title: A cell-based reporter assay for screening for EcR agonist/antagonist activity of natural ecdysteroids in Lepidoptera (Bm5) and Diptera (S2) cell cultures, followed by modeling of ecdysteroid-EcR interactions and normal mode analysis.
abstract: Ecdysteroid signal transduction is a key process in insect development and therefore an important target for insecticide development. We employed an in vitro cell-based reporter bioassay for the screening of potential ecdysone receptor (EcR) agonistic and antagonistic compounds. Natural ecdysteroids were assayed with ecdysteroid-responsive cell line cultures that were transiently transfected with the reporter plasmid ERE-b.act.luc. We used the dipteran Schneider S2 cells of Drosophila melanogaster and the lepidopteran Bm5 cells of Bombyx mori, representing important pest insects in medicine and agriculture. Measurements showed an EcR agonistic activity only for cyasterone both in S2 (EC50=3.3μM) and Bm5 cells (EC50=5.3μM), which was low compared to that of the commercial dibenzoylhydrazine-based insecticide tebufenozide (EC50=0.71μM and 0.00089μM, respectively). Interestingly, a strong antagonistic activity was found for castasterone in S2 cells with an IC50 of 0.039μM; in Bm5 cells this effect only became visible at much higher concentrations (IC50=18μM). To gain more insight in the EcR interaction, three-dimensional modeling of dipteran and lepidopteran EcR-LBD was performed. In conclusion, we showed that the EcR cell-based reporter bioassay tested here is a useful and practical tool for the screening of candidate EcR agonists and antagonists. The docking experiments as well as the normal mode analysis provided evidence that the antagonist activity of castasterone may be through direct binding with the receptor with specific changes in protein flexibility. The search for new ecdysteroid-like compounds may be particularly relevant for dipterans because the activity of dibenzoylhydrazines appears to be correlated with an extension of the EcR-LBD binding pocket that is prominent in lepidopteran receptors but less so in the modeled dipteran structure.
authors.affiliation: Department of Crop Protection, Ghent University, Coupure links 653, 9000 Ghent, Belgium; Department of Phytosanitary, FAEM, Federal University of Pelotas, P.O. Box 354, CEP, 96010-900 Pelotas, RS, Brazil; Department of Crop Protection, Federal University of Santa Maria, Santa Maria, Brazil. Electronic address: moises.zotti@ugent.be.
authors.forename: Moisés J
authors.initials: MJ
authors.lastname: Zotti
authors.forename: Ellen
authors.initials: E
authors.lastname: De Geyter
authors.forename: Luc
authors.initials: L
authors.lastname: Swevers
authors.forename: Antônio S K
authors.initials: AS
authors.lastname: Braz
authors.forename: Luis P B
authors.initials: LP
authors.lastname: Scott
authors.forename: Pierre
authors.initials: P
authors.lastname: Rougé
authors.forename: Josep
authors.initials: J
authors.lastname: Coll
authors.forename: Anderson D
authors.initials: AD
authors.lastname: Grutzmacher
authors.forename: Eder J
authors.initials: EJ
authors.lastname: Lenardão
authors.forename: Guy
authors.initials: G
authors.lastname: Smagghe
mesh_terms: D000818:Animals; D002460:Cell Line; D004175:Diptera; D026461:Ecdysteroids / Q000378:metabolism*; D007915:Lepidoptera / Q000378:metabolism*; D011485:Protein Binding; D011987:Receptors, Steroid / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D026461:Ecdysteroids; D011987:Receptors, Steroid; C055469:ecdysone receptor
keywords: Cell-based screening system; Ecdysteroid agonist; Ecdysteroid antagonist; Molecular modeling; Normal mode analysis; Steroids