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Dissertation
SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF
SARIKA SAHU
IBI2006010
M.Tech IT (Specialization Bioinformatics)
Under the Supervision of
Date: ___________________
WE
HEREBY
RECOMMEND
THAT
THE
THESIS
DEGREE
INFORMATION
OF
MASTER
OF
TECHNOLOGY
TECHNOLOGY
IN
(SPECIALIZATION-
DEAN (ACADEMIC)
Prof.U.S.Twari
THESIS ADVISOR
Prof.Krishna Misra
CERTIFICATE OF APPROVAL*
COMMITTEE ON FINAL
EXAMINATION FOR
EVALUATION OF THE
THESIS
DECLARATION
This is to certify that this thesis work entitled in-silico designing of novel
triazole derivatives as substitute for resistant fungicides is submitted by
me in partial fulfillment of the requirement for the completion of M.Tech in
Information Technology (specialization in Bioinformatics) to Indian Institute
of Information Technology, Allahabad comprises only my original work and
due acknowledgement has been made in the text to all other material used.
Sarika sahu
ACKNOWLEDGEMENT
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. U. S. Tiwary, Dean Academics, IIIT Allahabad for providing all the necess
ary requirements and for his moral support for this dissertation work as well during the whole co
urse of M. Tech.
The most notable source of guidance was my advisor, Prof. Krishna Misra, Professor, IIIT Alla
habad. I owe her a great deal of thanks for taking me under her wings and allowing me to soak u
p some of her knowledge and insight. She 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 throug
h the classical discussions we had in her office.
C.M. Bhandari, Coordinator of Indo Russian Center for Biotechnology for his honest dedication t
owards our education and career and for being with us in various levels of academic pursuits.
I am also grateful to assistant Dr. C.V.S. Siva Prasad, T. Lahiri, Mr. Vikram Katju, Mr. Pritish
Varadwaj, Mr. Manish Kumar and Ms. Anamika Singh for their support and motivation
throughout my research project work.
I also thankful to IPL, Lucknow for providing me the proposed structure of triazole compounds.
In completing this thesis work, I have disturbed, interrupted, Interrogated and discussed with
INDIAN INSTITUTE OF INFORMATION TECHNOLOGY, ALLAHABAD
a great variety of people, yet I have never once met anything but patience and politeness. I cannot
thank everybody by name, but I would like to record my great debts of gratitude to Dr. Bakul gohel,
Dr. Neera singh, Dr. Shallu kalia, Dr. Hrishikesh Mishra, Dr.Sandeep Kumar, Mr. Buksh, Ms.Mona
chaurasia, Mr.Upendra Kumar, Mr. Rajesh Kesharawani, Mr.Shakti Kumar and Mr.Manarshi Das.
For not only helping me in studies but also for making this batch a house of learning through their
hard work and dedication.
I am also thankful to rest of classmates and M.Tech. Friends for their cooperation 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.
Sarika sahu
Contents
List of figures
10
List of tables
11
Abbreviations
12
Abstract
13
1. Introduction
1.1
1.2
1.3
1.4
1.5
1.6
Motivation
Problem statement
Introduction to triazole
Target of triazole
Sterol 14-demethylase
Synthesis of egrosterol
2. Literature review
2.1 Introduction to fungi
2.1.1 Plant fungal disease
2.1.1.1 Symptoms and signs of powdery mildew
2.1.2 Human fungal disease
2.1.2.1 Symptoms and signs of Aspergillosis
2.2 Computer aided drug design
2.2.1
2.2.2
Introduction
Drug design cycle
General Procedures
2.4 Docking
14
14
15
16
17
17
18
21
21
21
22
23
25
26
26
28
29
30
30
Modeler program
3.1.1
3.4.2
3.5
Sphgen
3.5.1
3.5.2
3.5.3
3.6
32
33
34
34
34
34
34
37
41
41
42
42
42
42
43
43
43
Grid
3.6.1 Creating a box around the active site
3.6.2 Generating the Grid
Rigid and Flexible Ligand Docking
Flow chart
43
43
44
44
44
44
45
Results
47
48
48
3.7
3.7.1
3.7.2
5.1.2
57
8
61
61
62
Discussions
Conclusion
Future work
68
References
69
65
67
List of figures
F ig u r e 1 .
17
F ig u r e 2 .
18
F ig u r e 3
19
F ig u r e 4 .
synthesis of ergosterol
20
F ig u r e 5 .
22
F ig u r e 6 .
Aspergillus fumigatus
24
F ig u r e 7 .
35
graminis
3-D structure of sterol 14 demethylase of Blumeria graminis
modeled by MODELER9.0 program
Figure 9. Ramachandran plot for sterol 14 demethylase of Aspergillus
fumigatus
Figure 10. 3-D structure of sterol 14 demethylase of Aspergillus fumigatus
modeled by MODELER9.0 program
Main
programs in DOCK suite
F ig u r e 1 1 .
42
Figure 12. Bar graph showing energy comparison of top 10 ligands on docking
63
F ig u r e 8 .
36
36
37
Figure 13 Docking Result of virtual screening showing H-bond and Figure 13b 64
F ig u r e 1 4
10
List of tables
Table1.
38
Table 2.
48
Table 3.
57
Table 4.
61
Table 5.
62
11
Abbreviations
CYP51
RMSD
PDB
MW
Molecular Weight
H-bond
Hydrogen bond
HBa
HBd
DMI's
Demethylation inhibitors
HPR
ITC
Itraconazole
HIV
12
Abstract
Sterol 14 demethylase (CYP51) is an enzyme play important role in metabolism of endogenous
and xenobiotic substances. The ergosterols provide membrane structure, modulation of
membrane fluidity, and possibly control of some physiologic events. Inhibition of this vital
enzyme in the ergosterol synthesis cycle leads to the declining of ergosterol in the cell membrane
and increase of toxic intermediate sterols, causing increased membrane permeability and
inhibition of fungal growth.
As the site of action is very specific so most of the fungus has become resistant to the triazole
derivatives. Because of the resistant developed in fungi several triazoles fungicides have
disappeared from the market and they no longer provide benefit or advantage in a disease control
program. So, we have to develop some novel triazole derivative which fights against the fungus
which have become resistant. We screened some novel triazole drugs whose synthesis is feasible
in laboratory. It would be good for resistant variety of fungal species before
engaging in costly experiments.
13
1. Introduction
1.1 Motivation
Fungi are important multicellular organisms [1], some of them have economic value and others
participate in biological ecosystem. These degrade the dead organic material, the cycle of this
process is repeated through ecosystems. Most advanced (like mocot or dicot) plants need
association of fungi to grow such as Mycorrhizae that reside in the plant roots and provide
essential nutrients to the plant. Other useful fungi provide foods and antibiotic drugs (such as
penicillin). Apart from this beneficial aspect there are pathogenic fungus which causes number of
diseases in plants, human and animals. The absence of chlorophyll makes fungi totally dependant
on host and the similarity between the membranes of fungi and plant/animal is the main cause
why fungal infections are so stubborn. [2].
There are number of different type of fungicides available, the demethylation inhibitors (DMI's)
one of them best fungicides. It has many advantageous features; including far above the ground
fungicidal activity, very low toxicity to other organisms, defending and healing properties and
compatibility with integrated pest management. They share a related mode of action inhibiting
the formation of sterols, such as ergosterol, which are important in fungal cell walls. Each
compound may act in a slightly different part of the biochemical pathway to make sterols but the
result is a similar spectrum of activity against diverse diseases [3].due to the resistance of fungi to
some fungicides, it fails to control the fungal disease. The sterol demethylase (DMI's) have
become one of the important groups of fungicides and are being very much watched for symbols
that resistance might increase or developed. They are chemically diverse groups which all
prevent the same demethylation step in the synthesis of ergosterol, a critical substance of cell
INDIAN INSTITUTE OF INFORMATION TECHNOLOGY, ALLAHABAD
14
walls in many fungal organisms [4]. The reason for resistance of a plant to certain fungicides is
due to its overuse or misuse in some way or the other. This effects the genetic make up of the
plant which is inherited by the next generations.[5].
Computer aided drug design like structure based approach can be used
effectively against resistant fungal species. To develop novel drug which bind to target and
inhibit the synthesis of cell wall of the fungi. We have designed computationally new triazole
derivative which bind to active site of the receptor lead to inhibit the synthesis of cell wall.
This project will help in designing the new triazole derivatives drug. There is need for designing
new fungicides 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 wetlab with collaborations with some Laboratories around the world.
[6]
Ireland's, killed 1.5 million people, of Ireland's total population. The crop was vanished by a
fungal disease. There are at least 50,000 diseases of crop plants. Still New diseases are revealed
every year. About 15% of the total U.S. crop production is lost annually to infectious diseases
despite improved cultivars and disease control techniques.
15
Disease-causing organisms also know as pathogen, they reproduce and mutate quickly. These
organisms acquired genetic resistance to chemical controls and have the capability to pass on a
disease to new hybrids [7].
Currently most of the fungal organisms have become resistant to marketed triazole derivatives
because their site of action (active site) has mutated loosing the sensitivity for different triazoles.
Some of the triazole based drugs thus disappeared from the market. In this project work we try to
develop novel triazole derivative which effective against that fungal organism which becomes
resistant. They become resistance because the target sites become change so, we have to find
new target site and related drug. The most of the triazole derivative are toxic to rat and rabbit and
other mammalian [8]. These are marketed triazole drug Ketoconazole, Itraconazole, Fluconazole,
Triarimol,
Prothioconazole,
Terconazole,
Voriconazole,
Posaconazole,
Ravuconazole,
16
[9]
.These agents
inhibit the enzyme 14-demethylase, a cytochrome P450 enzyme that catalyses the synthesis of
ergosterol. Therefore, ergosterol synthesis is inhibited and membrane integrity and function is
affected.
17
18
The interaction with cytochrome P450-dependent enzymes present in human responsible for
drug interaction in human
19
20
2.Literature review
2.1. nroduction to fungi
fungi are amazing organisms which are neither plants, nor animals. Fungi cannot synthesize their
food from sunlight, water and carbon dioxide as plants do, in the process known as
photosynthesis. This is because they lack chlorophyll, its green pigment which plants use to
capture light energy. So, like other animals, they depend on the other organisms for food supply.
They do this in three ways. They may break down or 'rot' dead plants and animals. Organisms
which obtain their food this way are known as 'saprophytes'. Alternatively they may feed directly
off living plants and animals as 'parasites'. A third group is symbioiotic relationship with the
roots of plants; know as mycorrhizae
[13]
metabolite which is act as a weapon for self defense. So it becomes very difficult to fungi to
combat on plant tissue. Some fungi produce phytotoxic metabolite, which is toxic for plant [14].
2.1.1. Plant fungal disease
Plant pathology is the branch of science to study plant diseases. Fungal diseases are the most
important biotic factors limiting crop production [15]. The diversity of fungal pathogen leads to
different kind of resistance, which is a challenging task to develop newer active compounds.
Some common plant diseases are powdery mildew, rust, leaf spot, blight, root and crown rots,
damping-off, smut, anthracnose, and vascular wilts.
In this project I worked on Blumeria graminis which causes powdery
mildew disease, and is one of the most important foliar diseases of wheat over the world,
21
growing only on living tissue and whose spores destroy the leaf by germinate on surface of leaf
[16]
. Now days Blumeria graminis has become highly resistant to tridimenol, the most widely
used triazole in the late 1970s and early 1980s . The reason behind the resistance was pointed
to mutation in the CYP51gene, encoding a replacement of tyrosine for phenylalanine at position
136(Y 136 F), with resistance [17].
2.1.1.1. Symptoms and signs of powdery mildew
The symptoms of powdery mildew is white to gray spot on the surface of leave because fungal
grow mostly in upper leaf (epidermis layer) .some of the hyphae of fungi penetrate inside the
surface up to the cortex level and form a pustules [18]. Powdery mildew reproduce asexually and
produces conidia from conidiophores they form a chain like structure. The single spore look like
oval shape and colorless.
22
23
Taxonomic Classification
Kingdom:Fungi
Phylum:Ascomycota
Order:Eurotiales
FamilyTrichocomaceae
Genus: Aspergillus
24
There are point mutation at 220 position by methionine is substituted leads to the disruption
drug binding binding site. This substitution is responsible for that they become resistant, because
they involved in conformational changes associated with the catalytic cycle rather than in
residues that directly contact the drug [21].
Most studies concerning mechanisms of azole resistance in Aspergillus have been performed in
A. fumigatus and have demonstrated that resistance was associated with modification of the 14-
sterol demethylase target enzyme (CYP51), specifically mutations in the gene cyp51A.
Importantly, different mutations appear to result in resistance to posaconazole and itraconazole
versus voriconazole and ravuconazole Cross-resistance to itraconazole and posaconazole has
been associated with amino acid substitutions at glycine 54 (G54) whereas cross-resistance to
voriconazole and ravuconazole has been associated with amino acid substitutions at G448 It has
been postulated, based on molecular modeling studies, that a substitution at G54 in the Ahelix
of AF-CYP51A confers resistance to posaconazole and itraconazole by perturbing the binding of
the long side chain in the hydrophobic channel (channel 2) of the enzyme (20). Given that
voriconazole and ravuconazole lack a long side chain, substitutions at G54 would be predicted to
have no effect on the binding of these compact triazoles to the target [22].
25
breathing; chest pain; and fever and night sweats.. Aspergillus infection of the ear (called
otomycosis), can produce itching and a discharge, sometimes noticed as a spot on the pillow [23].
[26]
. Today, many
scientists are working on ways to try to predict the three-dimensional structure of a protein from
its one-dimensional amino acid sequence
[27]
26
27
28
. 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 stability
or novel functions [32].
One method that can be applied to generate reasonable models of protein
structures is homology modeling. 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
[33]
29
Identify homologous proteins and determine the extent of their sequence similarity with
one another and the unknown
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
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
30
[36]
. 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
novo drug design [37].
.
31
32
Chimera
analysis of molecular structures and related data, including density maps, supramolecular
assemblies, sequence alignments, docking results, trajectories, and conformational ensembles.
Chimera can read molecular structures and associated data in a large number of formats, display
the structures in a variety of representations, and generate high-quality images and animations
suitable for publication and presentation [38]. To search triazole target in fungi mainly in Blumeria
graminis, the target was carbon 14 sterol demethylase (CYP51). The 3-dimensional structure
of CYP 51 was not available in Protein Data Bank (PDB).
33
this project I used multiple-model default.py. Run MODELLER itself by typing the
34
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.
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.
Ramachandran plot: 86.9% core 11.4% allow 0.7% gener 1.0% disall
35
Ramachandran plot: 90.3% core 8.7% allow 0.5% gener 0.5% disall
36
3.3. Ligand
The existing known triazole drugs have become resistant, so there was need for new modified
triazole derivatives. The proposed structures used as ligand for docking were taken from R&D
department of IPL Lucknow .the synthesis of these structure is feasible in laboratories , so insilico approach was used to validate the binding of the proposed structure.
37
Proposed structure
S
N
N
2-((1,2,4)Triazole-1-carbothioyl)-3a,4,7,7a-tetrahydro-isoindole-1,3-dione
2-((1,2,4)Triazole-1-carbonyl)-3a,4,7,7a-tetrahydro-isoindole-1,3-dione
N
HS
N
N
(1,2,4)Triazole-1-thiol
2-([1,2,4]Triazole-1-carbothioyl)-isoindole-1,3-dione
O
2-([1,2,4]Triazole-1-carbonyl)-isoindole-1,3-dione
Cl
S
N
N
Cl
N
N
Cl
N
Cl
(1,2,4)Triazole-1-mercapto-trichloromethane
38
Triazole like drug also taken from Zinc Database for virtual screening, then filter
these molecule on the basis of drug like properties.
Drug-like filter
MIN_MOLWT 200
MAX_MOLWT 600
MIN_NUM_HVY 15
MAX_NUM_HVY 35
MIN_RING_SYS 0
MAX_RING_SYS 5
MIN_RING_SIZE 0
MAX_RING_SIZE 20
MIN_CON_NON_RING 0
MAX_CON_NON_RING 15
MIN_FCNGRP 0
MAX_FCNGRP 18
MIN_UNBRANCHED 0
MAX_UNBRANCHED 6
39
MIN_SUM_FORMAL_CRG -2
MAX_SUM_FORMAL_CRG 2
MIN_CHIRAL_CENTERS 0
MAX_CHIRAL_CENTERS 4
MIN_XLOGP -5.0
MAX_XLOGP 6.0
"Minimum solubility"
"Count S and P as polar atoms"
"Minimum 2-Dimensional (SMILES) Polar Surface
Area"
"Maximum 2-Dimensional (SMILES) Polar Surface
Area"
"Eliminate known aggregators"
"Eliminate predicted aggregators"
ELIMINATE_METALS Sc,Ti,V,Cr,Mn,Fe,Co,Ni,Cu,Zn,Y,Zr,Nb,Mo,Tc,Ru,Rh,Pd,Ag,Cd
\
40
3.4. DOCK
DOCK addresses the problem of "docking" molecules to each other. In general, "docking" is the
identification of the low-energy binding modes of a small molecule, or ligand, within the active
site of a macromolecule, or receptor, whose structure is known.
3.4.1. Dock working principle
Dock software is based on the force field energy scoring. This is including van der waals Force,
molecular mechanics and electrostatic energy. [42].
41
Receptor
coordinate
Sphegen
Site characterization
Negative image of the
site
Grid
Precompute
grid
for
evaluation
score
rapid
Dock
Screen molecule for complementarity
With receptor
Ligand
coordinate
42
3.5. Sphgen
Sphgen identifies the active site, and other sites of interest, and generates the sphere centers that
fill the site. The purpose of this document is to describe the steps required to prepare active site
spheres for a DOCK run [44].
3.5.1. Generate the molecular surface of the receptor
The molecular surface of the target is generated, According to the Richards and Connolly, the
surface of protein is determined by rolling a drop of water molecular whose radius about 1.4 A0
over the surface of protein which forms a van der waals force. This method used for calculating
each of sphere size.
3.5.2. Generate the spheres surrounding the receptor
Sets of overlapping spheres are used to create a negative image of the surface invaginations of
the target. To generate spheres from the molecular surface and the normal vectors, the program
sphgen that is distributed as an accessory with DOCK is used. Spheres are calculated over the
entire
surface,
producing
approximately
one
sphere
per
surface
point.
3.5.3. Select a subset of spheres to represent the binding site(s)
Use the largest cluster generated by sphgen.
3.6. Grid
This tutorial describes the generation of the grid used for grid-based scoring in DOCK.
3.6.1. Creating a box around the active site
43
The interactive program showbox is used to visualize and define the location and size of the grid
to be calculated using grid.
3.6.2. Generating the Grid
Grid creates the grid files necessary for rapid score evaluation in DOCK. Two Types of scoring
are available: contact and energy scoring.Within the DOCK suite of programs, the program
DOCK matches spheres (generated by sphgen) with ligand atoms and uses scoring grids (from
grid) to evaluate ligand orientation
44
4. Flow chart
Target protein
Target Search
Target
PDB
found?
Yes
Download PDB
No
Model the
protein
Is accuracy
>85%?
No
Loop Modeling
YES
Accept structure
45
Ligand
Accept Target
structure
DOCK
Final output
Exit
46
5. Results
Docking with proposed structure shows better H-bonding and binding energy with Target protein
CYP51 of both Blumeria graminis and Aspergillus fumigatus, while the docking with known or
existing fungicides showing poor binding energy and H-bonding.
Later virtual screening was carried out for finding novel inhibitor of CYP51 of both Blumeria
garminis and Aspergillus fumigatus. The 1049 triazole like molecule obtained from Zinc
Database, then filer the drug like molecule using open eye solution software for filtering, finally
667 triazole like molecule obtained then dock.
The
molecules
2-(1H-1,2,4-triazol-1-ylcarbonothioyl)-3a,4,7,7a-tetrahydro-1H-isoindole-
1,3(2H)-dione and 2-(1H-1,2,4-triazol-1-ylcarbonyl)-3a,4,7,7a-tetrahydro-1H-isoindole-1,3(2H)dione showing good binding energy and Hydrogen bond with target protein CYP51 of Blumeria
garminis.
47
Table 2a
48
Table 2b
49
Table 2c
50
Table 2d
51
Table 2e
52
Table 2f
53
Table 2g
54
Table 2h
INDIAN INSTITUTE OF INFORMATION TECHNOLOGY, ALLAHABAD
55
Table 2i
56
Table 3 A
Table 3B
INDIAN INSTITUTE OF INFORMATION TECHNOLOGY, ALLAHABAD
57
Table 3C
Table 3D
58
Table 3E
Table 3F
INDIAN INSTITUTE OF INFORMATION TECHNOLOGY, ALLAHABAD
59
Table 3G
Table 3H
INDIAN INSTITUTE OF INFORMATION TECHNOLOGY, ALLAHABAD
60
MoleDock score
Affinity
logP
MW
HBA
HBD
ZINC00560057
-142.479
-28.6438
2.42
332.381
ZINC00064573
-139.477
-31.0579
1.67
274.236
ZINC00570929
-138.766
-26.4399
2.1
293.323
ZINC00560047
-136.565
-38.0105
2.46
336.388
ZINC00115651
-134.905
-32.7638
3.52
331.416
ZINC00576263
-134.578
-29.4997
2.09
293.323
ZINC00541851
-134.205
-25.9191
3.29
324.423
ZINC00182689
-133.3
-34.015
2.82
332.404
ZINC00560030
-133.179
-33.706
2.24
336.413
ZINC00411662
-132.115
-34.9462
2.82
346.815
ZINC00360664
-131.55
-33.9106
3.27
324.377
ZINC00560066
-131.035
-29.5436
2.52
332.424
ZINC00299084
-130.959
-29.4402
0.25
315.327
ZINC00129663
-130.829
-26.5647
1.91
244.292
name
61
MoleDock score
Affinity
ZINC00406640
-147.227
-33.8897
ZINC00115651
-146.22
ZINC00268949
logP
MW
HBA
HBD
2.79
339.42
-37.2478
2.32
330.41
-145.349
-29.813
2.14
342.37
ZINC00032585
-143.004
-29.6872
1.15
344.39
ZINC00360663
-139.897
-33.4501
3.27
324.38
ZINC00411656
-139.812
-31.1697
2.5
325.39
ZINC00360584
-139.188
-35.0356
3.59
344.8
ZINC00182689
-133.3
-34.015
2.82
332.41
ZINC00115647
-138.607
-40.961
2.56
308.75
ZINC00246313
-137.954
-36.5279
2.26
318.38
ZINC00285763
-137.58
-29.3326
3.59
338.43
ZINC00285731
-136.537
-26.9243
1.02
317.32
ZINC00068477
-136.179
-33.2195
3.05
339.8
ZINC00081006
-135.895
-28.6802
3.16
335.38
name
62
Figure 12a.Bar graph showing energy comparison of top 10 ligands on docking (Aspergillus
fumigates)
Figure 12b.Bar graph showing energy comparison of top 10 ligands on docking (Blumeria
graminis)
63
Figure 13b
Figure 13a
Figure 13a Docking Result of virtual screening showing H-bond and Figure 13b triazole like
molecule (Blumeria graminis)
Figure 14b
Figure 14a
Figure 14a Docking Result of virtual screening showing H-bond and Figure 14b triazole like
molecule (Aspergillus fumigatus)
64
6. Discussion
The B. graminis and A. fumigatus belong to the kingdom Fungi. Fungi cannot synthesize their
own food from sunlight because of lack of chlorophyll, it is a green pigment in plant which help
in the synthesis of own food from sunlight and CO2 [1]. Fungi cause different diseases in plants
and humans. The target protein for triazole is carbon 14 sterol demethylase (CYP51), this
protein play important role in the synthesis of sterol in the membrane of fungi
[10]
. Nowadays
CYP51 protein has become resistant to the marketed antifungal triazole fungicides like
fluconazole, epoxoconazole, triadimol, itraconazole and Propiconazole, because of mutation in
target protein at the binding site. The reason behind mutation may be due to prolonged use of
these fungicides. IPL Lucknow has designed some triazole derivatives for commercial use. We
have studied in silico interaction of these compounds with the target protein.
The complete structure of
CYP51 is not available in protein data bank (PDB) so, the target
protein was modeled by comparative homology modeling using modeler software (9.0 version).
The accuracy of the model was 86.9 % of residue fall in core region and other 11.4 % in
allowable region in Ramachandran plot in case of B.graminis.In case of A.fumigatus modeled
accuracy is 90.3% of residue fall in core region and 7.3% in allowable region. We had screened
out best two molecules on the basis of Hydrogen bonding and binding energy from above
proposed compounds using Dock software. We also took 1049 triazole derivatives compound
from Zinc database. After filtering these compounds on the basis of drug like molecules by filter,
we obtained finally 667 compounds.
65
We had screened all these compounds and obtained top 10 molecules which show best binding
energy and hydrogen bonding with the same target protein present in
66
7. Conclusions
The
proposed
structures
isoindole-1,3(2H)-dione
2-(1H-1,2,4-triazol-1-ylcarbonothioyl)-3a,4,7,7a-tetrahydro-1H-
and
2-(1H-1,2,4-triazol-1-ylcarbonyl)-3a,4,7,7a-tetrahydro-1H-
isoindole-1,3(2H)-dione are showing best docking energy in case of B.graminis , while N,Ndimethyl-1H-1,2,4-triazole-1-carboxamide shows best binding energy in case of A.fumugatus .
After virtual screening with CYP51 of Aspergillus fumigatus, N-(3, 4-dimethylphenyl)-2-[[5-(4pyridyl)-2H-1, 2, 4-triazol-3-yl] sulfanyl] acetamide showing good docking score and Hydrogen
bonding. While Blumeria graminis shows best energy and Hydrogen bonding with Nisopropylideneamino-4-[2-(2H-1,
2,
4-triazol-3-ylsulfanyl)
acetyl]
amino-benzamide.
comparison of the screened compounds from zinc database and the proposed structure, the
former show better binding energy than proposed structure.
67
8. Future work
This project was aimed at finding novel fungicides for the inhibition of CYP51, an important
enzyme playing crucial role in sterol synthesis in fungi. We have found novel fungicides which
might be helpful for the inhibition of sterol synthesis. These novel fungicides compounds may
act as a potent and specific inhibitor for CYP51 enzyme; though their efficacy, toxicity and
pharmacokinetic properties need to be studied experimentally.
The following steps are for future work
We are planning to use different parameters, so that more result can be obtained from
Zinc database.
Spraying on infected plants and comparing proposed molecules with mutated fungicides.
68
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Flexible
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74