Triagem de compostos com base na ativação do receptor de ecdisteroides em culturas celulares e planejamento racional de moléculas inseticidas associado a ferramentas computacionais

Abstract

Applied cellular biotechnology is essential for Integrated Pest Management, because through several high performance techniques, it allows the rational planning of new insecticides. Nuclear receptors are important targets in the research for new insecticides, and the ecdysteroid receptor is one of the most important targets nowadays. This receptor is a protein whose artificial transactivation is mediated by molecules (ligands) that bind to its active site, thus causing an agonist or antagonistic effect on the hormone 20-hydroxyecdysone. Computational tools are of fundamental importance for the understanding of receptor/ligand interactions, thus enabling the design of new molecules. The aim of this research was to establish a protocol for transfection of insect cells with a plasmid sensitive to the activity of the ecdysone receptor, to compare the activity of ecdysteroids and dibenzoylhydrazines, and new organocalcogenic molecules (similar to dibenzoylhidrizines) understanding its mechanism of action with computational tools. Bioassays consisted of the transfection of S2 (Diptera) and SF9 (Lepidoptera) cell lines with the plasmid B.act.ere.luc., designed to express the luciferase enzyme against the activation of the receptor. The activity of the hormones 20-hydroxyecdysone and Ponasterone-A, the insecticide tebufenozide (dibenzoylhydrazine) and eleven new molecules of dibenzoylhydrazines in cell lines were evaluated. Dose response curves were prepared with the two hormones and tebufenozide. With the new molecules, the cytotoxicity, agonist and antagonist effect on ecdysteroids was investigated. To investigate the antagonistic activity of the new molecules on the ecdysteroids, the parameter established for a molecule have a considerable effect was the reduction of >50% of the activity, comparing to the action of the isolated hormone. Finally, using three-dimensional models, the interactions between ligands and the active site of the Diptera and Lepidoptera ecdysone receptor were investigated. It was concluded that it is possible to find new molecules with activity in the insect ecdysone receptor. In both cell lines, Ponasterone-A was the most active molecule, and in SF9, tebufenozide was more active than 20-Hydroxyecdysone. Among the new dibenzoylhydrazines tested, none showed an agonist effect. Antagonistic effect was observed only in Lepidoptera cells, but with different patterns according to the hormone used. The antagonistic activity observed from some new molecules can be due to the fitting of them in the binding site from the ecdysone rceptor, blocking important ligation among receptor and hormone.