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 Research & Reviews: A Journal of Bioinformatics
ISSN: 2393-8722 (online)
Volume 1, Issue 3
www.stmjournals.com

Insilico 3D Structure Modeling and Docking to Find

Potent Inhibitors for Thrombocytopenia
Pranati Swain1*, Jasnaik D2
1
Bioinformatics, Orissa University of Agriculture and Technology, Orissa, India
2
School of Biotechnology and Bioinformatics, DY Patil University, Maharashtra, India

Abstract
Lots of people are getting affected by various kinds of auto-immune disease in their day
to day life and thrombocytopenia is one of them, which occurs due to lack of
thrombocytes or platelets. Various kinds of vaccines, drugs are used to stop the disease,
but the effect varies from person to person depending upon the immune system. In
thrombocytopenia disease the integrin beta protein of human affects the persons own
immune system leading in the less production of thrombocytes. Here the main objective of
the study is to analyse the interaction between the disease stimulating proteins and the
ligands or drugs bind to the protein at its active site. The structural prediction along with
the stereo-chemical evaluation of protein helped to understand the function of the protein.
The active sites of protein show the ligand binding regions. 14 different drugs were
considered for the study. Out of which AC1NS627 drug showed best interaction with the
protein as its binding perfectly at the active site of the protein. So from the study, we can
conclude that AC1NS627 is the better drug which can be used for inhibiting the activity of
integrin beta protein.

Keywords: In silico homology modeling, thrombocytopenia, molecular docking

*Author for Correspondence E-mail: rosylora20@gmail.com

INTRODUCTION the symptoms of thrombocytopenia [2].

Platelets are also known as thrombocytes. Activation of thrombocyte is inhibited by
Thrombocytes are the blood cells which act as nitric oxide, endothelial-ADPase,
blood coagulating factor. Thrombocytes are and PGI2. Endothelial-ADPase. Drugs like
the fragments of cytoplasm without nucleus Aspirin, Valproic acid, Methotrexate,
derived from the megakaryocytes of bone Carboplatin, Interferon, Isotretinoin,
marrow. It reduces the chances of death due to Panobinostat causes thrombocytopenia by
hemorrhage during child birth. Thrombocytes inhibiting the function of cyclooxygenase-1
are dark purple in colour. The normal (COX1), and hence normal
thrombocyte count is 150,000 to 450,000 hemostasis. thrombopoietinmimetics,
platelets per microlitre(l) of blood. desmopressin, factor VIIa drugs stimulate the
Thrombocytes control hemostasis [1]. production of Thrombocytes.

Primary thrombocytes are formed by the Thrombocytopenia may be caused by less

formation of coagulation cascade with production of Vitamin B12 or folic acid and
resultant fibrin deposition. Thrombocyte thrombopoietin, Leukemia, Congenital
production is controlled by a amegakaryocytic thrombocytopenia, Grey
hormone thrombopoietin. Thrombopoietin is platelet syndrome, Bernard-Soulier syndrome,
produced in kidney and liver. WiskottAldrich syndrome [3]. Some other
Old Thrombocytes are destroyed by reasons of thrombocytopenia are
phagocytosis in liver and spleen. Decrease in pseudothrombocytopenia, Lyme Disease.
the count of thrombocytes causes Bone marrow biopsy is used for diagnosis
thrombocytopenia. Bleeding gums, nose bleed, of the disease. Idiopathic thrombocytopenic
and gastrointestinal bleeding; menorrhagia; purpura (ITP), also known as immune
and intraretinal and intracranial bleeding are thrombocytopenia, primary immune
thrombocytopenia, primary immune thrombo-

RRJoBI (2014) 31-40 STM Journals 2014. All Rights Reserved Page 31
Insilico 3D Structure Modeling And Docking
cytopenic purpura or autoimmune thrombo-
cytopenic purpura. This is an Autoimmune
diseases in which purpuri crashes, petechiae
are formed and lead to bleed [4]. As it is an
Autoimmune diseases the antibodies like
glycoproteins IIb-IIIa or Ib-IX in ITP anti-
ADAMTS13 in TTP, and HUS anti-
cardiolipin (anti-cardiolipin
antibodies), 2 glycoproteinI in Antiphosphol
ipid syndrome anti-HPA-1a, anti-HPA-5b, and
others in NAIT act against the persons own
immune system. The treatment is carried out
by the use of Steroids
dexamethasone or methylprednisolone. Anti
D, Immunosuppresants such
as mycophenolate mofetil and azathioprine,
Splenectomy, Platelet transfusion etc. Here the
main objective is to analyse the interaction
between the protein causing thrombocytopenia
and the drugs used for the treatment.
Thrombocytopenia is caused by one of the
glycoprotein GPIIIa. HPA produces HPA-1a,
HPA-1b, which further produce glycoprotein
GPIIIa. We considered the integrin beta
protein of homo sapiens as the target protein.
It combines with an extracellular or
intracellular messenger which brings changes
in the cellular activity. Itegrin beta proteins
cell surface has heterodimeric receptors which
mediates dynamic cell-to-cell as well as cell-
to-matrix adhesion. The integrin beta protein is
able to make rapid and reversible changes in
its adhesive function as it acts as
mechanochemical sensors and transducers by
modulating their ligand-binding affinity.
All the subunits possess a large N-terminal
extracellular domain followed by a
transmembrane domain and a short C-terminal
cytoplasmic region. Some subclasses of
integrins have common beta chain and
different alpha chains. The 3D structure of
integrin beta protein of human was constructed
by using MODELLEER9.12 tool. Best quality
model was checked by using PROCHECK,
PROSA, ERRAT, VERIFY 3D and WHAT-
IF. Among of all the models best model was
selected by using overall scores. Protein-
ligand docking was performed between the
disease stimulating protein and the drugs used
to inhibit the production of that stimulating
proteins. 11 different drugs were selected from
drug bank for analysis.
RRJoBI (2014) 31-40 STM Journals 2014. All Rights Reserved
Swain and Jasnaik
The protein-ligand interaction was studied by
using autodock and igemdock tools. Both the
results were analysed to find the best
interaction.
MATERIALS AND METHODS
Sequence Collection
The sequence of the disease stimulating
protein was retrived from uniprot
(http://www.rcsb.org/pdb/home/home.do). The
uniprot ID of the disease stimulating protein is
L7UUZ7, which is an integrin beta protein of
homo sapiens present on ITGB3 gene. The
sequences were downloaded in fasta format
for further study.
Sequence Alignment
The basic local alignment similarity search is
generally done to find out the matching amino
acid and nucleotide sequences. BLAST are of
five different types [5]. The collected sequence
was further subjected to BLAST
(http://blast.ncbi.nlm.nih.gov/Blast.cg) to find
the percentage of identity with other species.
Dimensional Protein Structure
Secondary structure prediction of protein gives
idea about structural pattern from the protein
sequence in terms of helix, sheets and coils
[6]. For this GOR method was used to predict
the secondary structure of HUMAN Integrin
beta (Figure 1).
3D Structure Prediction
The tertiary structures of the protein was
generated by using various bioinformatics
tools i.e. modeller 9.12, swiss-model and
phyre2 server. Three different models were
generated to select the best model by checking
the sterio-chemical evaluation.
Generating Model by using Modeller9.12
The FASTA format sequence were subjected
to BLAST against PDB. The first template
was selected which showed 99% similarity
with 3IJEB. Then the alignment files, model
evaluating files and model single files were
modoified depending upon the target and
template. This process is known as homology-
modelling. After running the commands on the
modeller screen it generates the best model.
The best model was selected on the basis of
lowest DOPE score. Only one model was
selected out of 25 models.
Page 32
Research & Reviews: A Journal of Bioinformatics
Volume 1, Issue 3
ISSN: 2393-8722 (online)

Genearating Model by using Swiss-model which all ligands are binding to that protein
This is a protein homology modeling tool [1517]. For this analysis 14 different drugs
(http://swissmodel.expasy.org/interactive). were collected from drug bank (Table 1).
This modeling server is accessible via the
expasy web server. It gives the clear tertiary Table 1: Lists of Ligands Considered for
structure of a protein model showing the Docking.
regions of alpha helix, beta sheets, beta turns, Sl. no Ligands/drugs SID
coils, helix etc. The FASTA format sequences 1 AC1NS627 113911626
were submitted in the work space and this
generated model [79]. 2 CCG205186 124750244
3 DO5819 47207480
Genearating Models by using Phyre2 4 HMS3229013 99302833
Here also the fasta format sequences were
5 LS_193151 50076383
given as an input. The phyre2 server
(http://www.sbg.bio.ic.ac.uk/phyre2/html/page 6 NSC-696819 525180
.cgi?id=index)also generates 3D model of a 7 QCR-86 164041897
protein [10].
8 RT-016226 204356888

Structure Validation 9 SEMAXANIB 135141433

The structure validation generally involves the 10 SEMAXINIB 53789746
sterio-chemical evaluation along with the 11 SU-5416 163686018
ramachandran plot analysis [11].
12 SUGEN-5416 5329098
PROSA((https://prosa.services.came.sbg.ac.at/
prosa.php) generally helps in the structural 13 UNII-71149535AJ 198984189
validation of a protein PDB file of the model is 14 VEGFR2 204356888
given as an input. It can also be applied to low
resolution structures and approximate models
Prediction of Active Site/ Binding Site of the
obtained early in the structure determination
Protein
process. Active site or binding site is the position at
which an enzyme binds to the substrate. The
The PROCHECK saves server cast P server (http://sts-
(http://services.mbi.ucla.edu/SAVES/Ramacha fw.bioengr.uic.edu/castp/calculation.php) was
ndran/) is generally used to find the region of used to find out the binding regions of the
allowed and disallowed regions of a protein. It protein.
also shows the phi-psi angles. It gives the
sterio-chemical evaluation. The model file is
Checking the Protein-ligand Interaction
generally uploaded in the server, which The interaction can be studied directly from
analyses the backbone conformation of a autodock vina or by using discovery studio
protein [12]. visualization tool. Then the interaction among
the protein-ligand was performed manually by
Protein-ligand Binding selecting the highest volume of the generated
Here the drugs were considered as ligands and model.
the disease stimulating protein was considered
as protein component. The interaction between
RESULT AND DISCUSSION
protein and lignad was studied by using
Secondary Structure of Protein
autodock vina. Both the pdb files of ligands
The GOR online server identified the
and proteins were uploaded and by setting the
secondary structure of HUMAN Integrin beta
parameters the interaction was studied. The
protein with distinct regions of helices and
best interaction was selected on the basis of
strands. Thus, overall the helix 18.65%, Strand
least binding energy [13,14].A docking study
20.81 and coils 60.53% was calculated
was conducted to find the binding sites of the
(Figure 1).
disease stimulating proteins and to check

RRJoBI (2014) 31-40 STM Journals 2014. All Rights Reserved Page 33
Insilico 3D Structure Modeling And Docking Swain and Jasnaik

Fig. 1: Secondary Structure Prediction of HUMAN Integrin Beta Protein.

Tertiary Structure swiss-model generated the best model by

Three different models were generated by
using phyre2 server, swiss-model server and
the modeller9.12 tool. The best model was
selected on the basis of the lowest dope score
from modeller9.12, however, the phyre2 and
refining the loops. The structures were then
visualized by using pymol, discovery studio
visualiser and swiss pdb viwer, respectively to
analyze the models more clearly.

Fig. 2: Structure Obtained from Phyre2. Fig. 3: Structure Obtained from Modeller9.12.

Fig. 4: Structure Obtained from Swiss Model.

RRJoBI (2014) 31-40 STM Journals 2014. All Rights Reserved Page 34
Research & Reviews: A Journal of Bioinformatics
Volume 1, Issue 3
ISSN: 2393-8722 (online)

Structural Evaluation which is modelled. Normally the allowed

The structural evaluation was performed by
using the PROCHECK saves server, rampage
server. The procheck saves server showed the
backbone conformation of protein and the
rampage showed the allowed regions,
disallowed regions and the residues lying in
the outer region. From the percentage of
allowed and disallowed regions we can
analyze which is the best protein structure
region lies between 7090%. The models
which generated using modeller 9.12 lies in
82.0% of the favored region, a model which
was generated using their lies in 75.6% of the
favored region, a model which was generated
using swiss-model lies in 74.00%. From this
observation it was found that the best model
was generated by using modeller9.12 tool
(Table 2).

Fig. 5: Structure from Modeller. Fig. 6: Structure from Phyre2.

Fig. 7: Structure from Swiss Model Server.

Table 2: Sterio-Chemical Evaluation of Protein.

Residues in Residues in Residues in Residues in Number of end Number Total
Model Favoured Additional Generously Disallowed Residues (excl. of proline No. of
Region Allowed Region Allowed Region Region Gly and Pro) Residues Residues
Modeller Output 564 (82.0%) 97 (14.1%) 13 (1.9%) 14 (2.0%) 2 40 788
Phyre2 server
459 (75.6%) 116 (19.1%) 18 (3.0%) 14 (2.3%) 1 36 695
Output
Swiss Model
449 (74.00%) 129 (21.3%) 16 (2.6%) 13 (2.1%) 2 36 695
server Output

RRJoBI (2014) 31-40 STM Journals 2014. All Rights Reserved Page 35
Insilico 3D Structure Modeling And Docking Swain and Jasnaik

Active Site Prediction volume was selected to get the better binding
From the active site prediction we got the sites.
binding regions of the protein. The highest

Fig. 8: Binding Site of Protein.

Docking was chosen as the best interaction. From the

The protein-ligand interaction was performed final output of the docking we can find the
by using Auto Dock tool which showed the residues of the interactions which are given
binding energies between the protein and below (Figures 922).
ligands (Table 3). The least binding energy

Fig. 9: Receptor-AC1NS627. Fig. 10: Receptor-CCG205186.

RRJoBI (2014) 31-40 STM Journals 2014. All Rights Reserved Page 36
Research & Reviews: A Journal of Bioinformatics
Volume 1, Issue 3
ISSN: 2393-8722 (online)

Fig. 11: Receptor+DO5819. Fig. 12: Receptor+HMS3229013.

Fig. 13: Receptor+LS_193151. Fig. 14: Receptor+NSC-696819.

Fig. 15: Receptor+QCR-86. Fig. 16: Receptor+RT-016226.

RRJoBI (2014) 31-40 STM Journals 2014. All Rights Reserved Page 37
Insilico 3D Structure Modeling And Docking Swain and Jasnaik

Fig. 17: Receptor+SEMAXANIB. Fig. 18: Receptor+SEMAXINIB.

Fig. 19: Receptor+SU-5416. Fig. 20: Receptor+SUGEN-5416.

Fig. 21: Receptor+UNII-71149535AJ. Fig. 22: Receptor+VEGFR2.

RRJoBI (2014) 31-40 STM Journals 2014. All Rights Reserved Page 38
Research & Reviews: A Journal of Bioinformatics
Volume 1, Issue 3
ISSN: 2393-8722 (online)

Table 3: Auto-Dock Result Analysis. Nurses and other Allied Healthcare

Name of the Lead AutoDock Binding Professionals. 2011 EBMT.
Compound Energy 2. Sekhon S. S., Roy V., Thrombocytopenia
AC1NS627 8.88 in Adults: A Practical Approach to
CCG205186 5.86 Evaluation and Management. Southern
Med J. 2006; 99(5): 491498p.
DO5819 8.18
3. information@lymphomas.org.uk;
HMS3229013 7.54
www.lymphomas.org.uk.
LS_193151 7.1 4. Scully M., Beverley J. Hunt, Sylvia
NSC-696819 8.49 Benjamin, et al. Guidelines on the
QCR-86 8.38 Diagnosis and Management of Thrombotic
RT-016226 8.55 Thrombocytopenic Purpura and other
Thrombotic Microangiopathies. Brit. J.
SEMAXANIB 8.07
Haematol. 2012; 13652141p.
SEMAXINIB 6.56 DOI:10.1111/j.
SU-5416 7.93 5. Altschul S.F., Madden T.L., Schffer A.,
SUGEN-5416 8.37 et al. Gapped BLAST and PSIBLAST: A
UNII-71149535AJ 8.01 New Generation of Protein Database
VEGFR2 6.75
Search Programs, Nucleic Acids Res.
1997; (25), 33893402p.
6. Garnier J., Gibrat J-F., Robson B. GOR
CONCLUSION Secondary Structure Prediction Method
In this study, we generated the best tertiary Version IV Methods in Enzymology. R.F.
structure of the protein by using the template Doolittle Ed., 266: 540553p.
3IJE with B chain This model has been 7. Arnold K., Bordoli L., Kopp J., et al. The
qualified using several validation methods, SWISS-MODEL Workspace: A Web-
including PROCHECK saves server, rampage, based Environment for Protein Structure
prosa. From the sterio-chemical evaluation of Homology Modeling. Bioinformatics,
tertiary structure of protein, it was easy to 2006; 22(2): 195201p.
study the function of the protein. DOI: 10.1093/bioinformatics/bti770
8. Schwede T., Kopp J., Guex N., et al.
All evidences suggested that the geometric SWISS-MODEL: An Automated Protein
quality of the backbone conformation, residue Homology-modeling Server. Nucleic
interaction and contact and the energy profile Acids Res. 2003; 31(13): 33813385p.
of the structure generated was well within the DOI: 10.1093/nar/gkg520.
limits. The protein-ligand docking study has 9. Kelley LA., Sternberg MJE. Protein
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