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All in-text references underlined in blue are linked to publications on ResearchGate, Available from: Santhoshkumar Muthu
letting you access and read them immediately. Retrieved on: 14 June 2016
Int. J. Curr. Res. Biosci. Plant Biol. 2015, 2(4): xx-xx
Department of Biochemistry, PSG College of Arts and Science, Coimbatore- 641 004,
Tamil Nadu, India
*Corresponding author.
A b s t r a c t K e y w o r d s
Molecular docking is a commonly used method in structure based rational
inhibitors and drug design. It is used for assessing the complex formation of
small ligand molecules with proteins to predict the potency of the bonding Acetylcholine esterase
forces and finding lead compounds. A series of nine compounds from Annona Annona muricata
muricata L. were investigated as potential acetylcholine esterase inhibitors.
The enzyme was modeled using SWISS-MODEL and the compounds were Anopheles gambiae
tested for their ability to inhibit acetylcholine esterase (AChE) of mosquito Docking
Anopheles gambiae. The mode of binding in the active site of AChE was
investigated by molecular docking with AutoDock 4.2. Only five of the Syepharine
selected compounds (Syepharine, Murisolin, Corrossolone, Coreximine and
Coclaurine) expressed significant AChE inhibitory activity.
Introduction
Mosquitoes transmit more diseases than any other agricultural pests. Chemical pesticides react with a
group of arthropods and affect public throughout the serine residue at the catalytic site, thereby disabling the
world. Anopheles gambiae and Anopheles stephensi function of AChE (Dengfeng et al., 2013). In both
are the major vectors that transmit malarial parasites mammals and in insects, Acetylcholinesterase (AChE)
among human beings. Mosquito control is essential to belonging to serine hydrolase enzyme regulates
prevent the proliferation of mosquito borne diseases acetylcholine action. This enzyme is being used as a
and improve quality of environmental and human target for pesticides (Mirjana et al., 2013). AChE has a
health (Anupam et al., 2012). One of the most deep and narrow active site, the bottom and the
effective and safer approach is to explore botanical opening regions of which are known as catalytic and
bio-pesticides that can paralyze the insects by peripheral sites respectively (Tougu et al., 2001). Since
inhibiting acetylcholine esterase enzyme. Anti- serine residue is present in mammalian AChEs, the
cholinesterase pesticides are developed for controlling application of such chemical pesticides is severely
wide spectrum of insects including mosquitoes and limited by their toxicity to mammals (Zaidi and
B. Durairaj and S. Muthu (2015) / Int. J. Curr. Res. Biosci. Plant Biol. 2015, 2(4): xx-xx 1
Int. J. Curr. Res. Biosci. Plant Biol. 2015, 2(4): xx-xx
Soltani, 2013). Even at low concentrations, pests are on target-template alignment. The crystal structure of
more sensitive to the chemicals than humans (Weill et mouse AChEs (1J07) was assigned as the template
al., 2004). The use of anti-cholinesterase pesticides has structure for the target sequence. The 1J07 was
also been limited by resistance problems caused by crystallized with an inhibitor 3,8-Diamino-5,10'-
mosquitoes possessing AChE mutants such as the (Trimethylammonium) Decyl-6-Phenyl Phenanthridinium.
G119S mutant that is insusceptible to current The homology model was generated using default
pesticides (Alout et al., 2012). Control of mosquito- settings without any manual adjustments. The target
borne diseases through the use of effective and safer protein sequence of the AgAChE was obtained from
pesticides requires thorough in silico investigation for NCBI (Genbank accession number: BN000066). The
conserved target sites that are particularly present in alignment between the target and template sequence
mosquito AChEs. Amino acid sequences in this region were generated by using the T-COFFEE web server.
can be used as specific target sites for designing new The aligned file was saved in FASTA format and used
pesticides that will not lead to mammalian toxicity and for the construction of the homology model. The final
reduce the pesticidal resistance problems (Rozengart et model was then built by manually docking
al., 2006).. Though three-dimensional (3D) model of acetylcholine into the active site of the homology
Anopheles gambiae AChE (AgAChE) has been model (Mariani et al., 2011; Robert et al., 2011).
reported in the previous studies, conserved and
mosquito-specific region of AgAChE has to be Ligand and protein preparation
modulated using molecular modeling software. This in
silico investigation needs sequence analysis of The phytoconstituents such as Annomuricatin B,
AgAChEs from GenBank and 3D model of AgAChE Annomuricin A, Annomuricin B, Annonacin,
generated by homology modeling (Pang, 2006). Coclaurine, Coreximine, Corrossolone, Murisolin, and
Syepharine are present in Annona muricata leaves. All
Annona muricata L. is an undersized, deciduous, and these plant derived ligands were downloaded from
roundish canopy-like tree. Height of this fruit bearing chemical databases PUBCHEM compound and
tree is measured to be in the range between 5 and 8 m. chemspider. The downloaded ligands were converted
Traditionally, the leaves are used for headaches, to PDB format using suitable software. The homology
insomnia, cystitis, liver problems, diabetes, and modeled receptor was used for docking. AutoDock
hypertension. The leaves were also found to have anti- uses an adapted AMBER force field and so the atoms
inflammatory, pesticides, antispasmodic and of the protein and the ligands have to be set up in
antidysenteric (Di Stasi et al., 2002, Orlando et al., accordance with this. The missing hydrogen atoms
2010). The phytochemicals isolated and characterized were added for the ligand and protein. A torsion search
from leaves of the plant include annonacin, was made to the ligand and the default number of
annocatalin, annomonicin, annomuricin, annomuricatin, torsion is set for each ligand. Then the ligand was
corossolone, epomuricenin, gigantetrocin, javoricin, saved as PDBQT file format for further analysis.
muricine, muricinine, muricapentocin, muricoreacin, Similarly for proteins the heteroatom was removed and
montanacin, montecristin, muracin, muricatalin, additional chains were deleted to get a monomer
muricin, murisolin, robustocin, and solamin (Zeng et (Morris et al., 2009).
al., 1996; Benkert et al., 2011). In the present study,
nine compounds of the Annona muricata leaves were Docking procedure
subjected to docking studies against the AChE of
Anopheles gambiae. Docking calculations were performed using AutoDock
software (version 4.2). Desired compounds were
Materials and methods docked into the active site of target enzyme AgAChE.
In order to assign the perfect grid of each ligand, grid
Homology model box values were obtained from trial and error and
previous studies. The implementing Lamarckian
The homology model of the Anopheles gambiae Genetic Algorithm (LGA), considered as one of the
acetylcholine esterase (AgAChE) was modelled using best docking methods available in AutoDock, was
SWISS-MODEL program. Swiss model is a web based adopted to perform the molecular docking studies. The
automatic protein structure modelling software based parameters for LGA were defined as follows: a
B. Durairaj and S. Muthu (2015) / Int. J. Curr. Res. Biosci. Plant Biol. 2015, 2(4): xx-xx 2
Int. J. Curr. Res. Biosci. Plant Biol. 2015, 2(4): xx-xx
maximum number of 250,000 energy evaluations; a Fig. 1: Image of the Homology Modeled Receptor
maximum number of generations of 27,000; and (AgAChE).
mutation and crossover rates of 0.02 and 0.8,
respectively. Both Autogrid and AutoDock
computations were performed on Cygwin and ten
independent docking runs were performed for each
compound. The docked conformations of each ligand
were ranked into clusters based on the binding energy
and the top ranked conformations were visually
analyzed. Hydrogen bonding and hydrophobic
interactions between docked potent agents and
macromolecule were analyzed using AutoDock
(version1.50) Tools (Dhananjayan et al., 2014;
Andrew et al., 2011). The proper coordinates were set
Fig. 2: Essential amino acids for ligand interaction.
so that the grid can be specific to the binding site of
the enzyme. The grids file was generated and saved in
GPF format with default setting (Pang et al., 2003).
B. Durairaj and S. Muthu (2015) / Int. J. Curr. Res. Biosci. Plant Biol. 2015, 2(4): xx-xx 3
Int. J. Curr. Res. Biosci. Plant Biol. 2015, 2(4): xx-xx
inhibition of the AChE enzyme. Instead, the do not have any reactive group. Hence, H Bond
covalent bond interaction with Serine 286 plays interaction of the compounds with the amino acids
important role for irreversible inhibition of the Arginine 337 and Serine 286 was extensively
enzyme (Bulbuli et al., 2013). A reactive group is investigated. Acetylcholine was used as standard for
considered to form a covalent bond in docking docking experiment and it forms H Bond interaction
experiments. The compounds chosen for the study with Serine 286 (Fig. 4).
The compounds Annomuricatin A, B, Annomuricin B, The oxygen atom of the hydroxyl group functioned as
and Annonacin did not bind with the enzyme target a donor and formed hydrogen bond with the hydrogen
site. Hence, these four compounds might be inactive atom of amino group of Arginine 339 (Fig. 5).
against AgAChE enzyme. The calculations reveal that
these compounds form H-Bond interaction with Fig. 5: Coclaurine, H-Bond interaction with close
Arginine 339 and glutamate 336. Hydrogen atom of contact amino acids.
the hydroxyl group or H-Bond donor might form
hydrogen bond interaction with oxygen atom of the
above mentioned amino acids.
Coreximine
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Int. J. Curr. Res. Biosci. Plant Biol. 2015, 2(4): xx-xx
Corrossolone
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Int. J. Curr. Res. Biosci. Plant Biol. 2015, 2(4): xx-xx
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Int. J. Curr. Res. Biosci. Plant Biol. 2015, 2(4): xx-xx
insecticide resistance is easily detectable in larvivorous fish, Gambusia affinis. Adv. Environ.
mosquito vectors. Insect Mol. Biol. 13(1), 1-7. Biol. 7(4), 605-613.
Pang, Y.P., 2006. Novel acetylcholinesterase target Zeng, L., Wu, F.E., Oberlies, N.H., McLaughlin, J.L.,
site for malaria mosquito control. PLoS ONE 1(1), Sastrodihardjo, S., 1996. Five new
e58. monotetrahydrofuran ring acetogenins from the
Zaidi, N., Soltani, N., 2013. Laboratory evaluation of leaves of Annona muricata. J. Nat. Prod. 59, 1035-
environmental risk assessment of pesticides for 1042.
mosquito control: Toxicity of dimilin on a
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