340

Insect Pest Management, A Current Scenario, 2011 (ed.), Dunston P. Ambrose,

Entomology Research Unit, St. Xaviers College, Palayamkottai, India, pp.340-345.

INHIBITION OF ACETYLCHOLINESTERASE IN THREE INSECTS OF ECONOMIC

IMPORTANCE BY LINALOOL, A MONOTERPENE PHYTOCHEMICAL
A.PRAVEENA AND K.P.SANJAYAN*
G.S. Gill research Institute, Guru Nanak College, Chennai - 600 042, Tamil Nadu, India.
email: kpsanjayan@yahoo.co.in
ABSTRACT
Monoterpenoids from plants have been shown to be an alternative to synthetic insecticides against
various insects. Inhibition of Acetylcholinesterase (AChE) activity has been opined as a possible
mode of action of monoterpenoids recently. However, it is necessary to gain knowledge of the mode
of binding of the monoterpenoids in the target region so as to facilitate an understanding of the
evolution of novel molecules for pest management. In the present study, the interaction of
monoterpene linalool with the AChE of Aedes aegypti (L.), Leptinotarsa decemlineata (Say) and
Spodoptera litura (F.) belonging to three taxonomic orders, Diptera, Coleoptera and Lepidoptera
respectively were studied using bioinformatics tools. The three-dimensional structure of the AChE
(targets) from the insects was modelled using the MODELLER9v8 software. The molecular
interaction of the linalool (ligand) with the modelled targets were analysed using the docking
concepts by iGEMDOCKv2.1 software. The interactions represent the conserved interacting
residues that often form binding pockets with specific physico-chemical properties to play the
essential functions of the target. Application of Tices Rule to evaluate the insecticidal property of
linalool, revealed that, there was no violation of the rule and linalool could be a potent insecticide.
The interaction of linalool with the targets was stable and the formation of intermolecular complex
could disturb the AChE. As per the calculated fitness energy scores, the interaction of linalool with
the AChE of these insects was in the following order: A.aegypti > L.decemlineata >S.litura. The
results presented here indicate linalool to be a potent insecticide and details of the molecular
interaction indicate that their effect varied with the species of target insects.
Key words: Monoterpenoids, linalool, AChE, Aedes aegypti, Leptinotarsa decemlineata, Spodoptera
litura, molecular modelling, docking.

The evolution of insecticide resistance in insects

tends to be rapid because selection is strong,
populations are large, and generation times are short.
Serious problem of genetic resistance in insect
species, widespread environmental hazards,
vertebrate toxicity and increasing cost of currently
using synthetic pesticides have directed to the
designing of effective biodegradable pesticides from
plants (Glenn et al., 1994; Ewete et al., 1996;
Guedes et al., 1997). Over 2000 species of plants
are known to possess some insecticidal activity, by
containing either antifeedant, repellent or insecticidal
compounds (Bouda et al., 2001; Klocke, 1989).
A chemical class conspicuous among plant
secondary compounds and containing chemicals
acting against insects are the terpenoids (Mabry and
Gill, 1979). The cyclic monoterpene, pulegone,
an irritant commonly found in mint oils, deters
* Corresponding author

feeding by the slug, Ariolimax dolichophallus

(Mead) and by the fall armyworm, Spodoptera
frugiperda (J.E. Smith) and repels the German
cockroach, Blattella germanica (L.) (Gunderson
et al., 1985).
Acetylcholinesterase (AChE; EC 3.1.1.7) is a
key enzyme of the cholinergic system because it
regulates the level of acetylcholine and terminates
nerve impulses by catalyzing the hydrolysis of
acetylcholine. Its inhibition causes death, so
irreversible inhibitors have been developed as
insecticides such as organophosphates and
carbamates (Aldridge, 1950). The first case of AChE
with a reduced sensitivity to pesticides was explained
by Smissaert (1964). There are huge number of
studies that are related to the AChE inhibitory
activity of monoterpenes, p-menthane skeleton in

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Mentha spp L. (Miyazawa et al., 1997) and oils of

Melissa officinalis L. and Rosmarinus officinalis
L. (Perry et al., 2000; Perry et al., 1996). Linalool,
a monoterpene compound reported to be one of
the major volatile components of the essential oils
of several aromatic species. A number of linalool
producing species are used in traditional medicine
systems to relieve symptoms and cure a variety of
ailments, both acute and chronic (Peana and Moretti,
2002). The linalool has various remarkable toxicity
properties against insects (Lopez, 2010).

MODELLER9v8 software. MODELLER

implements comparative protein structure modeling
by satisfaction of spatial restraints (Sali et al., 1993;
Fiser et al., 2000). The python script modelsingle.py was used to generate five models using
the template. The stereo quality of the generated
models was checked using the ProSA and
Ramachandran plot using the tool RAMPAGE
(Lovell et al., 2002). The modelled structures were
visualized using RASMOL (molecular graphics
visualisation Program).

In the present study, we analysed the binding

interaction of linalool with the AchE of three pests
belonging to different orders viz., Spodoptera litura
(Fab.) (Lepidoptera), Aedes aegypti (L.) (Diptera)
and Leptinotarsa decemlineata (Say)
(Coleoptera). The main aim of the present study is
in exploring the binding affinity and binding site
variations of linalool in the AchE of insect pests using
insilico approaches. Applying rational methods in
designing insecticides will be useful to overcome
problems in conventional methods.

Docking

MATERIALS AND METHODS

Target sequence collection
The acetycholinesterase protein sequences of L.
decemlineata (AAB00466.1), A. aegypti
(ABN09910.1) and S. litura (ACR47975.1) were
collected from the NCBI database.
Ligand search
The structure of Linalool (3,7-dimethylocta-1,6dien-3-ol; C10H18O) was downloaded from the
PUBCHEM database using the search option. The
insecticidal property of the ligand molecule was
evaluated using the physico-chemicals properties of
the compounds using Tice rules (Tice, 2001).
Template selection and Molecular modelling

The molecular interaction and the post dock

analysis were done using the default parameters in
iGEMDOCKv2.1 software (A Graphical
Environment for Recognizing Pharmacological
Interactions and Virtual Screening). GEMDOCK
uses an empirical scoring function and an
evolutionary approach. The GEMDOCK energy
function consists of electrostatic, steric, and
hydrogen-bonding potentials (Yang et al., 2004).
RESULTS AND DISCUSSION
The exact template for modelling the target
structure was short listed from BLASTP results using
the E-Value and the sequence identity between the
target and template (Table 1). There are five models
generated from the MODELLER software. Among
the five models, the top model was traced based on
the DOPE (Discrete Optimized Protein Energy)
score and GA341 score. GA341 score was used
to assess the overall fold quality of the modelled
structure. The models which have less DOPE score
was considered as the top model (Table 2, Figure
1). The ProSA results showed that the modelled
structure relies on the energy values of the template
(Figure 2). The Ramachandran plot showed that the
most of the residues present in the modelled
structures fall under the favoured region of the plot
(Table 3, Figure 3).

BLASTP program was used to select the correct

The structural properties of the linalool strictly
template for modelling the target structures.
Molecular modelling was done using the followed the Tice rules (Table 4). Thus, the linalool
could be a potent insecticide. The iGEMDOCK

342

results showed that the linalool have best interaction

with the AChE of the selected targets. Among the
three targets, the fitness energy value showed that
the intermolecular complex of acetylcholinesterase
of A. aegypti with linalool had best interaction
compared to S. litura and L. decemlineata (Table
5). Since molecules in nature have a tendency to be
found in their low energy form, the final configuration
of intermolecular complex should also be of low
energy (Pyne and Gayathri, 2005). The interaction
between the target and the ligand is due to van der
waals and Hydrogen bond interaction. Inhibition of
AChE activity by monoterpenoids were examined
against various pests (Lopez et al., 2009; Jukic et
al., 2007). Majority of the monoterpenoids such as
fenchone, S-carvone and linalool tested showed high
inhibition of the enzyme AChE (Lopez et al., 2009).
The post dock analysis explored the amino acids
involved in the intermolecular complex formation. A
common structural feature of terpenoids is their
hydrocarbon skeleton, which in turn confers upon
them a common property of hydrophobicity. Many
hydrophobic compounds are associated with
protein deactivation and enzyme inhibition, and
one enzyme particularly susceptible to hydrophobic
interactions is AChE (Hansch and Deutsch, 1966).

The docking results showed that linalool binds to

the target site at the hydrophobic region which
consist of hydrophobic amino acids, PHE, ILE,
TRP, LEU, GLY, SER, TYR. The results
showed that in all the targets (AChE of S. litura, A.
aegypti and L. decemlineata) GLY was the
common amino acid involved in the interaction.
Other than GLY, there were few more amino acids
such as GLU, ILE, TRP found commonly in the
interaction profile of A. aegypti and L.
decemlineata (Table 5, Figure 4). The binding
pocket comparison showed that the linalool binding
to the AChE in A. aegypti and L. decemlineata
were similar with overlapping amino acids in
comparison to S. litura. Further in-vivo studies on
the action of linalool against the AChE of A. aegypti,
L. decemlineata and S. litura could offer clearer
understanding of the insecticidal activity of linalool.
CONCLUSION
The above findings based on bioinformatic tools
prove that linalool has effective insecticidal property
against A. aegyptii, L. decemlineata and S. litura.
It inhibits acetylcholineesterase and the interaction
of ligand with the receptor.

Table 1. Templates used for the modelling of target structures.

Target:
Acetylcholinesterase
Leptinotarsa
decemlineata

Aedes aegypti

Spodoptera litura

Template

PDB
Id of
the template

E-Value

Sequence identity between

target and template

Chain A, Native
Acetylcholinesterase
from Drosophila
melanogaster

1QO9

0.0

59%

Chain A, Fasciculin
2-Mouse
Acetylcholinesterase
complex

1KU6

5e-151

49%

Chain X, ACheE in
complex with a Bis(-)-Nor-Meptazinol
derivative

2W6C

3e-107

46%

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Table 2. DOPE and GA341 scores of the top models obtained from MODELLER.
Target: Acetylcholinesterase

Dope score

GA341 score

Leptinotarsa decemlineata

-70433.67969

1.00000

Aedes aegypti

-68614.414063

1.00000

Spodoptera litura

-48003.48047

1.00000

Table 3. Stereo quality of the top models using Ramachandran plot.

Target: Acetylcholinesterase

Residues in favoured
region (%)

Residues in
allowed region (%)

94.6

5.1

0.3

Leptinotarsa decemlineata

92.2

5.6

2.2

Aedes aegypti

93.1

4.7

2.1

Spodoptera litura

Residues in
outlier region (%)

Table 4: Molecular property analysis of linalool.

Parameters

Tice rule

Linalool

Molecular Weight

<500

154.24932 [g/mol]

LogP

<5

2.7

H-bond donor

<3

H-bond acceptor

<12

Table 5. Docking results of the targets and linalool obtained from iGEMDOCK analysis.
Target-Ligand complex:
Acetylcholinesterase linalool

Total
fitness
energy

VDW
interaction

Spodoptera litura

-59.58

Aedes aegypti

Leptinotarsa decemlineata

H bond
interaction

Amino acid residues

involved in interaction

-54.06

-5.53

GLY 41, TYR 43, PHE

210, PHE 251

-72.87

-61.98

-10.89

THR 410, GLY 412,

GLU 415, TRP 408,
GLY 409, GLY 409,
GLY 412, ILE 413

-64.46

-53.46

-11

SER 588, GLY 589, LUE

619, GLU 592, ILE 620,
TRP 621

344

(A)

(B)

(C)

Figure 1. Three-dimensional structure of acetylcholinesterase modelled using MODELLER: A) Spodoptera

litura B) Aedes aegypti and C) Leptinotarsa decemlineata.

(A)

(B)

(C)

Figure 2. Energy plot of acetylcholinesterase generated by the tool ProSA. Light and dark lines indicate the
templates and targets: A) Spodoptera litura B) Aedes aegypti and C) Leptinotarsa decemlineata.

(A)

(B)

(C)

Figure 3. Ramachandran plot of acetylcholinesterase: A) Spodoptera litura B) Aedes aegypti and C)

Leptinotarsa decemlineata.

(A)

(B)

(C)

Figure 4. Interaction of linalool with the AChE of A) Spodoptera litura B) Aedes aegypti and C) Leptinotarsa
decemlineata.

345

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