CUCURBITACINS ARE INSECT STEROID-HORMONE ANTAGONISTS ACTING AT THE ECDYSTEROID RECEPTOR

Two triterpenoids, cucurbitacins B and D, have been isolated from seed s of Iberis umbellata (Cruciferae) and shown to be responsible for the antagonistic activity of a methanolic extract of this species in prev enting the 20-hydroxyecdysone (20E)-induced morphological changes in t he Drosophila melanogaster B-II permanent cell line, With a 20E concen tration of 50 nM, cucurbitacins B and D give 50% responses at 1.5 and 10 mu M respectively. Both cucurbitacins are able to displace specific ally bound radiolabelled 25-deoxy-20-hydroxyecdysone (ponasterone A) f rom a cell-free preparation of the B-II cells containing ecdysteroid r eceptors. The K-d values for cucurbitacins B and D (5 and 50 mu M resp ectively) 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 a ssay. CucB also prevents the formation of the Drosophila ecdysteroid r eceptor/Ultraspiracle/20E complex with the hsp27 ecdysteroid response element as demonstrated by gel-shift assay, This is therefore the firs t definitive evidence for the existence of antagonists acting at the e cdysteroid receptor. Preliminary structure/activity studies indicate t he importance of the Delta(23)-22-oxo functional grouping in the side chain for antagonistic activity. Hexanorcucurbitacin D, which lacks ca rbon 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 p ossesses weak antagonistic activity.