MOL2NET Molecular docking study of triterpenoid azadirachtin A on acetylcholinesterase of Drosophila melanogaster (Diptera: Drosophilidae)

Organic molecules of botanical origin can offer a source of compounds of pest management that are more environmentally acceptable and an efficient alternative to replace persistent synthetic insecticides. The molecular docking study using Molegro Virtual Docker software identified that the triterpenoid azadirachtin A showed stable conformations, with lower energy in the ligand-receptor complex of the compounds analyzed in this study, thus having a high affinity for the active site of the enzyme acetylcholinesterase, from a variety of interactions, which can determine its insecticidal potential against the species Drosophila melanogaster. _____________________________________________________________________________


Introduction
Pest control has mainly depended on insecticides.
Organophosphates, carbamates, pyrethroids and neonicotinoids show the development of insects resistant to various insecticides. To evaluate insecticide toxicities, Drosophila melanogaster is an interesting model (ARAIN et al., 2017). Organic molecules of botanical origin can offer a source of pest control compounds that are more environmentally acceptable and an efficient alternative to replace persistent synthetic insecticides. The increasing interest in the potential of secondary metabolites in pest control favors the search for new sources of biologically active natural products with low mammalian toxicity, low persistence in the environment, and biodegradability (CESPEDES et al., 2013). Bioinsecticides are safer than synthetic pesticides due to rapid degradation in the environment and low toxicity to vertebrates (DERE et al., 2015). Therefore, one of the alternatives is the use of botanical insecticides, Azadirachtin A, is a triterpenoid belonging to the class limonoids, which is present mainly in the seeds of the neem tree (Azadirachta indica) (MORGAN 2009) and is one of the most biologically natural insecticides SciForum MOL2NET, 2017, 3, 10.3390/mol2net-03-05054 2 http://sciforum.net/conference/mol2net-03/wrsamc active (BOULAHBEL et al., 2015).
Recently this compound was evaluated as a significant biopesticide and used for increasingly in pest control programs (BAJWA and AHMAD, 2012). Therefore, the objective of this work was to verify the interactions between triterpenoid molecules azadirachtin A, the active principle of a synthetic insecticide carbofuran and the PDB ligand in the active site of the enzyme acetylcholinesterase of the species Drosophila melanogaster, helping to understand the determining characteristics of the interaction ligandreceptor.

Results and Discussion
In the molecular docking study it was possible to verify that the activity of the selected triterpenoid Azadirachtin A, shows a greater affinity with the acetylcholinesterase enzyme than the commercial insecticide carbofuran and 9-nphenylmethylamino-tacrine (PDB ligand). In the Table 1, we can observe that the triterpenoid Azadirachtin A presented the lowest binding energy value, in relation to 9-nphenylmethylamino-tacrine (PDB ligand) and to the active principle, carbofuran. This demonstrates that Azadirachtin A presented more stable conformations, thus, as the greater number of interactions with the amino acid residues in the enzyme acetylcholinesterase. Analyzes of the interactions identified by the amino acid residue of the enzyme with the ligands under study were also performed.

Protein Data Bank (PDB)
The acetylcholinesterase enzyme was selected because of its importance in the nervous system of insects, because in order for nerve impulses to be transmitted through synapses, it is necessary for a neurotransmitter, acetylcholine (ACh), to transmit these impulses from one neuron to another, until it reaches the cell to be excited. After this excitation is performed the acetylcholine needs to return to the inside of the neuron where the nerve cell returns to the resting state and can be excited again. This return is accomplished by the enzyme acetylcholinesterase that breaks Acetylcholine into choline + acetate, which within the neuron rejoins acetylcholine for a new transmission. The active principles of insecticides, such as Carbofuran, which belong to the chemical group of organophosphates and carbamates and act by binding to the enzyme acetylcholinesterase inhibiting its action, resulting in an accumulation of acetylcholine in the synapse causing hyperexcitability due to continuous transmission and uncontrolled nervous impulses including tremors, seizures, collapse of the central nervous system and death (MATIAS, 2016). According to Cespedes et al. (2013), global agricultural systems consistently use pesticides of synthetic origin, such as carbamates and organophosphates. These active pesticide targets target acetylcholinesterase and have resulted in a generation of new insect strains resistant to the original pesticides. The development of resistance is related to the modification of receptors involved in the mechanisms and targets of action of a given molecule.

Docking
Initially, the crystallized structure of the acetylcholinesterase protein was obtained in the PDB (Protein Data Bank) under the code 1DX4, being this protein of origin of the species Drosophila melanogaster. The resolution of the crystallographic structure deposited in the PDB is 2.7Å. Anchoring of the molecules was performed using a 15Å GRID in the radius and 0.30Å resolution at the enzyme binding site with the structures. Molecular docking calculations were performed in the Molegro Virtual Docker v.6.0.1 software and the Algorithm Molde Score algorithm. The water molecule and cofactors were removed from the protein to aid in understanding the ligand-receptor interaction. A template was created on the enzyme using the 9-nphenylmethylamino-tacrine linker, which was obtained along with the pdb file. The Moldock score algorithm was used as a score function to predict the best interaction between ligand and receptor. Next, a docking wizard was created, in which the enzyme molecules and the ligands were inserted, to analyze the stability of the system through the interactions identified with the active site of the enzyme, taking as reference the value of MolDock Score energy.

Conclusion
In conclusion, the molecular coupling study using the Molegro Virtual Docker software identified that the triterpenoid azadirachtin A showed more stable conformations, with a lower energy in the ligand-receptor complex of the compounds analyzed in this study, thus having a high affinity for the active site of the enzyme acetylcholinesterase, from a variety of interactions, which may determine its insecticidal potential against the species Drosophila melanogaster. In relation to the active principle of carbofuran, this showed a lower binding affinity with the amino acid residues of the enzyme, although it is widely used as a commercial insecticide (Furadan). Therefore, docking studies have proved to be a useful tool capable of identifying electronic affinity and helping to understand the ligand-receptor interaction.