In-Silico Drug Designing Against Ebola Virus: A Genomic ...
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ISSN 2348-313X (Print) International Journal of Life Sciences Research ISSN 2348-3148 (online)
Vol. 3, Issue 3, pp: (125-132), Month: July - September 2015, Available at: www.researchpublish.com
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In-Silico Drug Designing Against Ebola Virus:
A Genomic Approach
1Harris Kaushik,
2Kishor Shende
1Department of Life Science, Institute of Applied Medicine & Research, Ghaziabad
2Department of Biotechnology and Bioinformatics Center, Barkatullah University, Bhopal
Abstract: Ebola hemorrhagic fever is a deadly disease caused by infection of negative-stranded RNA Ebola virus in
human and primates, belonging to family Filoviridae, genus Ebolavirus. The identified Ebola virus species are,
Zaire ebolavirus, Sudan ebolavirus, Taï Forest ebolavirus), Bundibugyo ebolavirus and Reston ebolavirus. Symptoms
are fever, severe headache, fatigue, muscle pain, weakness, diarrhea, vomiting, abdominal pain, and unexplained
hemorrhage. Viral evolutionary study has revealed the source of infection as infected animals, which can spread
through either direct contact or body fluid, but not by air. Currently there is no known cure for Ebola virus,
except good hospital treatment and patient’s immune system. Advancement in instrumentation and Bioinformatics
enable to characterize viral genome sequences and other fragment data of gene, genome and proteomics. Observed
potential drug target are VP40-matrix protein and VP35, protein involved in RNA polymerization, assembling,
interferon inhibition. Derivatives of acetic acid, propaonic acid and triazole carboxyamide were proposed to be
binding to VP40 and VP35. Monoclonal Antibodies (mAbs) against EBOV found to be effective. Two molecules, 1)
anti-sense phosphorodiamidate morphino oligomers and 2) Lipid nano-particle/small interfering RNA are in
clinical trial Phase-I (Source FDA). The current work is in progress and this abstract is aimed to provide support
to In-silico approaches that can provide insight into understanding the viral genome, host-viral interaction, viral
multiplication, assembly and release. It can facilitate screening of target, drug molecules, potential antigenic region
for vaccine development, strategies to block host-cell-viral interacting protein.
Keywords: Ebola virus, Filovirdae, In-silico therapeutics designing, Bioinformatics.
I. INTRODUCTION
Ebola virus identified is an envelope, single-stranded, negative-sense RNA virus that causes severe hemorrhagic fever in
humans and nonhuman primates. This virus is naturally resistant to various antibiotics, and there is no proper treatment
for infection caused by this pathogen. And hence there is a need for new drugs and approaches to combat the life
threatening infection caused by Ebola virus. Computer aided drug design is one of the powerful tools for discovering new
drug leads against important targets. The various proteins that are essential for pathogenesis of organism are selected as
targets. The viral proteins vp35 and vp40 are capable of eliciting protective immune responses to EBOV. The various
functions of these proteins in pathogenesis suggested them as potential drug targets to control EBOV infections. After the
proteins were selected as target, new leads were chosen from a subset of small molecules that scored well when docked in
silico against targets. The drug lead molecules were evaluated for their drug likeness using “Lipinski rule of five”. The
identification of appropriate drug lead molecules against these proteins has lead to a successful drug candidate against
Ebola virus infection.
There are five identified Ebola virus species. Four of the five have caused disease in humans: Ebola virus (Zaire
ebolavirus); Sudan virus (Sudan ebolavirus); Taï Forest virus (Taï Forest ebolavirus, formerly Côte d’Ivoire ebolavirus);
and Bundibugyo virus (Bundibugyo ebolavirus). The fifth, Reston virus (Reston ebolavirus), has caused disease in
nonhuman primates but not in humans.
ISSN 2348-313X (Print) International Journal of Life Sciences Research ISSN 2348-3148 (online)
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As such there is no drug available for treatment of Ebola virus infection. In silico approach is useful for discovering drug
lead candidate against Ebola virus fatal infection. The use of computer and computational methods allows, using all
aspects of drug discovery, forming core of structure based drug design and has advantage of delivering drug more quickly
and at economic cost. Classical drug discovery takes much time which may be 10-14 yrs or more to undergo target
discovery, lead generation, optimization, preclinical development, clinical trial, and FDA approval and finally bring to
market (Kantardjieff, et al., 2004).
II. MATERIALS AND METHODS
Selection of Traget Proteins:
Initially the target proteins were selected which are involved in the pathogenesis. The structural information of the target
proteins was obtained from PDB and their active sites were determined using PYMOL.
Screening of Lead Molecules:
After choosing the target protein the inhibitory drug compounds were chosen from pubchem and virtual screening was
done by creating database of these compounds in CHIMERA. These databases were fed to ARGUSLAB for screening the
best ligands with the target protein. The best ligands were chosen with low energy values, and virtual screening was done
using ZINC DATABASE, from which class of similar compounds were obtained, and again were fed to ARGUSLAB and
best compounds obtained were passed for docking studies.
Docking studies:
In docking studies the interaction between target and ligand was studied. The best ligands screened were loaded in to auto
dock and docking studies were carried out. Based on the binding energies and details from the histogram, the drug lead
compounds were determined.
Optimiziation of Lead Molecules:
The drug lead molecules selected were revaluated for their drug likeliness by using “Lipinski Rule of Five” to predict
which drug molecules would fail because of poor pharmaco kinetics
III. RESULTS
A study titled as “In-Silico Drug Designing Against Ebola Virus: A Genomic Approach” was performed with an objective
to find out the 3D structure of Ebola virus protein on the basis of protein structure of different Ebola virus species we
have try to find out the drug which will inhibit activity of Ebola virus which cause disease in humans . Study was further
extended for prediction of Ebola virus disease drug predication by the analysis of Ebola virus protein sequence from the
different species of Ebola virus (Zaire, Sudan, Tai- Forest, Buddibug and Reston).
Antiviral Targets in Ebola Virus:
The Ebola virus proteins evade the immune system and have important role in pathogenesis of the virus. This makes their
potential as drug target to combat Ebola virus infection. The viral proteins selected as a target are VP35 andVP40
Information about the targets:
1) VP40
VP40 is matrix protein and is key particle for maturation of virion. VP40 is known to posses three domains M, I, and L
domain. For interaction with specific cellular proteins and for virus host interaction viral L domain is thought to serve as
docking site and it facilitates virus budding. Homo-oligomerization is the key feature of viral matrix protein required for
efficient release of virus like particles and virions and also to interact directly with lipid membrane or host protein for
efficient budding. They bud and assemble from specialized domains known as lipid rafts which are present within the
plasma membrane. Thus, the matrix protein, Ebola virus VP40, plays a key role in viral assembly, budding, and virion
formation (Hoenen, et al., 2005).
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2) VP35
The Ebola VP35 protein is multifunctional, acting as a component of the viral RNA polymerase complex, a viral
assembly factor, and an inhibitor of host interferon (IFN) production. Mutation of selected basic residues within the C-
terminal half of VP35 abrogates its ds RNA-binding activity, impairs VP35-mediated IFN antagonism, and attenuates
EBOV growth in vitro and in vivo. The ds RNA binding activity mediated by the C terminus of VP35 is critical for viral
suppression of innate immunity and for virulence. The ds RNA binding cluster is centred on Arg- 312, a highly conserved
residue required for IFN inhibition. Mutation of residues within this cluster significantly changes the surface electrostatic
potential and diminishes ds RNA binding activity. Knockdown of VP35 leads to reduced viral amplification and reduced
lethality in infected mice. Therefore, a functional VP35 is required for efficient viral replication and pathogenesis (Weik,
et al., 2002).
Determination of Active Pocket Site of the Targets:
Protein molecules are the fundamental units of all living cells. These macromolecules have a vital role in various cellular
functions. Each protein has specific function in our body. The structure of the protein has a very important role in its
function. The binding of a protein with other molecules is very specific to carry out its function properly. For this reason
every protein has a particular structure. Proteins are the molecules, with a linear chain of amino acids initially, thus folds
into secondary, tertiary and quaternary structures. The tertiary structure of a protein is more important due to the folding
of the secondary structures, tertiary structures form the pockets or clefts, where the ligand or potential molecules, or small
atoms can bind to the protein molecule, where these sites can be predicted as active sites of the molecule (David, et al.,
2004).
The active sites are lined up with the amino acid residues. Molecules with appropriate shape and appropriate groups can
bind to the active site of the protein molecule. The mechanism is as simple as lock and key, as the particular lock opens
with the only particular key, the molecule or the substrate with perfect shape can only fit into the active site (Jeremy, et
al., 2002).
Active Pocket site of VP 40 by Pymol
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Active Pocket site of VP 35 by Pymol
Active pocket site analysis by the Pymol:
Table 1: Active Pocket sites of the target proteins
VP40 ASP 104,ALA 105, ARG 64, TYR 17
VP35 LYS 251, PRO 293, ASP 302, ARG 305, ALA 221, LYS 248, GLN244, GLN 241.
Screening of Ligands:
Around 22 antiviral compounds are taken from the Pub chem and draw their structure in Chem Sketch software. Using
these compounds new databases were made with the help of CHIMERA. This compound database was screened in
ARGUSLAB and 12 ligands were obtained having proper interaction with the target protein. By drawing structure of
these obtained compounds in inc database, similar compounds were obtained and were again fed in arguslab. Compounds
with low binding energies from ARGUSLAB were subjected to docking studies
Docking Studies
For VP40 with selected leads-
Nearly 10 ligands were obtained after virtual screening in ARGUSLAB and were docked in auto dock. Docking results
with VP40 with the best hits arrived six ligands, which could possibly inhibit the target protein. The ligands obtained are:
Amodiaquine
Clomiphene
Cytidine
Glucosidase Inhibitor
Isoflavone
Rimantadine
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The docking results are shown in Figure (1-6)
Fig. 1 - Interaction of VP40 with Amodiaquine Fig. 2 - Interaction of VP40 with Clomiphene
Fig. 3 - Interaction of VP40 with Cytidine Fig. 4 - Interaction of VP40 with Glycosidase Inhibito
Fig. 5 - Interaction of VP40 with Isoflavone Fig. 6 - Interaction of VP40 with Rimantadine
For VP35 with selected Leads
Ten ligands obtained after virtual screening with VP35 were docked in auto dock. Docking results
Of VP35 with the best hits arrived at six ligands, which may inhibit the target VP35 activities. The ligands obtained are:
Chloroquine
Favipiravir
Gleevec
Toremifene
Valacyclovir Hydrochloride
Brivudine
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The docking results are shown in Fig.ure (7-12)
Fig. 7 - Interaction of VP35 with Chloroquine Fig. 8 - Interaction of VP35 with Favipiravir
Fig. 9- Interaction of VP35 with Gleevec Fig. 10 - Interaction of VP35 with Toremifene
Fig. 11 - Interaction of VP35 with Val acyclovir Hydrochloride Fig. 12 - Interaction of VP35 with Brivudine
Ligands Docking Features:
This study was done on the basis of available compounds in market for finding the new drug which have proper
interaction with VP 40 and VP 35. Knowing the importance of threats of Ebola virus, this work was carried out on Ebola
virus .Total twenty hits were target on both VP 40 and VP 35 for finding the proper interaction of compound with the
target protein that is VP 40 and VP 35. In the study it was found that there are Clomiphene which shows the proper
interaction with VP 40 and shows minimum binding energy to the target protein (-13.74 kcal/mol) in compared with other
six compounds. While the VP35 protein which is multifunctional in nature shows the proper interaction with Gleevec and
shows the minimum binding energy as compared to other six compounds that is (- 8.34kcal/mol).So knowing the
importance of Ebola virus treatment in future the Clomiphene and Gleevec are the best available compounds which helps
to inhibits the virus action in protein.
The findings of this study are important as there is need for new drug to inhibit Ebola virus. The lead found out, could
possibly inhibit the infection. However, these leads should undergo various preclinical analysis and optimization process
before going into clinical trials.
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Table 2: Properties of leads for VP40
Compound
Molecular weight
[g/mol]
Pose
Ligand Energy Number of hydrogen
bonds
Best Ligand Pose Energy
(Dock Score)
Clomiphene 405.95962 127 -138..25 au 16 -13.74 kcal/mol
Glycosidase
Inhibitor
105.32422
144
-100.29 au 6 -11.24 kcal/mol
Amodiaquine 355.86118 143 -133.97au 14 -9.21kcal/mol
Isoflavone 222.2387 150 -92.72au 4 -8.5 kcal/mol
Rimantadine 179.30184 82 -63.254 8 -7.51 kcal/mol
Cytidine 243.21662 94 -100.36 au 4 -6.57 kcal/mol
Table 3: Properties of leads for VP35
Compound
Molecular weight
[g/mol]
Pose
Ligand Energy Number of hydrogen
bonds
Best Ligand Pose Energy
(Dock Score)
Gleevec 589.71255 128 -89.26 au 6 - 8.34kcal/mol
Chloroquine 319.87265 114 -128.29au 16 -6.15kcal/mol
Favipiravir 157.102563 130 -79.36 au 14 - 5.91kcal/mol
Val acyclovir
Hydrochloride
360.79664
93 -114.425 14 -5.26 kcal/mol
Toremifene 405.95956 76 -93.36au 14 - 5.12kcal/mol
Brivudine 333.13532 117 -97.25 12 -4.93 kcal/mol
For VP 40 it was found that there are Clomiphene which show the minimum binding energy (-138.25 au) to protein so
it can be considered for the further docking studies.
For VP 35 it was found that there are Gleevec which show the minimum binding energy (-89.26 au) to protein so it
can considered for the further docking studies.
IV. DISCUSSION
Currently the life threatening Ebola hemorrhagic fever caused by Ebola virus is untreatable with high mortality rates and
hence there is need to develop a proper treatment. The active pocket site of the VP 40 and VP 35 is predicted with the
help of pymol. One potential target is the VP40 matrix protein, the key viral protein that drives the budding process, in
particular by mediating the specific virus-host interactions to facilitate the efficient release of virions from the infected
cells. Key structural and functional domains of VP40 believed to be necessary for efficient budding of virions and virus-
like particles. The Ebola VP35 protein is multifunctional, acting as a component of the viral RNA polymerase complex, a
viral assembly factor, and an inhibitor of host interferon (IFN) production. Thus targeting these proteins, that are
important in growth and pathogenesis, this is appropriate way to treat the infection. Inhibition of these proteins can help to
prevent the growth and pathogenesis of Ebola virus. By screening Zinc Database followed by the docking studies, we
have identified leads for the three targets. ForVP40, six ligands are identified from AUTODOCK out of 10 hits obtained
from ARGUSLAB. For VP35 six ligands were obtained out 10 hits obtained from ARGUSLAB. All these molecules
follow the Lipinski Rule of five, showing their drug likeness. These molecules may constitute to control multidrug
resistant viral infections. For VP 40 it was found that there are Clomiphene which show the minimum binding energy -
13.6au to protein and For VP 35 it was found that there are Gleevec which show the minimum binding energy -8.3au to
protein. We considered these molecule for increasing the binding energy to the protein and change‟s the groups and atoms
of these molecules on the basics of their native charge and nature. so it was considered for the further docking study‟s.
V. CONCLUSION
The development of an effective vaccine against Ebola has progressed further than efforts to identify small molecule
therapeutics to treat infection. Although several vaccine platforms and drug have entered the early stages of clinical
testing, the longevity of the immune response elicited by each is not fully characterized. This is of some concern since
recurrent immunization programs for Ebola hemorrhagic fever seem unrealistic and costly since the disease burden is
currently limited to a specific region of the world. Therefore the current study titled as „In-Silico Drug Designing Against
Ebola Virus: A Genomic Approach‟, is an efforts to understand the genomic relationship of various from the conserved
ISSN 2348-313X (Print) International Journal of Life Sciences Research ISSN 2348-3148 (online)
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nature of Protein RNA dependent RNA polymerase, Nucleoprotein complex, one of the glycoprotein and structural
glycoprotein. Beside the deviation from each other is also found from variation in matrix protein and structural
glycoprotein, which are less conserved. Phylogenetical Sudan strain may evolve into separate clade keeping the link
between a cluster of Zaire, Tai forest, Bundibug strain and Reston strains. Reston strain is diverging from other strains.
Sudan is partially close both to the cluster. All the viral protein have potential antigenic region but matrix protein and
glycoprotein region can be further tested for vaccine development. Among the in-silico tested drug molecules,
Clomiphene and Gleevec found to be binding strongly with target protein VP40 Matrix Protein and VP35 Polymerase
Complex Protein. These drug molecules further can be modified to enhance the binding potential and drug safety. Thus
computational biology helps in understanding the properties, based on which, drug molecule can be designed through
computer aided drug designing reducing the time of therapeutics designing.strain of Ebola virus, protein 3D structure and
drug design. The strains are closely related as observed
REFERENCES
[1] Kantardjieff K and Rupp B. 2004 Structural Bioinformatics Approaches to the Discovery of New Antimycobacterial
Drugs. Curr Pham Des. 10: 1-7
[2] Hoenen T, Volchkov V, Kolesnikova L, Mittler E, Timmins J, Ottoman M, Reynard O, Becker S and Weissenhorn
W. 2005. VP40 octamers are essential for Ebola virus replication. J Virol. 79:1898–905
[3] Weik M, Modrof J, Klenk HD, Becker S and Muhlberger E. 2002. Ebola virus VP30-mediated transcription is
regulated by RNA secondary structure formation. J Virol. 76:8532-8539