A cell‐based high‐throughput screening system for detecting ecdysteroid agonists and antagonists in plant extracts and libraries of synthetic compounds

Abstract

Screening systems for ecdysteroid mimetic or antiecdysteroid substances in plant extracts or libraries of synthetic compounds are commonly based on the observation of morphological and/or growth responses in insect cell lines. Because these responses are slow and require careful monitoring, existing screening systems are considered limited regarding their applicability to analysis in high-throughput (HT) formats. Here we describe the generation of transformed silkmoth (Bombyx mori) cell lines that respond to the addition of ecdysone-like substances through the expression of the green fluorescent protein (GFP) and the appearance of green fluorescence. Because tests consist of three simple steps, i.e., 1) distribution of transformed cells in microtiter plates; 2) addition of compounds/extracts at different concentrations; and 3) quantification of fluorescence intensity by a fluorescence plate reader, they can be performed quickly and be easily adapted to a HT format. The generated reporter cell lines are used for the screening of extracts from available plant collections for the presence of compounds with ecdysone mimetic or antagonistic activities as well as for monitoring subsequent activity during enrichment and purification steps. The same cell lines are also used here for the determination of structure-activity relationships among available synthetic dibenzoylhydrazine derivatives. Finally, for the identified agonists, we show that their activity as determined by the cell-based screening assays parallels their bioactivity in growth inhibition and toxicity assays carried out on live insects.

Semi‐Synthetic Ecdysteroids as Gene‐Switch Actuators: Synthesis, Structure–Activity Relationships, and Prospective ADME Properties

“…In its best versions, it is characterised by extremely low basal transcription, high dynamic range of induction, and highly dose-responsive ligand sensitivity. Use of the EcR gene switch has been demonstrated both in cell and tissue culture (yeast, [51] plant, [52] insect, [53] mammalian [54] ) as well as in animal models (zebrafish, [55] mouse, [49,56] rat [57] ), both in a simple reporter gene format, as well as in functional genomics [52,58] and actual disease models. [59,60] The most potent ligands are represented by primarily two chemotypes: the synthetic diacylhydrazines and the natural, usually plant-derived, ecdysteroids.…”

Inhibition of Anopheles gambiae Odorant Receptor Function by Mosquito Repellents

“…To test the suitability of the expression system for use as screening platform for olfactory receptor agonists and antagonists in a single compound screen, as was previously reported for other ion channels (38), olfactory receptors (28), and nuclear receptors (39), cells co-expressing selected A. gambiae ORs with Orco and Photina were transferred to 96-well plates. Following coelenterazine loading, the cells were subjected to two cycles of compound additions (see Fig.…”

Cell-Based Screening Systems for Insecticides

“…The reporter plasmids typically consist of a basal reporter cassette (basal promoter followed by GFP or luciferase ORF and polyadenylation sequence) preceded by multiple copies of a high-af fi nity EcRE (for instance, seven copies of the EcRE identi fi ed in the hsp27 promoter of Drosophila , see Swevers et al 2004 ) (Fig. 6.1 ).…”

“…6.1 , cell-based screening systems were developed that are speci fi c for lepidopteran insects with the use of pEcRE/b.act/GFP or pEcRE/b.act/Luc reporter plasmids (based respectively on GFP and luciferase reporters). Good examples are cell lines that have been derived from lepidopteran insects (Bm5 and Sl2 cells derived from Bombyx mori and Spodoptera littoralis , respectively; Swevers et al 2004 ;Soin et al 2010a ) , dipteran insects (S2 cell line; derived from Drosophila melanogaster ; Soin et al 2010b ) , and coleopteran insects (Ag3C cell line; derived from Anthonomus grandis ; Soin et al 2009 ) . With the exception of Bm5 cells, EcRE-based reporter constructs were introduced by transfection and their presence in the cells in episomal form was only transient, therefore necessitating new transfections to be carried out for each new assay.…”