FINAl 3

Homology Modeling of Mycobacterium tuberculosis H37Rv gyrA Protein :
Design and Evaluation of novel gyrA Inhibitors
1.Introduction
1.1 Motivation
Mycobacterium tuberculosis (MTB) is an obligate aerobic prokaryote, can survive only in an
[1]
environment containing oxygen
. First discovered by the Robert Koch in 1882. M. tuberculosis
bacteria are not stained by any bacteriological stain (such as Methylene blue) due to high content
of lipid in its wall. So, staining of Mycobacteria requires specific staining techniques, such as
Ziehl-Neelsen staining and acid-fast, staining is used. M. tuberculosis comes from the
genus Mycobacterium, which is composed of approximately 100 recognized and proposed
species. Its unusual cell wall, rich in lipids (e.g., mycolic acid), is likely responsible for this
[2]
resistance and is a key virulence factor
. The most familiar of the species are Mycobacterium

[3]
tuberculosis and Mycobacterium leprae (leprosy) .
There are number of different type of inhibitor available, the Trovafloxacin and Moxifloxacin
inhibitors one of them best inhibitor. Trovafloxacin and Moxifloxacin are an antibiotic of the
fourth generation flouroquinolone family. Trovafloxacin (sold Trovan by Pfizer and Turvel
by Almirall
of supercoiled
Laboratories) is a broad spectrum antibiotic that inhibits the uncoiling

DNA
in
various
bacteria
by
blocking
the
activity
of
DNA
[4]
gyrase and topoisomerase IV
. Moxifloxacin is marketed worldwide under the brand
names Avelox, Avalox, and Avelon. Moxifloxacin is a broad-spectrum antibiotic that is active
against both Gram-positive and Gram-negative bacteria. It functions by inhibiting DNA gyrase a
[5]
type II topoisomerase and topoisomerase IV,
enzymes necessary to separate bacterial DNA,
thereby inhibiting cell replication.
Computer aided drug design like structure based approach can be used effectively against
resistant Mycobacterium species. To develop novel drug which bind to target and inhibit the
synthesis of DNA gyrase of Mycobacteria. We have designed computationally new
Trovafloxacin and Moxifloxacin derivative which bind to active site of the receptor lead to
inhibition of DNA gyrase activity.

Design and evaluation of novel gyrA inhibitors
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.
1.2 Problem statement
Tuberculosis is the second-largest cause of death from an infectious agent worldwidekilling

approximately 1.7 million people in 2003. The number of new cases appears to be growing, with
an estimated 8.8 million new cases in 2003,[6].Various antibiotics have been used to control the
diseases
caused
by the
Mycobacterium
tuberculosis
species.
The
fluoroquinolones
(Trovafloxacin and Moxifloxacin) are the most important group of antibiotics that are available
as bactericidal.. They are used to control the diseases caused by the bacteria. Single genetic
[7]
changes usually produce highly resistant strains of pathogens
. Disease-causing organisms also
know as pathogen, they reproduce and mutate quickly. These organisms acquired genetic
[8]
resistance to chemical controls and have the capability to pass on a disease to new hybrids
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
[9]
drugs has severe adverse effect
. These are marketed Quinolone
drug
Flerofloxacin,
Lemofloxacin, Pefloxacin, Levofloxacin, Sparfloxacin, OfLoxacin, Moxifloxacin, Norfloxacin,

Enoxacin, Dextrofloxacin, Nalidixic acid, Trovafloxacin, Ciprofloxacin etc.
2
School of Biotechnology and Center for Bioinformatics

1.3 Introduction to Trovafloxacin

Trovafloxacin is a broad spectrum antibiotic that inhibits the uncoiling of supercoiled DNA in
[10]
various bacteria by blocking the activity of DNA gyrase and topoisomerase IV
. It had better
gram-positive bacterial coverage and less gram-negative coverage than the previous
[11]
fluoroquinolones
.
O
H
F
H
O
N
H
H
H
H
H
N
H
Figure 1. Stucture of Trovafloxacin
1.4 Introduction to Moxifloxacin

[12]
Moxifloxacin is a synthetic fluoroquinolone antibiotic agent
. The bactericidal action of
Moxifloxacin results from inhibition of the enzymes topoisomerase II (DNA gyrase) and
[13]
topoisomerase IV
Figure 2. Structure of Moxifloxacin

3

1.4 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 agents inhibit the enzyme DNA gyrase and topoisomerase II[14]. Therefore,
replication of DNA is inhibited and bacteria gets kill.
1.5 DNA gyrase

DNA gyrase is an ATP-dependent enzyme that acts by creating a transient double-stranded DNA
break. It is unique in catalyzing the negative supercoiling of DNA and is essential for efficient
DNA replication, transcription, and recombination. DNA gyrase is a tetrameric A2B2 protein.
The A subunit carries the breakage-reunion active site, whereas the B subunit promotes ATP
hydrolysis [15].
2.Literature review
2.1. ntroduction to Mycobacterium tuberculosis Genome

The genome of the H37Rv strain was published
[16]
and sequenced in 1998[17]. Its size is 4
million base pairs, with 3959 genes; 40% of these genes have had their function characterised,
with possible function postulated for another 44%. Within the genome are also 6 pseudogenes.
The genome contains 250 genes involved in fatty acid metabolism, with 39 of these involved in
the polyketide metabolism generating the waxy coat. Such large numbers of conserved genes
show the evolutionary importance of the waxy coat to pathogen survival.
About 10% of the coding capacity is taken up by two clustered gene families that encode acidic,
glycine-rich proteins. These proteins have a conserved N-terminal motif, deletion of which
impairs growth in macrophages and granulomas.[18].Nine noncoding sRNAs have been
characterised in M. tuberculosis,[19] with a further 56 predicted in a bioinformatics screen
[20]
4

.
2.1.1. Disease caused by Mycobacterium
Tuberculosis
TB is responsible for 25% of adult deaths in the developing world- more than those caused by
diarrhea, malaria and AIDS combined. Tuberculosis (TB) is a bacterial infection caused by a
[21]
germ called Mycobacterium tuberculosis
. The bacteria usually attack the lungs, but they can
also damage other parts of the body. TB spreads through the air when a person with TB of the
[22]
lungs or throat coughs, sneezes or talks
.In 2007 there were an estimated 13.7 million chronic

[23]
active cases, 9.3 million new cases, and 1.8 million deaths, mostly in developing countries
In this project I worked on Mycobacterium tuberculosis which causes tuberculosis

disease, and is one of the most important throat and lung disease over the world.
Now days Mycobacterium tuberculosis has become highly resistant to available
fluoroquinolone. The reason behind the resistance was pointed to mutation in the gyrA gene,
[24]
encoding a replacement of Alanine for Serine at position 74(A74 S), with resistance
Figure 3: Mycobacterium tuberculosis colonies
5

2.1.2 Symptoms and Sign

The symptoms of disease does not notice until the disease is quite advance. In active pulmonary
TB, patients may have no symptoms, except not feeling well, anorexia, fatigue, and weight
loss, which develop gradually over several weeks, or they may have more specific symptoms.
Cough is the first symbol of the infection with M.tuberculosis [25]. At first, it may be minimally
productive of yellow or green sputum, usually on rising, but cough may become more productive
as the disease progresses. Hemoptysis occurs only with cavitary TB (sometimes due to fungal
growth in a cavity). Drenching night sweats are a classic symptom but are neither common in
nor specific for TB. Dyspnea may result from lung parenchymal damage, spontaneous
pneumothorax, or pleural TB with effusion.
Figure 4: Symptoms of M. tuberculosis in Human lung
6

2.2. Computer aided drug design

2.2.1 Introduction
Structure-based ligand or inhibitor design, or rational drug design, as it is sometimes called, aims
to identify chemical compounds or peptides that bind strongly to key regions of biologically
relevant molecules, e.g. enzymes or receptors, for which three-dimensional structures are known.
Designed compounds should be able to inhibit or stimulate the biological activity of their target
molecules. The rapid progress of the human genome project is providing an ever-increasing
number of potential protein drug targets. 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
[26]
discovery and pharmaceutical research
Computational methods are required to extract all of the relevant information from the available
structures and to use it in an efficient and intelligent manner to design improved ligands for the
target. Due to genome sequencing projects, the number of known sequences is increasing at a
[27]
rapid rate
New target identification strategies and associated bioinformatics technologies are being
[28]
developed to categorize this vast body of information
. Today, many scientists are working on
ways to try to predict the three-dimensional structure of a protein from its one-dimensional
[29]
amino acid sequence
. There is also a worldwide effort in functional genomics to determine as
many three-dimensional structures of proteins as possible or to develop computational

approaches to cluster sequences into families of related proteins and then select and solve the
three-dimensional structure of a representative sequence. Computational methods are needed to
exploit the structural information to understand specific molecular recognition events and to
elucidate the function of the target macromolecule. This information should ultimately lead to
the design of small molecule ligands for the target, which will block its normal function and
7

thereby act as improved drugs. Most of the drugs currently on the market have been found
through large-scale random screening of compounds for activity against a target, for which no
three-dimensional structural information was available. That is, thousands of compounds (in the
company database) are screened for activity. High-throughput robotic screening methods
accelerate this process. In the end, it is hoped that at least a small number of compounds will be
[30]
active against the target. A good lead compound is active at concentrations of 10 mM or less
The first example of structure-based design was reported by the group of Beddell and Goodford
in 1976 at Wellcome Laboratories in the United Kingdom [98]. Hemoglobin was selected as a
target, which at the time was the only example of pharmacological relevance with a known
crystal structure. The goal of the studies was to develop a ligand that acts similarly to the natural
allosteric effector diphosphoglycerate. This endogenous ligand binds to hemoglobin and
regulates its oxygen affinity. Taking this molecule as a reference, the Wellcome group designed
dialdehyde derivatives and related bisulfite adducts which, as expected, modify the oxygen
affinity to hemoglobin. Several years later the antihypertensive captopril, which inhibits the
angiotensin-converting enzyme, was introduced onto the market; this was the first drug to be
developed using structural information. The past 20 years of drug design have witnessed the
structural characterization of a tremendously number of therapeutically important targets. The
increasing number of successful applications of drug design has led to the discovery of new
therapeutics. The recent development of human immunodeficiency virus (HIV) protease
inhibitors has convincingly demonstrated the impact and the relevance of structure-based
[31]
approaches to the development of new drugs
2.2.2. 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
8

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
[32]
and, often, specificity for the target
2.3. Homology modeling

Now days there are number of technique developed in molecular biology that provide easy to
identification, sequencing of DNA, RNA or proteins. To determine the three-Dimensional
structure of protein is very difficult and time consuming task. In the field of structural biology
the main objective to find out the three dimensional structure of protein from the there
[33]
sequences.
. The ultimate goal of protein modeling is to predict a structure from its sequence
with an accuracy that is comparable to the best results achieved experimentally. This would
allow users to safely use rapidly generated in silico protein models in all the contexts where
today only experimental structures provide a solid basis: structure-based drug design, analysis of
protein function, interactions, antigenic behavior, and rational design of proteins with increased
[34]
stability or novel functions
One method that can be applied to generate reasonable models of protein structures is homology
modelling. In protein structure prediction, homology modeling, also known as comparative
modeling, is a class of methods for constructing an atomic-resolution model of a protein from its
amino acid sequence (the "query sequence" or "target").most of the homology modeling
technique based on the already known 3-D coordinates of the protein which we called template
structure or parent structure, it should be likely to resemble to the query sequence (whose
structure is not known). The model of our target protein is produced by the use of both sequence
alignment and template structure. Because protein structures are more conserved than protein
sequences, detectable levels of sequence similarity usually imply significant structural similarity
9

[35]
. The quality of the homology model is dependent on the quality of the sequence alignment
[36]
and template structure
2.3.1. General Procedures

[37]
The steps to creating a homology model are as follows
Identify homologous proteins and determine the extent of their sequence similarity with one
another and the unknown
Align the sequences
Identify structurally conserved and structurally variable regions
Generate coordinates for core (structurally conserved) residues of the unknown structure from
those of the known structure(s)
Generate conformations for the loops (structurally variable) in the unknown structure
Build the side-chain conformations
Refine and evaluate the unknown structure.
2.4. Docking
The application of computational methods to study the formation of intermolecular complexes
has been the subject of intensive research during the last decade. It is widely accepted that drug
activity is obtained through the molecular binding of one molecule (the ligand) to the pocket of
another, which is commonly a protein. In the binding conformations of a complex of a protein
with a therapeutic drug, the molecules exhibit geometric and chemical complementarities, both
of which are essential for successful drug activity. The computational process of searching for a
ligand that is able to fit both geometrically and energetically to the binding site of a protein is
called molecular docking. The docking problem is analogous to an assembly-planning problem
where the parts are actuated by molecular force fields and have thousands of degrees of freedom.
In general docking process can be divided in to two phases. One is the searching algorithm,
which finds possible binding geometries of the protein and its ligand. The other is the scoring
function, which ranks the searching results and selects out the best binding geometry based on
10

the energies of the of the complexes or, more theoretical value, Gbind, the binding free energy
[38]
difference between the bound and unbound states of the ligand and protein
. Ligand docking
and screening algorithms are now frequently used in the drug-design process, and have
additional application in the elucidation of fundamental biochemical processes. The purpose of
docking algorithms is now expanding beyond the original goal of fitting a given ligand into a
specific protein structure. Newer applications include database screening, lead generation and de
[39]
novo drug design
11

3. Materials and Tools Used

For our present study I used bioinformatics tools,biological databases like PubMed, Drug Bank,
PDB (Protein Data Bank) and softwares like MOE, ACD ChemSketch,Modeller.
3.1 : ACD/ChemSkecth
ACD/ChemSketch is the powerful all-purpose chemical drawing and graphics package from
ACD/Labs developed to help chemists quickly and easily draw molecules, and schematic
diagrams, calculate chemical properties, and design professional reports and presentations. ACD
Chemsketch can convert SMILES notations to Structure and vice versa.
3.2 : MOE(Molecular Operating Enviroment)

MOE is an Interactive Molecular Graphics Programing suite for calculating and displaying
feasible docking modes of pairs of protein and DNA molecules. MOE can also calculate ProteinLigand Docking, assuming the ligand is rigid, and it can superpose pairs of molecules using only
knowledge of their 3D shapes.MOE (Molecular Operating Environment), combines visualisation,
simulation and methodology development into a single integrated package. MOEs collection of
built-in applications, which include tools for protein modeling, molecular modeling, structurebased drug design.
3.3 : Drug Bank

Drug Bank is a unique Bioinformatics/Chemoinformatics resource that combines detailed drug
(i.e. chemical) data with comprehensive drug target (i.e. protein). Each Drug Card entry contains
greater than 80 data fields with half of the information being devoted to drug/chemical data and
[40]
the other half devoted to drug target or protein data
12

3.4 : Protein Data Bank

The PDB (Protein Data Bank) is the single world wide archive of Structural data of Biological
[41]
macromolecules,
established
in
Brookhaven National Laboratories (BNL) in 1971
.It
contains Structural information of the macromolecules determined by X-ray crystallographic,

NMR methods etc.
3.5 : Pubmed
PubMed is a free digital archive of biomedical and life sciences journal literature at the U.S.
National Institutes of Health (NIH), developed and managed by NIH's National Center for
Biotechnology Information (NCBI) in the National Library of Medicine (NLM). PubMed is
a free search engine for accessing the MEDLINE
database of citations and abstracts of
[42]
biomedical research articles
3.6 : Modeller 9v7

The Modeller is the freely available computer program for the production of homology models
of
protein tertiary structures. Modeller was maintained and written by Andrej Sali at the
University of California, San Fransisco.
13

4. METHODOLGY
Bioinformatics is seen as an emerging field with the potential to significantly improve how drugs
are found, brought to the clinical trials and eventually released to the marketplace. Computer
Aided Drug Design (CADD) is a specialized discipline that uses computational methods to
simulate drug receptor interactions. CADD methods are heavily dependent on bioinformatics
[43]
tools, applications and databases
4.1 :Sequence retrieval of gyrA gene

The protein sequence of
Mycobacterium tuberculosis H37Rv gyrA, Accession Number
NP_2145201 was retrieved from NCBI Reference Sequence database and the FASTA sequence
is used for our studies.The protein sequence is mutated at 74 position from Ala to Ser.
4.2 : Screening for best homologous templates
[44-45]
The target protein sequence was blasted using BLASTP
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.
4.3 : In silico Comparative Modeling of gyrA Protein
An alignment between the target and temple is performed.An in silico comparative modeling of
[46]
the gyrA protein was carried out by the MODELLER 9v7
. 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.
4.4 : Model refinement, validation and evaluation
[47]
The modeled protein is validated by PROCHECK
.The parameters include the covalent bond
distances, angles and torsion, stereocemical validation and atom nomenclature. The input to
PROCHECK is a single file containing the coordinates of your protein structure. This must be in
Brookhaven file format the input to PROCHECK is a single file containing the coordinates of
your protein structure. This must be in Brookhaven file format.
14

The outputs comprise a number of plots, together with a detailed residue-by-residue listing. The
plots are output in PostScript format, it shows Ramachandran plot.
4.4 : Submission of Modeled structure to PMDB
[48]
The modeled gyrA Protein was deposited to the Protein Model Data Base
.] and can be
downloaded with the PMID PM0077412.

4.5: Selection of Potential Drug Candidates against gyrA protein
[49]
The Pubchem
[50]
, KEGG
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.
15

Figure5. 3-D structure of gyrA protein modeled by MODELLER 9v7
16

Ramachandran plot: 88.7% core 10.1% allow 1.1% gener 0.0% disall
Figure 6: Ramachandran plot for gyrA protein of M.tuberculosis
4.6. 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.
17

Trovafloxacin Derivative Structure

F
F
F
F
OH
NH2
H
N
H 2N
H2N
OH
OH
H
Analog 2
Analog 1
F
OH
HO
OH
O
F
OH
O
N
OH
N
HO
OH
HO
H
N
N
OH
HO
NH2
HO
H
O
OH
HO
Analog 3
Analog 4
HO
OH
HO
O
OH
O
N
OH
HO
OH
N
HO
OH
HO
O
H
HO
Analog 5
OH
Analog 6
Table 1
18

Moxifloxacin Derivative Structure

O
CH3
CH3
HOOC
O
H3C
O
H
CH3
N
H
Analog 1
Analog 2
CHH
C
33
O
CH3
O
H
H
N
H3C
H2N
H
N
O
H
O
H
Analog 4
Analog 3
H3C
H2N
CH3
H
N
CH
H
N
N
O
Analog 6
Analog 5
Table 2
19

4.7. Docking in MOE

The purpose of the Dock application is to search for favorable binding configurations between
one or more small, flexible ligands and a rigid macromolecular target, which is usually a protein.
For each ligand, a number of configurations called poses are generated and scored in an effort to
determine favorable binding modes.
.
4.7.1 : Methodology Overview
The Dock application is divided into a number of stages. The stages are:
Conformational Analysis:
If ligand conformations are not supplied via a conformation database, Dock will generate
conformations from a single 3D conformation by applying a collection of preferred torsion
angles to the rotatable bonds. Bond lengths and bond angles will not be altered. For small
ligands, a systematic search is conducted which generates all combinations of angles. For larger
ligands, a (non-deterministic) stochastic sampling of conformations is performed.
Placement :
A collection of poses is generated from the pool of ligand conformations using one of the
placement methods available.The methods are Alpha Triangle(default), Triangle Matcher, Proxy
Triangle, Alpha PMI, None.
Pharmacore Filtering :
Optionally, the generated poses are constrained to satisfy an arbitrary pharmacophore query.
Such a query is used to bias the search towards known important interactions.
20

Rescoring(l) :
Poses generated by the placement methodology could be rescored using one of the available
methods. Typically, scoring functions emphasize favorable hydrophobic, ionic and hydrogen
bond contacts. All scoring methods should assign low scores to good poses.
Refinment:
Poses resulted from the placement stage could be refined using one of the methods. Depending
on the choice of methodology, explicit molecular mechanics forcefields or grid-based energetics
may be used.
Rescoring(2):
Poses resulted from the refinement stage could be rescored using one of the scoring schemes.
21

5. Results
Docking with proposed structure of Trovafloxacin and Moxifloxacin shows better H-bonding
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.
22

5.1. 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
23

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
3D
1.40
Log P
Table 3b
24

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
25

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
26

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
27

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
28

5.2. 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
29

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
30

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
Ligand Interaction
Table 4c
31

Ligand
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
32

Ligand
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
33

Ligand
Active Site
Interaction Data
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
34

6. 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.
35

7. Conclusions
The Protein-Ligand interaction plays a significant role in structural based drug designing.In the
present work I have taken the DNA gyrase as receptor and identified the drugs that were used as
inhibitor. When the receptor (DNA gyrase) was docked with the drug the
energy
value
obtained was; Trovafloxacin (-11.4397), Moxifloxacin (-11.3729 ). When the modified drugs
were docked against the same receptor the energy value obtained was Trovafloxacin Analog 6 (12.4770), Maxifloxacin Analog 6(-11.9563). From this we can conclude that some of the
modified drugs are better than the commercial drugs available in the market.
36

8. Future work
In future research work the ADME/T (Absorption, Distribution, Metabolism, Excretion /

Toxicity) properties of these compounds can be calculated using the commercial ADME/T tools
available thus reducing the time and cost in drug discovery process.
37

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Homology Modeling of Mycobacterium tuberculosis H37Rv gyrA Protein :

Design and Evaluation of novel gyrA Inhibitors

environment containing oxygen

. First discovered by the Robert Koch in 1882. M. tuberculosis

resistance and is a key virulence factor

. The most familiar of the species are Mycobacterium

tuberculosis and Mycobacterium leprae (leprosy) .

Laboratories) is a broad spectrum antibiotic that inhibits the uncoiling

gyrase and topoisomerase IV

. Moxifloxacin is marketed worldwide under the brand

type II topoisomerase and topoisomerase IV,

enzymes necessary to separate bacterial DNA,

thereby inhibiting cell replication.

Homology Modeling of Mycobacterium tuberculosis H37Rv gyrA Protein :

1.2 Problem statement

Tuberculosis is the second-largest cause of death from an infectious agent worldwidekilling

changes usually produce highly resistant strains of pathogens

. Disease-causing organisms also

drugs has severe adverse effect

. These are marketed Quinolone

Lemofloxacin, Pefloxacin, Levofloxacin, Sparfloxacin, OfLoxacin, Moxifloxacin, Norfloxacin,

Homology Modeling of Mycobacterium tuberculosis H37Rv gyrA Protein :

1.3 Introduction to Trovafloxacin

various bacteria by blocking the activity of DNA gyrase and topoisomerase IV

Figure 1. Stucture of Trovafloxacin

1.4 Introduction to Moxifloxacin

Moxifloxacin is a synthetic fluoroquinolone antibiotic agent

. The bactericidal action of

Figure 2. Structure of Moxifloxacin

Homology Modeling of Mycobacterium tuberculosis H37Rv gyrA Protein :

1.4 Target of Trovafloxacin and Moxifloxacin

1.5 DNA gyrase

2.1. ntroduction to Mycobacterium tuberculosis Genome

and sequenced in 1998[17]. Its size is 4

Homology Modeling of Mycobacterium tuberculosis H37Rv gyrA Protein :

2.1.1. Disease caused by Mycobacterium

germ called Mycobacterium tuberculosis

. The bacteria usually attack the lungs, but they can

lungs or throat coughs, sneezes or talks

.In 2007 there were an estimated 13.7 million chronic

In this project I worked on Mycobacterium tuberculosis which causes tuberculosis

Figure 3: Mycobacterium tuberculosis colonies

Homology Modeling of Mycobacterium tuberculosis H37Rv gyrA Protein :

2.1.2 Symptoms and Sign

Figure 4: Symptoms of M. tuberculosis in Human lung

Homology Modeling of Mycobacterium tuberculosis H37Rv gyrA Protein :

2.2. Computer aided drug design

discovery and pharmaceutical research

developed to categorize this vast body of information

. Today, many scientists are working on

amino acid sequence

. There is also a worldwide effort in functional genomics to determine as

many three-dimensional structures of proteins as possible or to develop computational

Homology Modeling of Mycobacterium tuberculosis H37Rv gyrA Protein :

approaches to the development of new drugs

2.2.2. The drug design cycle

Homology Modeling of Mycobacterium tuberculosis H37Rv gyrA Protein :

and, often, specificity for the target

2.3. Homology modeling

stability or novel functions

Homology Modeling of Mycobacterium tuberculosis H37Rv gyrA Protein :

and template structure

2.3.1. General Procedures