Dr. Triloki Pant: Asst. Professor IIIT-Allahabad

Homology Modeling of Mycobacterium tuberculosis H37Rv gyrA protein:
Design and Evaluation of novel gyrA inhibitors

A MASTERS THESIS
Submitted in the partial fulfillment
of the requirements for the award of the degree
of
MASTER OF TECHNOLOGY
in
INFORMATION TECHNOLOGY
(M.Tech. in IT)
Submitted by
Anurag Yadav
IBI2011002
Under the Guidance of:
Dr. TRILOKI PANT

Asst. Professor
IIIT-Allahabad
July 2013
INDIAN INSTITUTE OF INFORMATION TECHNOLOGY

ALLAHABAD 211012 (INDIA)
May, 2013
CANDIDATES DECLARATION
I do hereby declare that the work presented in this thesis entitled Homology Modeling of
Mycobacterium tuberculosis H37Rv gyrA protein : Design and Evaluation of novel gyrA
inhibitors, submitted in the partial fulfillment of the degree of Masters of Technology (M.Tech), in
Bioinformatics at Indian Institute of Information Technology, Allahabad, is an authentic record of
my original work carried out under the guidance of Dr. Triloki Pant due acknowledgements have
been made in the text of the thesis to all other material used. This thesis work was done in full
compliance with the requirements and constraints of the prescribed curriculum.
Date: 14-08-2013
Anurag Yadav
CERTIFICATE FROM SUPERVISOR

Date:
I do hereby recommend that the thesis work prepared under my supervision by Anurag Yadav titled
Homology Modeling of Mycobacterium tuberculosis H37Rv gyrA protein : Design and
Evaluation of novel gyrA inhibitors be accepted in the partial fulfillment of the requirements of
the degree of Master of Technology in Information Technology for Examination.
Guides name & Signature

Faculty designation, IIITA
Date:
Place: Allahabad
Countersigned by Dean (A) ____________________
_______________________
____________________
_______________________
CERTIFICATE OF APPROVAL
The forgoing thesis is hereby approved as a credible study in the area of Information Technology
and its allied areas carried out and presented in a manner satisfactory to warrant its acceptance as a
prerequisite to the degree for which it has been submitted. It is understood that by this approval the
undersigned do not necessarily endorse or approve any statement made, opinion expressed or
conclusion drawn therein but approve the thesis only for the purpose for which it is submitted.
Signature &Name of the Committee members
___________________________
On final examination and approval of the thesis
___________________________
ACKNOWLEDGEMENTS
I am highly grateful to our honorable Director, IIIT-Allahabad, Prof. M.D. Tiwari for, his ever
helping attitude and encouraging us to excel in studies. Besides, he has been a source of inspiration
during my entire period of M.Tech at IIIT Allahabad.
I am thankful to Prof. G.C. Nandi, Dean Academics, IIIT Allahabad for providing all the necessary
requirements and for his moral support for this dissertation work as well during the whole course
of M. Tech.
The most notable source of guidance was my advisor, Dr. Triloki Pant, Assistant Professor, IIIT
Allahabad. I owe his a great deal of thanks for taking me under his wings and allowing me to soak
up some of her knowledge and insight. He has not only made me to work but guided me to orient
towards research. I thank her for teaching me the ability to think critically and analytically through
the classical discussions we had in her office.
Dr. C.V.S Siva Prasad, HOD, Divisional of Applied Sciences and IRCB, IIITA ,
Indo Russian Center for Biotechnology for his honest
Coordinator of
dedication towards our education and
career and for being with us in various levels of academic pursuits.

I am also grateful to Dr. G. Satyavani , Dr. Ashutosh Mishra, Dr. Amit Prabhakar
for
their support and motivation throughout my research project work.
I am also thankful to rest of classmates and M.Tech. friends for their co-operation during my
work. I am also thankful to them for helping me in my project work and also some kind of
discussion regarding my work which helped me to understand the concept regarding my work.
This acknowledgement will not complete until I pay my respectful homage to my family
especially my parents, my brother and my sister, whose enthusiasm to see this work complete was
as infectious as their inspiration. I am grateful to my parents for their efforts in building my
career, cheerfully bearing with my whims and for letting me make my own decisions.
Finally, I thank God for giving me an opportunity to thank all these people.
Place: Allahabad
Date:
Anurag Yadav
Contents
1 : Introduction1
1.1
Currently Existing Technologies
1.2
Drug Design Cycle

1.2.1 Homology Modeling
1.2.2 Docking
1.2
Analysis of Previous Research
1.3
Problem Definition and Scope
1.4
Formulation of present problem

1.4.1 Introduction to Trovafloxacin
1.4.2 Introduction to Moxifloxacin
1.4.3 Target for Trovafloxacin and Moxifloxacin
1.4.4 DNA gyrase
1.5
Organisation of thesis
2: Description of Software and Tools Used.10

2.1
2.2
2.3
2.4
2.5
2.6
ACD/ ChemSktech
MOE (Molecular Operating Environment)
Drug Bank
PubMed
Modeller 9.12
Protein Data Bank
3: Theoretical Tools- Analysis and Development..12

3.1: Sequence retrieval of gyrA genes
3.2: Screening for best homologous templates
3.3: In silico Comparative Modeling of gyrA protein
3.4: Model refinement, validation and evaluation
3.5: Submission of Modeled structure to PMDB
3.6: Selection of potential drug candidate against gyrA

3.7: Ligand
3.8: Docking in MOE
3.8.1: Methodology Overview
4: Result21
4.1 : Docking result with Trovafloxacin Derivative
4.2 : Docking result with Moxifloxacin Derivative
5: Discussion..34
6: Conclusion..35
7: Future work..36
8: References.37
List of figures
Figure 1: Colonies of Mycobacterium tuberculosis

Figure 2: Symptoms of M. tuberculosis in Human lung
Figure 3: Structure of Trovafloxacin
Figure 4: Structure of Moxifloxacin
Figure 5: Sequence alignment between Target and Template
Figure 6: 3-D structure of Modeled protein
Figure 7: Ramachandran plot of target protein
List of tables
Table 1:
Structure of Trovafloxacin derivatives
Table 2:
Structure of Moxifloxacin derivatives
Table 3:
Docking result with Trovafloxacin derivatives
Table 4:
Docking result with Moxifloxacin derivatives
Abbreviations
gyrA
Dna gyrase subunit A
RMSD
Root Mean Square Deviation
E_conf
Energy of Conformer
Final Energy
PDB
Protein Data Bank
PMDB
Protein Model Database
MW
Molecular Weight
H-Bond
Hydrogen Bond
H-acc
Hydrogen Bond Acceptor
H-don
Hydrogen Bond donor
T.B
Tuberculosis
MDR
Multi Drug Resistance
MTB
Mycobacterium tuberculosis

Abstract
The present study focuses on determining and validating the structure of a protein target
and conducting a preliminary in
silico
study on
Trovofloxacin
and Moxifloxacin
compounds for the design of a putative drug.
gyrA
encodes
Dna
gyrase subunit A,
important
for cell
and multiplication of bacteria. The crystal structure of gyrA is not
DNA
replication
available in any
structural database. In this study we modeled a 3D structure of gyrA by X-ray crystal

structure of N-terminal domain of Dna gyrase subunit A of Mycobcterium tuberculosis
H37Rv (PDB ID: 3IFZ, Chain A), used as the template.
The receptor was docked to the gyrA inhibitor drugs and final energy value of each
confomer obtained
as
follows
Gatifloxacin
(-11.2837),
Ciprofloxacin
(-11.2814),
Norfloxacin (-9.7557), Moxifloxacin (-11.3729), Trovafloxacin (-11.4397), Gemifloxacin

(-11.0109) and Tamifloxacin (-8.4628) using the docking suite of MOE. Trovafloxacin and
Moxifloxacin are chosen as best drugs on the basis of their binding energies. We
improve the binding efficiency and steric compatibility of these
tried to
two drugs namely
Trovafloxacin and Moxifloxacin.
Several modifications were made to the probable functional groups which were interacting
with the receptor molecule. Analogs of these drug molecule were prepared using ACD
ChemSketch and docked using MOE docking . Trovafloxacin Analog 6 and Moxifloxacin
analog 6 were detected with significant energy values.
Indian Institute of Information Technology, Allahabad

1.Introduction
1.1 Currently Existing Technologies
Aim of the structure based drug design or rational drug design, is to find out the chemical
compounds, that can bind to the biological molecule. Designed molecule should be able to
activate or inhibit the target molecule. Due to the rapid progress in the field of Human
Genome Project, newer drug targets are available. Together with advances in structural
determination techniques such as nuclear magnetic resonance, crystallography and even
homology modeling, structure-based design of ligands or inhibitors has emerged as
an important tool in drug discovery and pharmaceutical research [1] .
Several insilico methods are available to extract the relevant information from the known
structure of any compound and used it for the design of novel drug molecule. Due to rapid
progress in sequencing projects all over the world newer target are identified for the different
diseases
[2]
. So, to handle these vast amount of data generated through these sequencing
projects several bioinformatics software and databases are developed [3].
Nowadays scientists are working to develop new software and server to predict the threedimensional structure of protein from its one-dimensional amino acid sequence
[4]
. To
understand the function of target macromolecule several computational methods are develop
to extract the information from the three-dimensional structure of macromolecule. These
extracted information can be used for the design of small ligand molecule for the target,
which block the target molecule and act as improved drug.
Most of the available drugs in the market are found through large scale random screening of
compound for which no structural information was available, that is, lakhs of compound are
screened from the database for its activity. At the end of the high-throughput screening small
number of compounds are found which are active against the target. A good lead compound
is active at concentration of 10mM or less [5].
First case of the structure based drug designing was reported in 1976 at Welcome
laboratories in United Kingdom by Beddell and Goodford. Hemoglobin was selected as the
1

Design and evaluation of novel gyrA inhibitors
target molecule of this project is to develop a drug molecule that act as similar to the
naturally occurring allosteric effector di-phosphoglycera.
1.2 The Drug Design Cycle
The process of structure-based drug design is an iterative one and often proceeds
through multiple cycles before an optimized lead goes into phase I clinical trials. The first
cycle includes the cloning, purification and structure determination of the target protein or
nucleic acid by one of three principal methods: X-ray
crystallography, NMR, or
homology modeling. Using computer algorithms, compounds or fragments of compounds

from a database are positioned into a selected region of the structure. These compounds are
scored and ranked based on their steric and electrostatic interactions with the target site
and the best compounds are tested with biochemical assays. In the second cycle,
structure determination of the target in complex with a micromolar inhibition in vitro,
reveals sites on the compound that can be optimized to increase potency. Additional cycles
include synthesis of the optimized lead, structure determination of the new target-lead
complex, and further optimization of the lead compound. After several cycles of the drug
design process, the optimized compounds usually show marked improvement in binding
[6]
and, often, specificity for the target
14

1.2.1 Homology Modeling
Now days there are number of technique developed in molecular biology that provide easy to
identification,
sequencing
of
DNA,
RNA
or
proteins. Three-dimensional structure
determination of protein by experimental methods is very costly and time consuming.. In the field
of structural biology the main objective to find out the three dimensional structure of
protein from the there sequences
[7]
. Homology modeling is technique in the field of drug
design used to predict the tertiary structure of protei from its primary structure. These insilico
generated protein models, are similar in all content with experimental structures and provide
a concrete result for analysis of protein structure and function, interaction with other
molecule, and structure based drug designing with increased stability [8].
General Procedures
The steps to creating a homology model are as follows [9]:
Identification of the target protein through protein Blast
Alignment of the query sequence and template sequence
Co-ordinate generation for the structurally conserved region
Co-ordinate generate conformations for the loops regions
Build the side-chain conformations
Refine and evaluate the unknown structure
1.2.2 Docking
Several computational methods has been developed during the last decade to study the
formation of intermolecular complexes. It is well known that the activity of any drug molecule
is dependent on its binding with the pocket of the another molecule, which is commonly
protein. Docking process involves the searching of a ligand conformation which is
geometrically and energetically fit for the binding site of a protein.
Docking between ligand and protein molecule is a two-step process:
15

Search Algorithm :
Used to find the best possible binding mode between the protein and its ligand.
Scoring Function:
Used to rank the search result and find out the best binding mode on the basis of energies of
the complexes or on the basis of energy differences between the bound and the unbound states
of the ligand and protein
[10]
. The energy difference is called binding free energy. Beyond the
original goal of the docking algorithm to find out the best fit between the ligand and its specific
protein, it also has newer application like denovo drug design, database searching and
generation of lead molecule [11].
1.2 Analysis of Previous Research
Mycobacterium tuberculosis (MTB) is a prokaryotic bacteria and survive only in aerobic

conditions[12]. Bacteria was first time discovered by Robert Koch in 1882. Due to the high
content of lipid in its cell wall , M. tuberculosis do not get stained by any bacteriological stain,
such a Methylene blue. So, the staining of Mycobacteria requires specific techniques such as
acid-fast and Ziehl-Neelsen staining.
Figure 1: Colonies of Mycobacterium tuberculosis
16

Tuberculosis is the disease caused by the Mycobacterium tuberculosis. Tuberculosis was
ranked second in death from any infectious disease world-wide, Killing approximately 1.3
million in 2007. The primary site for infection is lung (pulmonary tuberculosis), but other
organs may also be affected. In 25%cases of pulmonary tuberculosis, person do not develop
any specific symptoms. The patient only show symptoms of not felling well, fatigue, and
weightness.
.
Figure 2: Symptoms of M. tuberculosis in Human lungs
1.3 Problem Definition and Scope
Currently most of the Mycobacterium organisms have become resistant to marketed

Quinolone derivatives because their site of action (active site) has mutated loosing the
sensitivity for different Quinolone. Some of the Quinolone based drugs thus disappeared
from the market. In this project work we try to develop novel Quinolone derivative which
effective against various Mycobacterium species which becomes resistant. They become
resistance because the target sites become change so, we have to find new target site and
related drug. Quinolone derivative drug has severe adverse effect [13].
These are marketed Quinolone drug
Flerofloxacin, Lemofloxacin, Pefloxacin,
Levofloxacin, Sparfloxacin, Ofloxacin, Moxifloxacin, Norfloxacin, Enoxacin, Dextrofloxacin,

Nalidixic acid, Trovafloxacin, Ciprofloxacin etc.
17

In this project I worked on Mycobacterium tuberculosis which causes tuberculosis disease,
and is one of the most important throat and lung disease.
Now days Mycobacterium tuberculosis has become highly resistant to available
fluroquinolone drugs. Resistance is develop due to a point mutation in gyrA gene, so
Alanine was replaced by Serine at position 74(A74S), with resistance [14].
1.4 Formulation of Present Problem
Computer aided drug design like structure based approach can be used effectively
against resistant Mycobacterium
inhibit the synthesis
computationally
of
species. To develop novel drug which bind to target and
DNA
gyrase
of
Mycobacteria.
We
have
designed
new Trovafloxacin and Moxifloxacin derivative which bind to active site
of the receptor lead to inhibition of DNA gyrase activity.
This project will help in designing the new Trovafloxacin and Moxifloxacin derivatives drug.
There is need for designing new bactericides agents as the problem of resistance developed in
the strains of the pathogens and also sensitivity of some patients with some drugs. Particularly
this project is important for me as it is giving me opportunity to work on live projects
whose predictions will be validated in wet-lab with collaborations with some Laboratories
around the world.
1.4.1: Introduction to Trovafloxacin
Trovafloxacin is a broad spectrum antibiotic which belongs to the family of fluoroquinolone.

Trovafloxacin was sold under the name Trovan. Trovafloxacin checks the growth of DNA
gyrase and topoisomerase IV [15]. DNA gyrase is the enzyme which play an important role in
the bacterial DNA replication by uncoiling the supercoiled DNA.
18

H
F
H
O
N
H
H
H
H
H
N
H
Figure 3. Stucture of Trovafloxacin
1.4.2 Introduction to Moxifloxacin

Moxifloxacin is also antibacterial agent. It belongs to the fourth generation fluoroquinolone
family. It is sold worldwide under the name Avelox and active against Gram-positive and
Gram-negative bacteria. Moxifloxacin checks the growth of bacteria by inhibiting the activity
of DNA gyrase a type II topoisomerase and Topoisomerase IV [16].
Figure 4. Structure of Moxifloxacin
1.4.3 Target of Trovafloxacin and Moxifloxacin
The main target enzyme of Trovafloxacin and Moxifloxacin antibiotics , the DNA gyrase
and topoisomerase II. DNA gyrase is a replicative
enzyme
which is responsible for
replication of DNA. These drug inhibit the enzyme DNA gyrase and topoisomerase II
[17]
. Therefore, replication of DNA is inhibited and bacteria gets kill.
19

1.4.4 DNA gyrase
Bacterial DNA gyrase is a type II topoisomerase and well known target to check the bacterial
growth. DNA gyrase is a tetrameric protein consist of two chains, A and B, that is, it is a type
of A2B2 protein. Active site for Breakage and reunion of DNA molecule is present on the A
subunit while energy for this process is provided by hydrolysis of ATP molecule by subunit B.
DNA gyrase is an ATP dependent molecule that is it require ATP for its functioning and creates
a transient break in the ds DNA [18].
1.5 Organisation of Thesis
In the first chapter we have discussed about the concepts involved in the process of drug
designing. First, we discussed about the in silico method for protein three-dimensional
prediction, called homology modeling or comparative homology modeling. Thereafter, we also
discussed about another step of drug designing which is called Docking.
In the next chapter we talk about the previous research done which has to be done in the field of
tuberculosis. We also talked about the Mycobacterium tuberculosis, causative agent for the
tuberculosis, its DNA gyrase enzyme and the mutations produced in the enzyme, available
inhibitors drugs for the inhibition of DNA gyrase activity.
In the third chapter, we discussed about the softwares, biological databases and online tools
used in this project. We mainly used modeler, SAVS (Structural Analysis and Verification
Server), MOE (Molecular Operating environment), ACD/ChemSketch, Drug bank.
The next chapter which is theoretical tools analysis and development in which we have given the
each and every detail of our work. It includes the sequence retrieval of the mutated protein,
Screening of the homologous template, homology modeling of DNA gyrase, Model refinement,
Validation and evaluation, Submission of modeled structure to the PMDB and the selection of
potential Drug candidates against gyrA protein.
In the result and the analysis chapter we have given the detailed of result obtained through the
docking of newly sketched drugs with the target protein. The result include final score, E score,
H-bond, H-acc, H-don, MW, log-P value
20

.
In the conclusion part we discuss the final result and the outcome of the thesis.
Finally, the last chapter which is recommendation and future work we discussed how we move
further for the experimental proof of our study and chemical synthesis of the designed molecule
in the laboratory and its effect on the mutated DNA gyrase protein.
21

2: Desription of the Software Used

In this project we used different bioinformatics software like (MOE, ACD/ChemSKetch,
Modeller), Online tools (Structural Analysis and Verification Server), Biological databases
(Pubmed, Drugbank, PDB ).
2.1 ACD/ChemSketch
ACD/ChemSketch is chemical drawing and graphic designing tool to draw molecules, calculate
chemical properties an d preparing reports and presentation.
2.2 MOE (Molecular Operating Enviroment)

MOE is tool widely us dint the field of bioinformatics. MOE programming suite can be used for
the structure based drug design, Protein Modeling, Chemiinformatics and High throughput
screening.
2.3 Drug Bank
Drug bank is a unique resource to obtain the details of drug (chemical) data with their drug
target(protein). Each entry of the drug bank contains more than 80 different data fields. Drug
Bank file provide information about the drug as well as information about the target for the
drug[19].
2.4 PubMed
PubMed is free search engine develop by the NCBI. PubMed provide free access to MEDLINE
(Medical Literature Analysis and Retrieval System Online) a bibliographic database which is
develop by NLM, the worlds largest medical library. PubMed is also abstracts of the biomedical
research articles [20].
22

2.5 Modeller9.12
Modeller program was written by Andrej Sali at the University of California, San Francisco.
Modeller works on the principle of percentage similarity between target and template sequence.
2.6 Protein Data Bank
The PDB (Protein Data Bank) is the single worldwide archive of structural data of Biological
macromolecules, established in Brookhaven National Laboratories (BNL) in 1971
[21]
It
contains structural information of the macromolecules determined by X-ray crystallographic,

NMR methods etc.
23

3: Theoretical Tools-Analysis and Development

Bioinformatics is an emerging field which can help to design novel drugs and to improve the
features of existing drugs. CADD is the specialized field of the bioinformatics for studying drugrecptor interaction. Methodologies used in the field of CADD are highly dependent on the
bioinformatics databases, tools and application[22]
3.1 Sequence retrieval of gyrA gene
The protein sequence of
Mycobacterium
tuberculosis
H37Rv
Number NP_2145201 was retrieved from NCBI Reference
gyrA,
Accession
Sequence database and the
FASTA sequence is used for our studies. The protein sequence is mutated at 74 position from
Ala to Ser.
3.2 Screening of best homologus template.

The target protein sequence was blasted using BLASTP
[23-24]
across Protein Data Bank to
obtain the most identical structures based on the percentage of identity, similarity,
expectation values
and alignment scores which could be considered as templates in the
modeling procedure.
3.3 In silico Comparative Modeling of gyrA Protein

An alignment between the target and temple is performed. An in silico comparative modeling
of the gyrA protein was carried out by the MODELLER 9.12
[25]
. The best homolog identified
earlier through BLASTP was used to generate alignment, atom and the script files for
modeling. The modeled protein was visualized by MOE.
3.4 Model refinement, validation and evaluation

The modeled protein is validated by PROCHECK
[26]
. The parameters include the covalent
bond distances, angles and torsion, stereochemical validation and atom nomenclature. The
input for the PROCHECK is a single file containing the co-ordinates of protein structure.
Format for the input file is PDB file file format. The output of PROCHECK, show detailed
information of every protein residue. The plots are output in PostScript format, it shows
Ramachandran plot.
24

3.5 Submission of Modeled structure to PMDB
The modeled gyrA Protein was deposited to the Protein Model Data Base [27] downloaded with
the PMID PM0077412.
3.6 Selection of Potential Drug Candidates against gyrA protein

The Pubchem
[28]
, KEGG
[29]
, and Drug Bank databases provides collection of drugs that help to
inhibit the function of DNA gyrase. The * .mol structure files of these drugs were obtained from
the Drug Bank and used in our study. The DrugBank database has a wide collection of bacterial
antibiotics and the drugs were directly obtained in .mol format for docking based on the literature
studies.
25

Figure 5 : Sequence Alignment between Target and Template
26

Figure 6 : 3-D structure of Modeled protein
27

Ramachandran plot: 88.7% core 10.1% allow 1.1% gener 0.0% disall
Figure 7: Ramachandran plot of Target protein
3.7. Ligand
The existing known Quinolones
drugs have become resistant, so there was need for new
modified Quinolines derivatives. The derivatives of Trovafloxacin and Moxifloxacin are used
as ligand.These ligands are sketched using ChemSkecth.
28

Trovafloxacin Derivative Structure
F
F
F
F
OH
N
N
NH 2
H
N
H 2N
H2 N
OH
OH
H
Analog 2
Analog 1
F
OH
HO
OH
O
F
OH
O
N
OH
N
HO
OH
HO
H
N
N
NH2
OH
HO
HO
H
O
F
HO
OH
Analog 4
Analog 3
HO
O H
H O
OH
O
N
OH
O
N
N
HO
H O
OH
O
H
O H
H O
Analog 5
H O
O H
Analog 6
Table 1
29

Moxifloxacin Derivative Structure
CH3
CH3
HOOC
O
H3C
O
H
CH3
N
H
Analog 1
Analog 2
CHH
33C
O
CH3
O
H
H
N
H3C
H2N
H
N
N
O
H
H
F
Analog 4
Analog 3
H3C
H2N
CH3
H
N
CH3
H
N
N
O
Analog 6
Analog 5
Table 2
30

3.8. Docking in MOE

Docking is done between a flexible ligand and rigid macromolecule target to search for a unique
conformation for a ligand. Different number of conformation are generated and scored for each
ligand molecule to determine best binding mode. The different configurations of the ligand
molecule are called poses.
.
3.4.1: Methodology Overview
In MOE, the docking process is done in the following stages:
Conformational Analysis:
If the conformation database does not contain the ligand conformation Dock will generate the
different number of conformation for the ligand molecule by applying allowable torsions angles
to rotable bonds. Bond angle and bond length of the ligand molecule will not altered. Foe small
ligands different conformations are generated by a systematic search. For large ligand,
conformations are generated by stochastic search.
Placement :
Different placement methods are available for the generation of different conformation of
ligand molecule. Following placement methods are available, Alpha Triangle(Default), Triangle
Matcher, Proxy Triangle, Alpha PMI, None.
Pharmacore Filtering :
Generated conformations for the ligand molecule need to satisfy a pharmacophore query.
Such type of queries are important to direct search for known important interactions.
31

Rescoring(l) :
Poses generated through the different placement method are rescored using different
rescoring methods. Good poses are low by all the available scoring methods.
Refinment:
Generated poses are refined using molecular mechanics force field or grid-based energetic.
Rescoring(2):
After refinement, selected poses are rescored using by using available scoring schemes.
32

4. Results
Docking with proposed structure of Trovafloxacin and Moxifloxacin shows better Hbonding and binding energy with Target protein gyrA , while the docking with known
or existing bactericides showing poor binding energy and H-bonding.
Trovafloxacin analog 6 and Moxifloxacin analog 6 show better H-bonding and binding
energy with the target protein then its predecessor.
33

4.1. Docking result with Trovafloxacin derivatives
Ligand
Active Site
Interaction Data
Final Score
9.9646
E_conf
0.0000
H-bond
No
H-acc
No
H-don
No
Amino acid
No
M.W
417.14
Log P
0.99
Active SiteWith Ligand
Ligand Interaction
Table 3a- Docking result with Trovafloxacin Analog 1
34

Ligand
Active Site
Interaction Data
Final Score
-9.6316
E_conf
1.0000
H-bond
H-acc
No
H-don
Amino acid
GLY(353)
M.W
446.12
Active Site with Ligand
Ligand Interaction
1.40
Log P
Table 3b- Docking result with Trovafloxacin Analog 2
35

Active Site
Ligand
Interaction Data
Final Score
7.5610
E_conf
0.0000
H-bond
H-acc
No
H-don
Amino acid
MET(35)
M.W
418
Log p
1.98
Ligand Interaction
Table 3c- Docking result with Trovafloxacin Analog 3
36

Active Site
Ligand
Interaction Data
Final Score
-11.137
E_conf
2.4023
H-bond
H-acc
No
H-don
Amino acid
TYR(32)
M.W
447.06
Log P
-0.20
Ligand Interaction
Table 3d- Docking result with Trovafloxacin Analog 4
37

Active Site
Ligand
Interaction Data
Final Score
-9.2662
E_conf
0.8000

H-bond
1
H-acc
1
H-don
No
Amino acid
Ser14
M.W
413.10
Log P
0.99
Ligand Interaction
Table 3e- Docking result with Trovafloxacin Analog 5
38

Active Site
Ligand
Interaction Data
Final Score
-12.4770
E_conf
2.6023
H-bond
H-acc
No
H-doc
Amino acid
Asp30 Val183
Gly351 Gly351
M.W
378.03
Ligand Interaction
-0.43
Log P
Table 3f- Docking result with Trovafloxacin Analog 6
39

4.2. Docking result with Moxifloxacin derivatives
Active Site
Ligand
Interaction Data
Final Score
-9.7132
E_conf
0.6406
H-bond
No
H-acc
H-don
1
Amino acid
Asp350
M.W
415.23
Log P
1.85
Ligand Interaction
Table 4a- Docking result with Moxifloxacin Analog 1
40

Ligand
Active Site
Interaction Data
Final Score
-9.0229
E_conf
1.4161
H-bond
No
H-acc
No
H-doc
No
Amino acid
No
M.W
415.23
Log P
2.47
Ligand Interaction
Table 4b- Docking result with Moxifloxacin Analog 2
41

Ligand
Active Site
Interaction Data
Final Score
-9.3648
E_conf
2.3992
H-bond
No
H-acc
No
H-doc
NO
Amino acid
No
M.W
400.19
Log P
1.57
Active
Ligand Interaction
Table 4c- Docking result with Moxifloxacin Analog 3
42

Ligand
Active Site
Active site
Interaction Data
Final Score
-9.8935
E_conf
2.6224
H-bond
H-acc
No
H-doc
Amino acid
Ser54 Gly352
M.W
373.18
Log P
2.40
Ligand Interaction
Table 4d- Docking result with Moxifloxacin Analog 4
43

Ligand
Act
Active site
Interaction Data
Final Score
-9.7361
E_conf
3.0156
H-bond
H-acc
No
H-don
Amino acid
Val283 Gly352
M.W
400.23
Log P
2.13
Ligand Interaction
Table 4e- Docking result with Moxifloxacin Analog 5
44

Ligand
Active Site
Interaction Data

Active site with Ligand
Final Score
-11.9563
E_conf
1.1755
H-bond
H-acc
H-don
Amino acid
Gly351 Ser34
Ser34
M.W
343.21
Log P
3.76
Ligand Interaction
Table 4f- Docking result with Moxifloxacin Analog 6
45

5. Discussion
The Mycobacterium tuberculosis belongs to the Kingdom Bacteria. M. tuberculosis is highly

aerobic and requires high level of oxygen for its survivel. M. tuberculosis is the causative
agent of Tuberculosis. Tuberculosis is a highly infectious disease and primarly affect lungs.
Due to presence of waxy coating (mycolic acid) around the cell membrane, making its cell
impervious to Gram staining, so staining requires special techniques such as acid fast. So, M.
tuberculosis is neither Gram positive or Gram negative.
The Trovafloxacin and Moxifloxacin drugs affect the replication process of tuberculosis bacteria
by the inhibition of DNA gyrase enzyme. DNA gyrase is an ATP dependent molecule (requires
ATP), relaxes the negative supercoiling of DNA molecule. M .tuberculosis, DNA gyrase has
become resistant to the available drugs, because of the mutation at binding site. The possible
reason behind the mutation is prolonged use of the drugs. So, We design novel derivatives of the
drugs and perform in silico study of these drugs with the DNA gyrase.
The complete structure of gyrA is not available in protein data bank (PDB) so, the target protein
was modeled by comparative homology modeling using modeller software (9v7 version). The
accuracy of the model was 88.7 % of residue fall in core region and other 10.1 % in allowable
region in Ramachandran plot. We
have screened out best two molecules on the basis of
Hydrogen bonding and binding energy from above proposed derivative compounds using MOE
docking software.
46

6. Conclusions
Interaction between the drug and receptor molecule is a key factor in the field of drug designing.
For Trovafloxacin and Moxifloxacin drugs the target protein is bacterial DNA gyrase, this
enzyme is essential for the replication of bacterial DNA.
Nowadays, the bacterial DNA gyrase become resistance to available inhibitors due to
mutation int the active site of the protein. Mutation may be caused due to prolonged use of
bactericides. We designed some novel derivatives of Trovafloxacin and Moxifloxacin and
studied insilico interaction between the derived compound and the mutated DNA gyrase.
3D structure of the mutated DNA gyrase is not available in PDB. To generate the threedimensional structure of the mutated protein Modeller software is used . The accuracy of the
model was 86.9% with residue fall in core region and other 11.4% in the allowable region of
the Ramachandran plot.
When the receptor (DNA gyrase) was docked with the drug the
energy
value
obtained was; Trovafloxacin (-11.4397), Moxifloxacin (-11.3729 ). After docking different

analog of Trovafloxacin and Moxifloxacin energy value obtained was Trovafloxacin Analog 6 (12.4770), Maxifloxacin Analog 6(-11.9563). From these docking energy obtained through the
Trovafloxacin and Moxifloxacin analog we can conclude that these analog may be better drugs
than the available drugs.
47

7. Future work
Our project aim is to find out the novel bactericides for inhibition of DNA gyrase, an important
enzyme required for the replication of the bacterial DNA. Through our study we find novel
bactericides which might be helpful for the inhibition for replication of bacterial DNA.
These newly designed compounds may act as inhibitor for the bacterial DNA gyrase enzyme,
through their drug likeness property like efficacy, toxicity and pharmacokinetic properties need
to be studied through wet lab.
For future work following steps need to be taken:
1. To obtain more results from the Zinc database, different parameters will be used.
2. Chemical synthesis of proposed molecule in laboratotry.
3. Evaluating the result of proposed molecule with mutated Mycobacterium tuberculosis species.
48

8. References
[1]
health-cares.net(http://respiratory-lung.health-ares.net/aspergillosis.php July18,2005
[2]
Zeng, Mini-Review Computational Structure-Based Design of Inhibitors that

Target Protein surfaces Combinatorial Chemistry and High Throughput Screening , 3,
355-362 Jun, 2000.
[3]
Andrade, M. A., & Sander, C. Bioinformatics: from genome data to biological

knowledge Curr Opin Biotechnology 8, 675683 1997.
[4]
Kingsbury, D. T. Dev Res Bioinformatics in drug discovery Drug, 41, 120128 1997.
[5]
Dunbrack, R. L., Gerloff, D. L., Bower, M., Chen, X. W., Lichtarge, O., & Cohen, F.
E. Meeting review The Second Meeting on the Critical Assessment of Techniques
for Protein Structure Prediction (CASP2) , Protein structure prediction. Curr Opin
Biotechnology 9, 383389.1997.
[6]
Gerhard, Klebe, Recent developments in structure-based drug design , J Mol Med,

78:269281, 2000
[7]
Anderson,
A.C.,
Review,
The
process
of
structure-based
drug
design ,
Chemistry & Biology Vol. 10, 787797, 2003.
[8]
Vriend G. Homology modelling for beginners introduction June 21 2000.
[9]
Chung SY, Subbiah S A structural explanation for the twilight zone of

protein sequence homology Structure , 4: 112327 1996.
[10] David R. Bevan Molecular modeling of proteins and nucleic acids , 445-485.
[11]
Tao, Peng and Lai, Luhua, Protein ligand docking based on empirical method
for binding affinity estimation , Journal of computer-aided molecular design. 15

49

:429-426, 2001.
[12]
http://en.wikipedia.org/wiki/Mycobacteria.
[13]
http://www.drugbank.ca/drugs/DB00685.
[14]
Lau RW, Ho PL, Kao RY, Yew, Functional analysis of gyrA mutation at position
74, Journal of Medical and Allied Science, 443-478.
[15]
http://en.wikipedia.org/wiki/Trovafloxacin.
[ 1 6 ] http://www.wikipedia.org/wiki/Moxifloxacin.
[17] Infect Disord Drug Targets, ( Jun 2007 ), Page159-168
[18]
Mdluli K, Ma Z, Mycobacterium tuberculosis DNA gyrase as a target for

drug discovery.
[19]
Drug Bank; a comprehensive resource for in silico drug discovery andexploration,

Nucleic Acids Research, 2006, Vol.34, Database Issue.
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PubMed:
Online Search Engine for
science and
biomedical
articles,www.pubmedcentral.nih.gov.
[21]
The Protein Data Bank, Nucleic Acids Research 2000, Vol. 28, Oxfords
Universty Press.
[22]
Computational Biology and Drug Discovery: From single network Drugs, Current
Bioinformatics, (2006).
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Mark
Johnson.
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better
web
interface,
Nucleic
Acids
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[24]
Sagl B Needleman, Christus D, Wuksch A. General Method Applicable to the

Search of similarities in the Amino Acid Sequence of two Protein , J. Mol Bwl. 1970
; Page 443-453.
[25]
Sali A, Blundell TL. Comparative protein modelling by satisfaction of spatial

restraints. J Mol Biol. 1993; 234: Page 779-81.
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of protein structures, J. Appl. Cryst. 1993; 26:Page 283291.
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51

Dr. Triloki Pant: Asst. Professor IIIT-Allahabad

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Homology Modeling of Mycobacterium tuberculosis H37Rv gyrA protein:

Design and Evaluation of novel gyrA inhibitors

Under the Guidance of:

Dr. TRILOKI PANT

INDIAN INSTITUTE OF INFORMATION TECHNOLOGY

CERTIFICATE FROM SUPERVISOR

Guides name & Signature

Signature &Name of the Committee members

On final examination and approval of the thesis

dedication towards our education and

career and for being with us in various levels of academic pursuits.

their support and motivation throughout my research project work.

Currently Existing Technologies

Drug Design Cycle

Analysis of Previous Research

Problem Definition and Scope

Formulation of present problem

2: Description of Software and Tools Used.10

3: Theoretical Tools- Analysis and Development..12

3.6: Selection of potential drug candidate against gyrA

3.8.1: Methodology Overview

Figure 1: Colonies of Mycobacterium tuberculosis

Figure 4: Structure of Moxifloxacin

Figure 5: Sequence alignment between Target and Template

Figure 6: 3-D structure of Modeled protein

Figure 7: Ramachandran plot of target protein

Structure of Trovafloxacin derivatives

Structure of Moxifloxacin derivatives

Docking result with Trovafloxacin derivatives

Docking result with Moxifloxacin derivatives

Dna gyrase subunit A

Root Mean Square Deviation

Protein Data Bank

Protein Model Database

Hydrogen Bond Acceptor

Hydrogen Bond donor

Multi Drug Resistance

Homology Modeling of Mycobacterium tuberculosis H37Rv gyrA protein:

compounds for the design of a putative drug.

and multiplication of bacteria. The crystal structure of gyrA is not

structural database. In this study we modeled a 3D structure of gyrA by X-ray crystal

Norfloxacin (-9.7557), Moxifloxacin (-11.3729), Trovafloxacin (-11.4397), Gemifloxacin

two drugs namely

Trovafloxacin and Moxifloxacin.

Indian Institute of Information Technology, Allahabad

Homology Modeling of Mycobacterium tuberculosis H37Rv gyrA protein:

1.1 Currently Existing Technologies

projects several bioinformatics software and databases are developed [3].

Homology Modeling of Mycobacterium tuberculosis H37Rv gyrA protein:

1.2 The Drug Design Cycle

homology modeling. Using computer algorithms, compounds or fragments of compounds

and, often, specificity for the target

Indian Institute of Information Technology, Allahabad

Homology Modeling of Mycobacterium tuberculosis H37Rv gyrA protein:

proteins. Three-dimensional structure

. Homology modeling is technique in the field of drug

Identification of the target protein through protein Blast

Alignment of the query sequence and template sequence

Co-ordinate generation for the structurally conserved region

Co-ordinate generate conformations for the loops regions

Build the side-chain conformations

Refine and evaluate the unknown structure

Docking between ligand and protein molecule is a two-step process: