Binding affinity of nonsteroidal ecdysone agonists against the ecdysone receptor complex determines the strength of their molting hormonal activity

N-tert-Butyl-N,N'-dibenzoylhydrazine and its analogs are nonsteroidal ecdysone agonists that exhibit insect molting hormonal and larvicidal activities. The interaction mode of those ecdysone agonists with the heterodimer of the ecdysone receptor and ultraspiracle has not been fully elucidated. We expressed the ecdysone receptor B1 and the ultraspiracle of the lepidopteran, Chilo suppressalis, using an in vitro transcription/translation system and confirmed, using gel-shift assays, that the proteins function as ecdysone receptors. We also analyzed their ligand-binding affinity. A potent ecdysteroid, ponasterone A, specifically bound to the ecdysone receptor with low affinity (K-D = 55 nM), and the specific binding was dramatically increased (K-D = 1.2 nM) in the presence of the ultraspiracle. For seven nonsteroidal ecdysone agonists and five ecdysteroids, the binding activity to the in vitro-translated ecdysone receptor ultraspiracle complex was linearly correlated with the binding activity to the inherent receptor protein in the cell-free preparation of C. suppressalis integument. The binding to the ecdysone receptor-ultraspiracle complex for a series of compounds was highly correlated with their molting hormonal activity, indicating that the binding affinity of nonsteroidal ecdysone agonists to the ecdysone receptor-ultraspiracle complex primarily determines the strength of their molting hormonal activity.