products database with more than 4,000 compounds to obtain FP-2 enzyme inhibitors and identified ten promising molecules
(Figure 71). These molecules showed moderate inhibitory activities against FP-2 with IC50 values ranging from 3.18 to 68.19
µM. While one of the inhibitors (358) also exhibits in vitro antiplasmodial activity against chloroquine sensitive strain (3D7)
and chloroquine resistant strain (Dd2) of Plasmodium falciparum in the micromolar range (IC50s = 5.54 µM and 4.05 µM
against 3D7 cells and Dd2 cells, respectively).

Fig. (71). Structures of the positive control (364) and FP-2 inhibitors identified[245]
Currently, the first-line malaria treatments comprise five major artemisinin based combination therapies (ACTs) as guided
by World Health Organization (WHO). However, ACTs could become ineffective in the near future considering that the rise
and spread of artemisinin resistance in Plasmodium falciparum has already been reported in several places in Asia. Thus, the
identification of potential drug targets as well as development of novel antimalarial chemotherapies with unique mode of
actions due to drug resistance by Plasmodium parasites are inevitable. Falcipains (falcipain-2 and falcipain-3) of Plasmodium
falciparum, which catalyse the haemoglobin degradation process, are validated drug targets. Previous attempts to develop
peptide based drugs against these enzymes have been futile due to the poor pharmacological profiles and susceptibility to
degradation by host enzymes. Musyoka and colleagues[246] performed a study aimed to identify potential non-peptide
inhibitors against falcipains and their homologs from other Plasmodium species. Structure based virtual docking approach was
used to screen a small non-peptidic library of natural compounds from South Africa against 11 proteins. A potential hit, 5α-
Pregna-1,20-dien-3-one (5PGA), with inhibitory activity against plasmodial proteases and selectivity on human cathepsins was
identified. A 3 D similarity search on the ZINC database using 5PGA identified five potential hits based on their docking
energies (Figure 72). The key interacting residues of proteins with compounds were identified via molecular dynamics and free
binding energy calculations. Overall, this study provides a basis for further chemical design for more effective derivatives of
these compounds. Interestingly, as these compounds have cholesterol-like nuclei, they and their derivatives might be well
tolerated in humans.

4.13. Obtaining candidates for antitrypanosomal drugs

Among the diseases caused by Trypanosoma microorganisms, Human African Trypanosomiasis (HAT), also known as
“sleeping sickness”, is perhaps the most neglected of all, because it is a disease restricted to the African continent, reaching the
poorest population and aged between 15 and 45 years old, living in rural areas and working in agriculture or fishing. When
untreated this disease is always fatal. The pathogen responsible for causing this disease is Trypanosoma brucei. T. brucei enters
the bloodstream of the host after the vector bite, the tsé-tsé fly or Aniel Rossi, of the genus Glossina, and it is estimated 20,000
annual deaths[247]. In an attempt to solve this problem and seeking new agents to combat this disease, Herrmann et al[247]
performed an extensive screening of natural product (NP) databases against a multitude of protozoan parasite proteins. They
screened a database of NPs from a commercial supplier, AnalytiCon Discovery (Potsdam, Germany), against Trypanosoma
brucei glyceraldehyde-3-phosphate dehydrogenase (TbGAPDH), a glycolytic enzyme whose inhibition deprives the parasite of
energy supply. NPs acting as potential inhibitors of the mentioned enzyme were identified using a pharmacophore-based virtual
screening and subsequent docking of the identified hits into the active site of interest. In a set of 700 structures chosen for the
screening, 13 (1.9%) were predicted to possess significant affinity towards the enzyme and were therefore tested in an in vitro
enzyme assay using recombinant TbGAPDH. Nine of these in silico hits (69%) showed significant inhibitory activity at 50 μM,
of which two geranylated benzophenone derivatives proved to be particularly active with IC50 values below 10 μM. The most
active inhibitors, both predicted to bind to the G-3-P site of TbGAPDH, were Compounds (371) and (372). It is interesting to
note that they are structurally very closely related, representing regioisomers in which only the aldehyde and carboxylic acid
substituents are exchanged (Figure 73). These compounds also showed moderate in vitro activity against T. brucei rhodesiense
and may thus represent interesting starting points for further optimization.

 
Fig. (72). Structures of South African natural compound and its analogs. Marked with asterisk is the South African hit used for
structure similarity search on the ZINC database.

 
Fig. (73). Structures of compounds (371) and (372) which were the most active inhibitors of TbGAPDH.
Scotti et al[248] performed a virtual screening in an in-house databank (SistematX), of 469 Apocynaceae indole alkaloids,
using models developed with fragment descriptors using Support Vector Machines (SVM) and Decision Trees (DT) were
performed. A dataset 545 agrochemicals selected from ChEMBL database was used to generate both models and the prediction
performance was tested using a small set of 44 alkaloids with the antitrypanosomal activity. From 469 Apocynaceae alkaloids,
the SVM model selected, as actives, 5 similar alkaloids, from 2 species of the Aspidosperma genus (excelsum, marcgravianum),
and the DT model selected 3 alkaloids from 3 species (gilbertii, nigracans, and subincanum) of the same genera from the
SistematX database. The values of Moriguchi octanol-water partition coefficient for these structures are between 2.3 to 5.3, and
5 alkaloids, passed the Lipinski alert index filter and Drug Like Score consensus (> 0.7), which indicate that these compounds
are good candidates to become a drug. These structures (Figure 74) might be an interesting starting point for antitrypanosomal
studies. The methodology, applying fragment descriptors and machine learning, was rapid and can be applied for virtual
screening for bigger databases.
Chagas disease is an endemic disease caused by Trypanosoma cruzi, which affects more than eight million people, mostly
in the Americas. Acevedo and colleagues[1] performed a search for new drugs to control and eliminate this disease.
Sesquiterpene lactones (SLs) are an interesting group of secondary metabolites characteristic of the Asteraceae family that have
presented a wide range of biological activities. From the ChEMBL database, they selected a diverse set of 4452, 1635, and
1322 structures with tested activity against the three T. cruzi parasitic forms: amastigote, trypomastigote, and epimastigote,
respectively, to create random forest (RF) models with an accuracy of greater than 74% for cross-validation and test sets.
Afterward, a ligand-based virtual screen of the entire SLs of the Asteraceae database stored in SistematX (1306 structures) was
performed. In addition, a structure-based virtual screen was also performed for the same set of SLs using molecular docking.
Finally, using an approach combining ligand-based and structure-based virtual screening along with the equations proposed in
this study to normalize the probability scores, they verified potentially active compounds (Figure 75).

 
Fig. (74). Selected alkaloids by Support Vector Machine and Decision Tree models with potential antitrypanosomal activity

 
Fig. (75). Potentially active sesquiterpene lactones (best five ranked) identified using a ligand-based virtual screen of each
parasitic form of T. cruzi; p=active probability value[1].

CONCLUSION
This work has made an important compilation of studies showing the importance of natural sources, of different types of
living beings, as raw material for obtaining new drugs, as well as several ways in which computational tools (Structure-based
(SB) or Ligand-Based (LB)) and molecule databases can aid and expedite the discovery process. We selected relevant papers
describing the obtaining of potential drug candidates that were distributed in 15 classes, of which the anticancer, antibacterial
and anti-inflammatory hits were the most abundant, in addition, there are also described works showing efforts to search for
new molecules against various other diseases in distinct biological systems. In this way, this work shows an overview of several
methodologies and we hope they can help and inspire the development of new research to improve people's quality of life.
It is important to mention that due to the focus given to this work, to show studies that selected molecules from databases of
natural products by using computational tools, some molecules used in therapy were not included, for example Artemisin, which is an
innovative antimalarial drug obtained from the Chinese plant Artemisia annua L. and that allowed the Chinese Youyou Tu to be
awarded the Nobel Prize[249, 250]. On the other hand, we recommend that future researchers who are searching for new compounds
in databases should pay close attention to Randomized Controlled Trials (RCTs), especially those of Traditional Chinese Medicine
(TCM), because Wang and collaborators[251] conducted a study that evaluated the quality of RCTs reports in TCM and used a
modified version of the Consolidated Standards of Reporting Trials (CONSORT) statement and the Jadad scale and concluded that
the quality of the reports from TCM's RCTs improved, but remains poor, as they have problems such as: wrong choice of sample size
of patients, failure to carry out double-blind randomized trials, among others. Also in this sense, Teschke et al[252] carried out an
interesting study in which they show a practical guide for correctly selecting possible substances for the treatment of gastrointestinal
disorders from the TCM databases. They searched traditional databases using the TCM terms based on plants and gastrointestinal
disorders and the results were analyzed for the type of study, inclusion criteria and outcome parameters. They found that the quality of
the randomized, double-blind, placebo-controlled clinical trial was poor, mostly neglecting rigorous evidence-based diagnostic and
therapeutic criteria. It has been found that the use of herbal TCM to treat various diseases is based on an interesting philosophy with a
long history but has received growing skepticism due to lack of evidence-based efficiency as demonstrated by high-quality trials.
Thus, new researchers must pay special attention to these details so that their work becomes more robust.
Another aspect that must be considered when using natural products in therapy is the question of the hepatoxicity of these
substances. There are several reports that some herbal products in medicinal plants of traditional and modern medicine are at risk of
herb induced liver injury (HILI) with a severe or potentially lethal clinical course, and the requirement for a liver transplant, for
example, with the use of green tea, obtained from the leaves of Camellia sinensis (L.) Kuntze (Fam. Theaceae) which despite being
widely used with potential health benefits such as reduced risk of cardiovascular disease and weight reduction, it is suspected of
inducing liver damage due to its content of Catechins which are inhibitors of the microsomal liver enzymes CYP2C8, CYP2B6,
CYP3A4, CYP2D6 and CYP2C19 and may increase plasma levels of several other drugs[253, 254]. Therefore, a rigorous evaluation
of the use of these products in patients with suspected HILI is necessary. To assess the hepatoxicity of natural products, the CIOMS
scale (Council of International Organizations for Medical Sciences) can be used and analytical methods can also be introduced to
identify cases of intrinsic hepatoxicity and onomic technologies, including enomics, proteomics, metabolomics and micro- Serum
RNA of some patients with intrinsic hepatoxicity. Thus, to improve their globalization, herbal medicines should be universally
marketed under strict regulatory surveillance, in analogy to the medicines approved by regulation, proving a positive risk/benefit
profile by imposing evidence-based clinical trials and excellent quality of herbal medicines[255]. It is also important to mention that
with the aging of the world population, the use of natural products, such as food or medicine, is highly stimulated to increase people's
longevity. For example, the population of the island of Okinawa in Japan has a high life expectancy and this increase is related to
genetic factors associated with a diet low in calories and rich in vegetables with high levels of phytochemicals, mainly antioxidants,
phenolic acids, essential oils and fatty acids that interfere in the production of ROS which can contribute significantly to human
longevity. So, it is important to stimulate studies to obtain compounds that can act in the regulation of ROS production, as well as in
the cell cycle control pathways, such as the caspase pathway[256].
To conclude, it is necessary that all citizens who carry out research and who are opinion makers are concerned with the sustainable
development of our planet. A plan of action for people, planet and prosperity was proposed with the tittle ‘The United Nations 2030
Agenda for Sustainable Development’, in particular the Sustainable Development Goal 3 (SDG3) which aims to ensure a healthy life
and promote well-being for all people of all ages, in particular ensure the lasting protection of the planet and its natural resources,
creating conditions for sustainable, inclusive and economically sustained growth, shared prosperity and decent work for all, taking into
account the different levels of development and national capacities, in addition to ending the epidemics of AIDS, tuberculosis, malaria
and other neglected tropical diseases, fighting hepatitis, waterborne diseases, among others[257-259]. In protecting natural resources,
special attention is needed to our tropical forests, oceans and reducing greenhouse gas emissions, in addition to reducing the disposal
of plastic particles in the oceans, as they interfere with marine biodiversity and this care is of fundamental importance, because as
described in this work, they are very important sources of new drugs and integrated attitudes of all people in society are essential for
us to make our planet a better place to live.

ACKNOWLEDGEMENT
None

CONSENT FOR PUBLICATION

Not applicable.

CONFLICT OF INTEREST
 The authors declare no conflict of interest, financial or otherwise.

FUNDING DETAILS
None

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