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. Insect Biochem Molec Biol

Laboratory of Agrozoology, Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, Ghent, Belgium.
Insect biochemistry and molecular biology (Impact Factor: 3.45). 07/2009; 39(8):523-34. DOI: 10.1016/j.ibmb.2009.06.003
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ABSTRACT 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.

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Available from: Guy Smagghe, Sep 28, 2015
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    • "ochemical and molecular effects of tebufenozide ( Mikitani , 1996 ) , methoxyfenozide , halofenozide , or chromafenozide and other BAH EAs ( Dhadialla et al . , 2005 ; Farkas and Slama , 1999 ; Mosallanejad et al . , 2008a , b ; Nakagawa , 2005 ; Nakagawa et al . , 2002a , b , c ; Smagghe and Swevers , 2013 ; Smagghe et al . , 1996 , 2000 , 2002 ; Soin et al . , 2009 , 2010a , b ) . Cytosolic and / or nuclear extracts from 20E - responsive cells and tissues containing functional EcRs , and bacterially expressed EcRs and USPs from different insects , also have been used to determine the relative binding affinities of BAH insecticidal compounds or to screen for new chemistries with a similar mode of a"
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    ABSTRACT: In this chapter, we review five members of a novel class of chemistry, the non-steroidal bisacylhydrazine (BAH) compounds that are true agonists of the steroidal insect moulting hormone, 20-hydroxyecdysone. Also referred to as ecdysone agonists (EAs), the five BAH compounds have been commercialized for the control of lepidopteran and coleopteran larvae. Of these, four compounds (methoxyfenozide, tebufenozide, chromafenozide, and fufenozide) are predominantly toxic to lepidopteran larvae, while the fifth compound, halofenozide, is active on both lepidopteran and coleopteran larval pests in turf. The evidence for the basis of this insect selective toxicity is reviewed. The nonsteroidal EA BAH insecticidal compounds are important tools in integrated pest management and insect resistance management programmes because of their selective insect toxicity, novel mode of action, and reduced risk for eco- and mammalian toxicology. In reviewing these BAH insecticides, there is greater emphasis on methoxyfenozide, the most widely used insecticide in this class of chemistry.
    Advances in Insect Physiology, Vol. 43, 1st edited by Tarlochan S. Dhadialla, 10/2012: chapter 2: pages 163-249; Academic Press, Elsiever Ltd.., ISBN: 978-0-12-391500-9
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    • "RGA is convenient because the experiment can be done without using radioactive materials, but its use has been mainly limited to lepidopteran and dipteran cell lines so far. Only one report of an RGA exists that is based on cells derived from a coleopteran species [25], despite the abundance of important pests of agricultural products within this order. The establishment of RGAs using coleopteran cell lines would be helpful for the development of new insecticides with selective toxicity against such pests. "
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    ABSTRACT: A novel reporter gene assay system using BCIRL-Lepd-SL1, a cell line from the coleopteran Colorado potato beetle (Leptinotarsa decemlineata), was established. Cells were transiently transfected with the reporter plasmid that was composed of a firefly luciferase gene with upstream ecdysone response elements, and an internal control plasmid that constitutively produces Renilla reniformis luciferase. Transfected cells were incubated with various molting hormone agonists, and the activity of these agonists was quantitatively determined by measuring luminescence emission. Transcription-inducing activity for ecdysone, 20-hydroxyecdysone and ponasterone A in terms of EC50 (50% effective concentration) were determined to be 1 μM (pEC50 = 5.99), 68 nM (pEC50 = 7.17) and 1.3 nM (pEC50 = 8.88), respectively. Among tested diacylhydrazine (DAH)-type compounds, 11 compounds were active (pEC50 = 3.56 ∼ 6.41), but two compounds were inactive. The EC50 values were linearly correlated to their binding affinity except for one compound. While several ecdysone reporter systems were developed before that employ dipteran and lepidopteron cell lines, the assay system described here is only the second one that employs a coleopteran cell line. This reporter system will allow screening in high-throughput format for new and more potent molting accelerating compounds that specifically target coleopteran pests.
    Pesticide Biochemistry and Physiology 09/2012; 104(1):1–8. DOI:10.1016/j.pestbp.2012.06.009 · 2.01 Impact Factor
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    • "It is worth mentioning that the residue Val92, conserved in lepidopteran insects but replaced by methionine in other insects, is considered to be essential for the specificity of BYI06830 to Lepidoptera (Fig. 6). As previously shown by Soin et al. (2009) this residue interacts via hydrophobic interaction with tebufenozide docked in the EcR-LBD model of the coleopteran A. grandis. Similarly, this "
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    ABSTRACT: Understanding how variations in genetic sequences are conveyed into structural and biochemical properties is of increasing interest in the field of molecular evolution. In order to gain insight into this process, we studied the ecdysone receptor (EcR), a transcription factor that controls moulting and metamorphosis in arthropods. Using an in silico homology model, we identified a region in the lepidopteran EcR that has no direct interaction with the natural hormone but is under strong evolutionary constraint. This region causes a small indentation in the three-dimensional structure of the protein which facilitates the binding of tebufenozide. Non-Mecopterida are considered much older, evolutionarily, than Lepidoptera and they do not have this extended cavity. This location shows differences in evolutionary constraint between Lepidoptera and other insects, where a much lower constraint is observed compared with the Lepidoptera. It is possible that the higher flexibility seen in the EcR of Lepidoptera is an entirely new trait and the higher constraint could then be an indication that this region does have another important function. Finally, we suggest that Try123, which is evolutionarily constrained and is up to now exclusively present in Lepidoptera EcRs, could play a critical role in discriminating between steroidal and non-steroidal ligands.
    Insect Molecular Biology 07/2012; 21(5):488-501. DOI:10.1111/j.1365-2583.2012.01154.x · 2.59 Impact Factor
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