The discovery of novel non-steroidal ecdysone agonists, belonging to the bisacylhydrazine class of chemistry, as environmentally safe pest selective insecticides has generated a lot of interest in understanding the molecular basis of action of these compounds. While there is substantial evidence that these compounds manifest their toxic effect in susceptible insects (mainly lepidopteran larvae) via interaction with the ecdysone receptor (EcR) protein as a heterodimer with ultraspiracle (USP) protein, it is still not clear where in the ligand binding domain (LBD) of EcR these compounds bind. The interaction of these compounds relative to that of the insect molting hormone, 20-hydroxyecdysone (20E), within the EcR LBD is also unknown. In an attempt to understand the molecular interaction of the lepidopteran active bisacylhydrazine insecticides (tebufenozide and methoxyfenozide) with lepidopteran EcR, we have synthesized a tritiated photoaffinity labelled (PAL) analog of the two insecticidal compounds. In ligand binding assays using tritiated ponasterone A as the radioligand and bacterial fusion proteins of EcR and USP from the spruce budworm, Choristoneura fumiferana, the unlabeled PAL displaces tritiated ponasterone A. The tritiated PAL binds to CfEcR/CfUSP in a specific and saturable manner and has been used to demonstrate its binding to CfEcR by analysis of binding reactions by SDS-PAGE followed by fluorography. Experiments are in progress to generate proteolytic fragments of PAL labeled CfEcR LBD for identification of the contact amino acid residues by LC/MS analysis.

Index Terms: Ecdysone receptor, photoaffinity ligand, spruce budworm