In-Silico Drug Designing Against Ebola Virus: A Genomic

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

Page | 125 Research Publish Journals

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.




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).




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




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




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




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.




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




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

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In-Silico Drug Designing Against Ebola Virus: A Genomic