Virtual screening for ligands of the insect molting hormone receptor.

Insect growth is regulated by the orchestrated event of ecdysteroids and their receptor proteins. Agonists/antagonists of ecdysteroid receptor are predicted to disrupt normal growth, providing good candidates of new insecticides. A database of over 2 million compounds was subjected to a shape-based virtual screening cascade to identify novel nonsteroidal hits similar to the known EcR ligand ponasterone A. Testing revealed micromolar hits against two strains of insect cells. Docking experiments against EcR were used to support the predicted binding mode of these ligands based on their overlay to ponasterone A.

Towards Coleoptera-specific high-throughput screening systems for compounds with ecdysone activity: development of EcR reporter assays using weevil (Anthonomus grandis)-derived cell lines and in silico analysis of ligand binding to A. grandis EcR ligand-binding pocket.

Molting in insects is regulated by ecdysteroids and juvenile hormones. Several synthetic non-steroidal ecdysone agonists are on the market as insecticides. These ecdysone agonists are dibenzoylhydrazine (DBH) analogue compounds that manifest their toxicity via interaction with the ecdysone receptor (EcR). Of the four commercial available ecdysone agonists, three (tebufenozide, methoxyfenozide and chromafenozide) are highly lepidopteran specific, one (halofenozide) is used to control coleopteran and lepidopteran insects in turf and ornamentals. However, compared to the very high binding affinity of these DBH analogues to lepidopteran EcRs, halofenozide has a low binding affinity for coleopteran EcRs. For the discovery of ecdysone agonists that target non-lepidopteran insect groups, efficient screening systems that are based on the activation of the EcR are needed. We report here the development and evaluation of two coleopteran-specific reporter-based screening systems to discover and evaluate ecdysone agonists. The screening systems are based on the cell lines BRL-AG-3A and BRL-AG-3C that are derived from the weevil Anthonomus grandis, which can be efficiently transduced with an EcR reporter cassette for evaluation of induction of reporter activity by ecdysone agonists. We also cloned the almost full length coding sequence of EcR expressed in the cell line BRL-AG-3C and used it to make an initial in silico 3D-model of its ligand-binding pocket docked with ponasterone A and tebufenozide.

High-throughput screening of ecdysone agonists using a reporter gene assay followed by 3-D QSAR analysis of the molting hormonal activity.

In this study, 172 diacylhydrazine analogs were examined for their ability to activate an ecdysone (molting hormone)-dependent reporter gene in a silkworm (Bombyx mori) cell-based high-throughput screening assay. The measured EC(50) values (concentration required to cause an effect in 50% of the cells) were used to construct a 3-D QSAR model that describes the ecdysone agonist activities of the diacylhydrazine analogs. Of these compounds, 14 exhibited no activity and were excluded from the 3-D QSAR analysis. The resulting equation described approximately 74% of the activity for 158 compounds. The final equation consisted of 42% electrostatic and 58% steric effects (r(2) = 0.74 and q(2) = 0.45). Comparative molecular field analysis (CoMFA) was used to visualize the steric and electrostatic potential fields that were favorable and unfavorable for biological activity. Of particular interest was the observation that the hydrophobic parameter (logP) was not necessary for describing the observed activities, although previous studies have cited the importance of hydrophobic parameters in both classical and 3-D QSAR analyses of these compounds. Modeling studies of the B. mori ecdysone receptor supported the observed physicochemical parameters required for activity reported by the CoMFA models. Comparison of the present analysis with those performed using other lepidopteran assay systems evidenced a high degree of correlation (r(2) = 0.81 for a Sf-9 cell-based assay and r(2) = 0.89 for a Chilo suppressalis integument-based assay), indicating that it is valid to compare the results generated with the B. mori cell-based system to those generated with previous lepidopteran assays. This novel assay system is amendable to a high-throughput screening format and should greatly increase our ability to discover novel agonists of molting hormone (ecdysone) activity.

Cloning and development expression of Choristoneura hormone receptor 75: a homologue of the Drosophila E75A gene.

Cloning and characterization of a cDNA of the spruce budworm, Choristoneura fumiferana, that showed high amino acid similarity with the deduced amino acid sequences of E75 cDNAs cloned from Manduca sexta, Galleria melonella, and Drosophila melanogaster are described. Initially, a cDNA fragment and then a full length cDNA were cloned from C. fumiferana. The longest open reading frame of this cDNA had 690 codons and its deduced amino acid sequence had all five domains typical of a steroid hormone nuclear receptor. The deduced amino acid sequence of this cDNA showed the highest identity with the deduced amino acid sequence of E75A cDNAs cloned from M. sexta, G. melonella, and D. melanogaster, and is therefore named Choristoneura hormone receptor 75A (CHR75A). The CHR75A cDNA probe detected a 2.6 kb mRNA that was abundant at the time of the ecdysteroid peaks during molting in the embryonic, larval and pupal stages. In the sixth instar larvae, CHR75 mRNA was detected in the epidermis, fat body and midgut, and maximum expression was observed during the prepupal peak of ecdysteroids in the hemolymph. CHR75 mRNA was induced in ecdysone treated CF-203 cells and in the midgut, fat body and epidermis of larvae that were fed the non-steroidal ecdysteroid agonist, RH-5992. In vitro transcription and translation of the CHR75A cDNA yielded a 79 kDa protein that bound to the retinoic acid receptor related orphan receptor response element (RORE).