inhibition of acetylcholinesterase by linalool

8/10/2019 INHIBITION OF ACETYLCHOLINESTERASE by linalool

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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: [email protected]

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 theevolution of novel molecules for pest management. In the present study, the interaction of

monoterpene linalool with the AChE ofAedes 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

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 ofstudies that are related to the AChE inhibitory

activity of monoterpenes, p-menthane skeleton in

* Corresponding author

Insect Pest Management, A Current Scenario, 2011 (ed. ), Dunston P. Ambrose,Entomology Research Unit , St. Xavier s College, Palayamkottai, India, pp.340-345.


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MenthasppL. (Miyazawa et al.,1997) and oils ofMelissa officinalisL. andRosmarinus 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).

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 Lept ino tarsa deceml ineata (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 overcomeproblems in conventional methods.

MATERIALS AND METHODS

Target sequence collection

The acetycholinesterase protein sequences ofL.

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-1,6-

dien-3-ol; C10

H18

O) 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

BLASTP program was used to select the correcttemplate for modelling the target structures.

Molecular modelling was done using the

MODELLER9v8 software. MODELLERimplements comparative protein structure modeling

by satisfaction of spatial restraints (Sali et al.,1993;

Fiser et al.,2000). The python script model-

single.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).

Docking

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 (Yanget 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).

The structural properties of the linalool strictly

followed the Tice rules (Table 4). Thus, the linalool

could be a potent insecticide. The iGEMDOCK


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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

ofA. aegypti with linalool had best interaction

compared to S. litura andL. 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 (Lopezet al.,2009).

The post dock analysis explored the amino acids

involved in the intermolecular complex formation. A

common structural feature of terpenoids is theirhydrocarbon 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 inA. aegypti andL. decemlineata

were similar with overlapping amino acids in

comparison to S. litura. Further in-vivostudies on

the action of linalool against the AChE ofA. 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

againstA. aegyptii,L. decemlineataand S. litura.

It inhibits acetylcholineesterase and the interaction

of ligand with the receptor.

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

Leptinotarsa

decemlineata

Aedes aegypti

Spodoptera litura

Target:

Acetylcholinesterase

PDB

Id ofthe template

Sequence identity between

target and template

E-ValueTemplate

Chain A, Native


from Drosophila

melanogaster

1QO9 0.0 59%

Chain A, Fasciculin

2-Mouse


complex

1KU6 5e-151 49%

Chain X, ACheE incomplex with a Bis-

(-)-Nor-Meptazinol

derivative

2W6C 46%3e-107


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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 Residues in Residues in

region (%) allowed region (%) outlier region (%)

Spodoptera litura 94.6 5.1 0.3

Leptinotarsa decemlineata 92.2 5.6 2.2

Aedes aegypti 93.1 4.7 2.1

Table 4: Molecular property analysis of linalool.

Parameters Tice rule Linalool

Molecular Weight


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(A) (B) (C)

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

lituraB)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 lituraB)Aedes aegypti and C)Leptinotarsa decemlineata.

(A) (B) (C)

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

Leptinotarsa decemlineata.

(A) (B) (C)

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

decemlineata.


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inhibition of acetylcholinesterase by linalool

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