Ailanthone exerts insecticidal activity as a non-orthosteric 20E antagonist by promoting ecdysone receptor degradation.

Ailanthone, a natural quassinoid from Ailanthus altissima, has been reported to exhibit insecticidal activity, yet its molecular mechanism remains elusive. Here, we show that ailanthone acts as a non-orthosteric inhibitor of 20-hydroxyecdysone (20E) signaling by promoting ubiquitin-proteasome-dependent degradation of the ecdysone receptor (EcR). In both Drosophila melanogaster and Ostrinia furnacalis, ailanthone treatment inhibited insect growth and metamorphosis. Mechanistically, in D. melanogaster, these developmental defects were attributable to disruption of 20E signaling, as evidenced by suppressed expression of 20E early response genes. We further found that ailanthone suppresses 20E signaling not through competitive binding to the EcR ligand-binding pocket. Instead, it promotes ubiquitination and proteasomal degradation of EcR, leading to EcR depletion. This degradation is associated with reduced interaction between EcR and the chaperone Hsp83. These results identify a novel mode of 20E antagonism and highlight ailanthone as a promising lead compound for further development as an insect growth regulator.