The document describes research on developing an in-silico approach to drug design against the Ebola virus. The researcher conducted various genomic and protein structure analyses of the Ebola virus, including multiple sequence alignment of proteins, phylogenetic analysis to study evolutionary relationships between virus species, identification of epitopes and protein structures, and determination of potential drug targets. Molecular docking simulations were then performed to screen compounds from a drug library and identify ligands that could potentially bind to and inhibit key viral proteins.

1. SUBMITTED BY
HARRIS KAUSHIK
Ingt.M.Tech.Bio-Tech(Xth sem)
Department of Life Science
INSTITUTE OF APPLIED MEDICINE & RESEARCH
“IN-SILICO DRUG DESINGING AGAINST EBOLA VIRUS: A GENOMIC APPROACH”

2. INTRODUCTION
Ebola Virus Disease are found in African countries (Guinea ,Liberia ,Sierra etc).
Discover in 1976 Near Ebola River , now the Democratic Republic of the Congo.
Ebola Virus Previously Know as Hemorrhagic Fever.
Five Identical Species, In Which Four Cause Disease in Humans.
Zaire Virus
Sudan Ebola Virus
Tai Forest Ebola Virus
Bundibigyo Ebola Virus
Reston Ebola Virus **
Total No of Deaths - 11,300
(As of 30 August 2015Report by WHO,2015.)
NOTE- Reston Ebola Virus do not cause Disease in humans*
0 20 40 60 80
Zaire virus
Sudan
Tai Forest
Bundibigyo
Reston **
Death Rate%

3. TRANSMISSON
Ebola Virus Transmission cycle (www.independent.co.uk/)
Ebola spread by contact with any surfaces, objects, and materials that have been in
contact with body fluids from a sick person.
Ebola then spreads through human-to-human transmission via direct contact (through
broken skin or mucous membranes) with the blood, secretions, organs or other bodily fluids
of infected people, and with surfaces and materials (e.g. bedding, clothing)
NOTE: Ebola Virus is not spread through the air, by water, or in general, by food
.

4. TARGET IN THE BODY
Ebola Virus Targets in Body. (WHO/emedicine.medscape.com/CDC/Medicine.Net.com/Midterms.)
 It Attacks every organ and tissue of the human body except the bones and skeletal muscles.
Ebola virus attacks on tissue multiplying rapidly in collagen virus causes small blood clots to
form in the bloodstream of the patient.
 The blood thickens and the blood flow slows down. vessels forming red spots on the patient
skin. Also, blood clots does not allow a proper blood supply to many organs such as the liver,
brain, lungs, kidneys, intestines, breast tissue, testicles, etc

5. SYMPTOMS OF EBOLA
 Fever
Headache
 Muscle pain
 Weakness
 Fatigue
 Diarrhea
 Vomiting
 Stomach pain
Symptoms may appear anywhere from 2 to 21 days after exposure to Ebola, but the average
is 8 to 10 days
VACCIENE AND TREATMENT
At this time, there are no vaccines to protect against EVD. Clinical trials for several
vaccines are in various phases and stages. eg (VSV-EBOV, Ad26-EBOV, MVA-EBOV etc)
Ebola virus Symptoms(WHO, Fact SheetAugust2015, http://www.who.int/mediacentre/factsheets/fs103/en/).
LATEST UPDATE
CNN NEWS - VSV-EBOV new Ebola vaccine is highly effective (Laura and Smith.,03,Aug,2015 ).
WIRED NEWS -Vaccines Weren’t Ready for Ebola. We Can Do Better Post by (Kendall Hoyt.,27,Aug,2015)

6. OBJECTIVES
1) Genomic Variation of the different strain present in Ebola Virus
2) Conserved nature of similar protein sequence of different Ebola virus strain
3) Phylogeny analysis of Ebola virus based on each individual protein sequence.
4) Evolutionary accept of Ebola virus strain
5) Epitopes Identification
6) Epitopic region validation through protein structure analysis
7) Identification of Drug Target
8) Drug Designing with respect to target

7. MATERIALAND METHODS
DATABASES
NCBI (www.ncbi.nlm.nih.gov)
Provides numerous free databases and molecular search tools.
Contains several free computerized information processing methods of biological information.
SEQUENCE RETRIEVEAL (www.ftp:ncbi.nlm.nih.gov/genome)
Contain’s all know species genome sequence where access freely
The genomic data files were comprised of “.rpt”, “.ffn”, “.rnt”, “.ptt”, “.faa”, “and .ffn.
PubChem (www.pubchem.ncbi.nlm.nih.gov)
Contain’s database of chemical molecules and their activities against biological assays.
More than 80 database vendors contribute to the growing PubChem database.
InterPro and Inter ProScan (www.ebi.ac.uk/interpro)
Protein sequences are searched using the InterProScan software and checkthem.
InterPro member databases: Pfam, PROSITE, PRINTS, ProDom, Gene3D etc
SOFTWARE
Bioedit (www.mbio.ncsu.edu/BioEdit)
Sequence alignment editor and sequence analysis softaware .
It offers several modes of easy hand-alignment, split-windows views, user defined colors, etc.
Clustal-Omega (www.ebi.ac.uk/Tools/msa/clustalo)
Multiple sequence alignment (MSA) program for proteins.
It produces high quality MSAs and is capable of handling data-sets of hundreds of thousands of
sequences.

8. MEGA6 (www.megasoftware.ne/)
Comparative analyses of DNA and protein sequences .
Building sequence alignments, inferring phylogenetic histories etc.
PHYRE2SERVER (www.sbg.bio.ic.ac.uk/phyre2 /)
Protein structure prediction.
Most powerful and accurate methods for detecting and aligning remotely related sequences .
EPITOPES BCPRES-B CELL PREDICTION SERVER
Identification and characterization of B-cell epitopes play an important role in vaccine design.
Allow’s user to identifi epitopes by three methods.
ARGUS LAB (www.arguslab.com/ArgusLab.html)
Molecular modeling, graphics, and drug design program .
Interactive molecular drawing and geometry optimization.
CHEMSKETCH (www.acdlabs.com/resources/freeware/chemsketch)
ACD/Chem Sketch is a drawing package that allows you to draw chemical structures.
Chem sketch is a molecular modelling software.
DISCOVERY STUDIO (www.accelrys.com)
Drug design and protein modelling research software.
Discovery Studio contains both established gold-standard applications
AUTO DOCK (www.autodock.scripps.edu)
Automated docking tools.
Structure-based drug design.

9. No. Protein Bundibug yo Reston Sudan Tai Forest Zaire
1 Nucleo Protein 1 1 1 1 1
2 Polymerase C.P 1 1 1 1 1
3 Matrix Protein 1 1 1 1 1
4
Spike
Glycoprotein 1 0 1 1 1
5
Second
Secreted
Glycoprotein 1 1 1 1 1
6
Small Secreted
Glycoprotein 1 0 0 1 1
7
Minor
Nucleoprotein 1 1 1 1 1
8
Membrane-
Associated
Protein 1 1 1 1 1
9
RNA-
Dependent
RNA
polymerase 1 1 1 1 1
10
Structural
Glycoprotein 0 1 0 0 0
GENOMIC INFORMATION OF EBOLA VIRUS SPECIES
Ebola is a member of the negative-stranded RNA virus family Filoviridae14,000 nm in
length and average 80 nm in diameter
3'-untranslated region
Nucleoprotein
viral structural protein
VP35-Polymerase Complex Protein
VP40- Matrix Protein
VP30-Minor Nucleoprotein
VP24-Membrane Associated Protein
polymerase(L)
5'-untranslated region
Negative-stranded RNA linear genome, about 18-19 kb size(David,. et al., 2007.)
Ebola virus Protein

10. MULTIPLE SEQUENCE ALIGNMENT
Ebola Virus Species Matrix Protein
Ebola Virus Species Membrane-Associated
Protein
Ebola Virus Species Minor Nucleoprotein
Determination of the consensus sequence of several aligned sequences.
Help’s to prediction of the secondary and tertiary structures of new sequences.
Preliminary step in molecular evolution analysis using Phylogenetic methods for
constructing phylogenetic trees.

11. Nucleoprotein Polymerase Complex Protein RNA-dependent RNA polymerase
Second Secreted Glycoprotein
Small Secreted Glycoprotein Structural Glycoprotein

12. Showing the entire result of the MSA
In Multiple sequence alignment Ebola virus protein. It was found that-
40% - 50 % identical region found – MP , MAP , MnP and PcP .
50% - 70 % identical region found - NP and SmSGP.
70% - 80% highly identical region – RNAP , SnSGP and SGP.
20 % or more Conserved region - PcP and SmSGP .
Rest all the protein are less than 18% CR .
40% or more Semi Conserved region - MP and SGP.
Rest all protein are less than 15 % SCR.
30% - 45% Gap region found - MP,MAP and MnP.
Rest all are less than 10%. GR

13. BundibugyoMP
TaiForestMP
ZaireEVMP
RestonMP
SudanMP
99
99
0.06
0.11
0.11
0.06
0.10
0.04
0.04
0.02
0.000.020.040.060.080.100.12
Matrix protein
PHYLOGENETIC ANALYSIS OF EBOLA VIRUS SEQENCES
We concentrate here on the analysis of Ebola virus protein sequences.
Comparisons of more than two sequences
Analysis of gene families, including functional predictions
Estimation of evolutionary relationships among Ebola virus species.
Membrane-associated protein Minor Nucleoprotein
BundibugyoMAP
TaiForestMAP
RestonMAP
SudanMAP
ZaireMAP
100
65
0.06
0.13
0.16
0.06
0.97
0.08
0.03
0.81
0.00.20.40.60.8
BundibugyoMNP
TaiForestMNP
ZaireMNP
SudanMNP
RestonMNP
99
100
0.07
0.20
0.18
0.07
0.12
0.05
0.06
0.02
0.000.050.100.150.20
BundibugyoNP
TaiForestNP
ZaireNP
RestonNP
SudanNP
100
100
0.11
0.20
0.21
0.11
0.15
0.03
0.05
0.01
0.000.050.100.150.20
Nucleoprotein
BundibugyoPCP
TaiForestPCP
ZairePCP
RestonPCP
SudanPCP
100
100
0.14
0.20
0.20
0.14
1.25
1.12
1.16
0.11
0.00.20.40.60.81.01.2
Polymerase complex protein
BundibugyoSSP
TaiForestSSP
ZaireSSP
SudanSSP
RestonSSP
100
100
0.07
1.28
0.24
0.07
0.15
0.08
0.09
1.04
0.00.20.40.60.81.01.2
Small secreted glycoprotein
BundibugyoSGP
TaiForestSGP
ZaireSGP
SudanSGP
100
0.15
0.30
0.15
0.22
0.07
0.08
0.000.050.100.150.200.250.30
Spike glycoprotein
BundibugyoRNAP
TaiForestRNAP
ZaireRNAP
RestonRNAP
SudanRNAP
100
100
0.09
0.39
1.40
0.09
0.12
0.03
0.27
1.02
0.00.20.40.60.81.01.21.4
RNA-dependent RNA polymerase
ROOT
FLY
NODE
CLADE
BRANCH
Sudan species of Ebola virus are distant related with all other five Ebola virus.

14. EPITOPES IDENTIFICATION
Play an important role in vaccine design.
An epitope, also known as antigenic determinant, is the part of an antigen that is
recognized by the immune system, specifically by antibodies, B cells, or T cells.
Matrix protein Membrane-associated protein Minor nucleoprotein Nucleoprotein
Polymerase complex protein RNA-dependent RNA polymerase Second secreted glycoprotein Small secreted glycoprotein
Spike glycoprotein Structural glycoprotein

15. PROTEIN STRUCTURE PREDICTION
Structure of glycoprotein Envelope glycoprotein
The 3D structure of target proteins helps to find out active site, binding site, possible
conformational changes.
Use in Molecular docking, molecular dynamics simulations etc.
Envelope glycoprotein Envelope glycoprotein
Matrix protein Membrane associated protein Minor nucleoprotein protein Polymerase cofactor

16. QMEAN Server for Model Quality Estimation
Validate the protein 3D structure
 Check protein structure stability
Check protein structure physical and chemical properties
PROTEIN STRUCUTRE VALIDATION
Minor nucleoprotein= QMEAN score= 0.752
Model quality estimation Minor nucleoprotein
Matrix protein, =QMEAN Score=0.648
Model quality estimation Matrix protein
Model quality estimation Nucleoprotein
Nucleoprotein, QMEAN Score=0.628
Model quality estimation Structural glycoprotein
Structural glycoprotein, QMEAN Score=0.427

17. Spike protein, QMEAN Score=0.46
Model quality estimation Spike protein
Small secreted protein, QMEAN Score=0.455
Model quality estimation Small secreted protein
Second Secreted glycoprotein, QMEAN Score=0.475
Model quality estimation Second Secreted glycoprotein Model quality estimation Polymerase complex protein
Polymerase complex protein, QMEAN Score=0.886
Model quality estimation Membrane associated protein
Membrane associated protein, QMEAN Score=0.62

18. ANTIVIRAL TRAGETS IN EBOLA VIRUS
Information about the targets
VP40-VP40 is matrix protein and is key particle for maturation of virion.
Have three domains M, I, and L domain
plays a key role in viral assembly, budding, and virion formation
VP35-is multifunctional in nature -acting as a component of the viral RNA polymerase
complex, a viral assembly factor, and an inhibitor of host interferon (IFN) production
DETERMINATION OF ACTIVE POCKET SITE OF THE TARGETS
Tertiary structures form the pockets, 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.
Active pocket site analysis by the Pymol
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.

19. Active Pocket site of VP 40 by Pymol
Active Pocket site of VP 35 by Pymol
Active pocket
site
Active pocket
site

20. SCREENING OF LIGANDS
22 antiviral compounds are taken from the Pub chem
Draw their structure in Chem Sketch software
Compound database was screened in ARGUSLAB
12 ligands were obtained having proper interaction with the target protein
Compounds with low binding energies were subjected to docking studies.
Antiviral Drug Library

22. 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

23. Water
molec
Amodiaquine
Interaction of VP40 with Amodiaquine
Interaction of VP40 with Clomiphene
Interaction of VP40 with Cytidine
Interaction of VP40 with Glycosidase Inhibitor
Interaction of VP40 with Isoflavone
Interaction of VP40 with Rimantadine
Rimantadin
e
chain
Isoflavone
Glycosidase Inhibitor
Water moleculeProtein chain
Water molecule
Protein chainWater moleculeClomiphene
Protein chain Protein chain Water molecule
Protein chain
Cytidine
Water molecule

24. 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

25. Interaction of VP35 with Chloroquine
Interaction of VP35 with Favipiravir
Interaction of VP35 with Gleevec
Interaction of VP35 with Toremifene
Interaction of VP35 with Val acyclovir Hydrochloride
Interaction of VP35 with Brivudine
Chloroquine
Water molecule
Favipiravi
r
Water molecule
Gleevec
Protein chain Water molecule
Toremifene
Water molecule
Val acyclovir Hydrochloride
Protein chain
Water molecule
BrivudineWater molecule

26. LIGANDS DOCKING FEATURES
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
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
Properties of leads for VP40

27. 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
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)
Properties of leads for VP35
The Clomiphene and Gleevec are the best available compounds which helps to inhibits
the virus action in protein and so it can considered for the further docking studies.

28. CONCLUSION
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 strain of Ebola virus
To Predict Protein 3D structure and
Drug design.
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.

29. REFERENCE
WHO/emedicine.medscape.com/CDC/Medicine.Net.com/Midterms.
David M, Knipe, Peter M, Howley and Fields. 2007. Virology. 5th edition. Lippincott
Williams & Wilkins. USA
Ebola virus Symptoms(WHO, FactSheetAugust2015,
WHO: Trials show new Ebola vaccine is 'highly effective' Laura and Smith, CNN, August
,03,2015
Vaccines Weren’t Ready for Ebola. We Can Do Better Post by (Kendall
Hoyt.,27,Aug,2015) by wired .com