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Insect Pest Management, A Current Scenario, 2011 (ed.), Dunston P. Ambrose, Entomology Research Unit, St. Xavier’s 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:
[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 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 Tice’s 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
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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 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
