title: Cucurbitacins are insect steroid hormone antagonists acting at the ecdysteroid receptor.
abstract: Two triterpenoids, cucurbitacins B and D, have been isolated from seeds of Iberis umbellata (Cruciferae) and shown to be responsible for the antagonistic activity of a methanolic extract of this species in preventing the 20-hydroxyecdysone (20E)-induced morphological changes in the Drosophila melanogaster BII permanent cell line. With a 20E concentration of 50 nM, cucurbitacins B and D give 50% responses at 1.5 and 10 microM respectively. Both cucurbitacins are able to displace specifically bound radiolabelled 25-deoxy-20-hydroxyecdysone (ponasterone A) from a cell-free preparation of the BII cells containing ecdysteroid receptors. The Kd values for cucurbitacins B and D (5 and 50 microM respectively) are similar to the concentrations required to antagonize 20E activity with whole cells. Cucurbitacin B (cucB) prevents stimulation by 20E of an ecdysteroid-responsive reporter gene in a transfection assay. CucB also prevents the formation of the Drosophila ecdysteroid receptor/Ultraspiracle/20E complex with the hsp27 ecdysteroid response element as demonstrated by gel-shift assay. This is therefore the first definitive evidence for the existence of antagonists acting at the ecdysteroid receptor. Preliminary structure/activity studies indicate the importance of the Delta23-22-oxo functional grouping in the side chain for antagonistic activity. Hexanorcucurbitacin D, which lacks carbon atoms C-22 to C-27, is found to be a weak agonist rather than an antagonist. Moreover, the side chain analogue 5-methylhex-3-en-2-one possesses weak antagonistic activity.
authors.affiliation: Department of Biological Sciences, University of Exeter, Washington Singer Laboratories, Perry Road, Exeter, Devon EX4 4QG, U.K.
authors.forename: L
authors.initials: L
authors.lastname: Dinan
authors.forename: P
authors.initials: P
authors.lastname: Whiting
authors.forename: J P
authors.initials: JP
authors.lastname: Girault
authors.forename: R
authors.initials: R
authors.lastname: Lafont
authors.forename: T S
authors.initials: TS
authors.lastname: Dhadialla
authors.forename: D E
authors.initials: DE
authors.lastname: Cress
authors.forename: B
authors.initials: B
authors.lastname: Mugat
authors.forename: C
authors.initials: C
authors.lastname: Antoniewski
authors.forename: J A
authors.initials: JA
authors.lastname: Lepesant
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000166:cytology / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D004441:Ecdysterone / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D019286:Endopeptidase K / Q000378:metabolism; D017344:Genes, Insect; D017930:Genes, Reporter; D008968:Molecular Conformation; D010936:Plant Extracts / Q000494:pharmacology; D011987:Receptors, Steroid / Q000037:antagonists & inhibitors*; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000037:antagonists & inhibitors; D014162:Transfection; D014315:Triterpenes / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*
chemical_list: D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D010936:Plant Extracts; D011987:Receptors, Steroid; D014157:Transcription Factors; D014315:Triterpenes; C066030:USP protein, Drosophila; C034808:ecdysteroid receptor; C041246:cucurbitacin B; D004441:Ecdysterone; C038105:cucurbitacin D; D019286:Endopeptidase K