Molecular analysis of the mode of action of RH-5992, a lepidopteran-specific, non-steroidal ecdysteroid agonist

The dibenzoyl hydrazine, RH-5992, induces precocious molting in all lepidopteran larvae tested including the tobacco hornworm, Manduca sexta and the spruce budworm, Choristoneura fumiferana. Synthesis of a new cuticle including formation of a new head capsule that is incompletely sclerotized and lack of ecdysis are some of the major phenotypic effects. Similar to 20E, RH-5992 induces Manduca hormone receptor 3 (MHR3) mRNA and suppresses 14 kDa larval cuticular protein (LCP-14) transcript levels in 4th instar epidermis of Manduca, cultured in vitro. The ED₅₀ for induction of MHR3 was 1.4 × 10^-7 M, 10 times less than that of 20E. When the epidermis was exposed to 20E for 17 h and then cultured in hormone-free medium for a further 48 h, the LCP-14 mRNA level went up to nearly 60% of the maximal level reached in the untreated control. However, when RH-5992 was used, the level remained low even after 48 h. Dopa decarboxylase (DDC) transcription normally occurs at the end of the molt in preparation for sclerotization and in the epidermis cultured in vitro requires initial exposure to 20E for 17 h followed by its removal. Both in vivo and transient in vitro treatment with RH-5992 prevented DDC expression for up to 48 h after the removal of the compound. Thus, RH-5992 mimics the action of 20E on the expression of these 3 genes, but its effects persist in the tissue much longer than 20E.