昆虫蜕皮激素受体及其类似物的杀虫机制研究进展

昆虫的蜕皮、变态和繁殖受到蜕皮激素的严格调控。蜕皮激素作用靶标由蜕皮激素受体（ecdysteroid receptor, EcR）和超气门蛋白（ultraspiracle protein, USP）组成,蜕皮激素与EcR/USP作用启动蜕皮级联反应过程。昆虫EcR具有种类或类群的特异性,研究其结构、功能和调控机理在开发环境友好型新药剂和基因调控开关等方面具有重要指导作用。该文介绍了昆虫EcR的结构和功能特点,蜕皮激素及其类似物与EcR/USP的分子作用方式,以及基于EcR/USP的新杀虫剂创制和基因调控开关设计等方面的重要进展。     

Highly flexible ligand binding pocket of ecdysone receptor: a single amino acid change leads to discrimination between two groups of nonsteroidal ecdysone agonists

The insect steroid hormone 20-hydroxyecdysone works through a ligand-activated nuclear receptor, the ecdysone receptor (EcR), which plays critical roles in insect development and reproduction. The EcR has been exploited to develop insecticides to control pests and gene switches for gene regulation. Recently reported crystal structures of the EcR protein show different but partially overlapping binding cavities for ecdysteroid (ECD) and diacylhydrazine (DAH) ligands, providing an explanation for the differential activity of DAH ligands in insects. 1-Aroyl-4-(arylamino)-1,2,3,4-tetrahydroquinoline (THQ) ligands were recently discovered as ecdysone agonists. Mutagenesis of the EcR (from Choristoneura fumiferana, CfEcR) ligand binding domain followed by screening in a reporter assay led to the identification of CfEcR mutants, which responded well to THQ ligands but poorly to both ECD and DAH ligands. These mutants were further improved by introducing a second mutation, A110P, which was previously reported to cause ECD insensitivity. Testing of these V128F/A110P and V128Y/A110P mutants in a C57BL/6 mouse model coactivator interaction assay and in insect cells showed that this mutant EcR is activated by THQ ligands but not by ECD or DAH ligands. The CfEcR and its V128F/A110P mutant were used to demonstrate simultaneous regulation of two reporter genes using THQ and DAH ligands.     

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.     

Effect of ecdysone agonists on vitellogenesis and the expression of EcR and USP in codling moth (Cydia pomonella)

The effects of tebufenozide and methoxyfenozide on vitellogenin (Vg) synthesis/release in the fat body, translocation in hemolymph, uptake by the ovary, and the expression of the ecdysone receptor (EcR) and its heterodimer partner, ultraspiracle protein (USP) in fat body, were investigated in Cydia pomonella. The results indicated that both ecdysone agonists significantly increased the Vg level in the adult hemolymph when the moths were exposed to agonist-treated surfaces. However, these agonists did not affect Vg release from the fat body nor Vg deposition in the first batch oocytes. Western blot analysis revealed that the expression of EcR and USP was significantly increased in tebufenozide- and methoxyfenozide-treated samples compared to the control, suggesting that ecdysone agonists regulated the Vg synthesis via the EcR and USP proteins complex.     

Structure-based virtual screening for insect ecdysone receptor ligands using MM/PBSA

The ecdysone receptor (EcR) is an insect nuclear receptor that is activated by the molting hormone, 20-hydroxyecdysone. Because synthetic EcR ligands disrupt the normal growth of insects, they are attractive candidates for new insecticides. In this study, the Molecular Mechanics/Poisson–Boltzmann Surface Area (MM/PBSA) method was used to predict the binding activity of EcR ligands. Validity analyses using 40 known EcR ligands showed that the binding activity was satisfactorily predicted when the ligand conformational free energy term was introduced. Subsequently, this MM/PBSA method was applied to structure-based hierarchical virtual screening, and 12 candidate compounds were selected from a database of 3.8 million compounds. Five of these compounds were active in a cell-based competitive binding assay. The most potent compound is a simple proline derivative with low micromolar binding activity, representing a valuable lead compound for further structural optimization.     

Expression and purification of the hormone binding domain of the Drosophila ecdysone and ultraspiracle receptors

Escherichia coli vectors were constructed for the production of a protein complex that mimics the native ecdysone receptor (EcR) isolated from Drosophila. The two steroid receptors, ultraspiracle (USP) and EcR, were expressed as truncations, retaining primarily the hormone binding domains. The recombinant receptor complex was able to mimic the pharmacology of the native receptor with respect to both synthetic and natural agonists. USP and EcR fusion proteins could be expressed in separate cell lines and then recombined following isolation to yield a ligand binding preparation with a dissociation constant (K(D)) for Ponasterone A of 1.5 nM and a total yield of 1.9 pmol ligand binding sites/mg protein. Alternatively, the simultaneous coexpression of both receptors increased yields by several orders of magnitude to 6 nmol ligand binding sites/mg protein with a K(D) of 0.6 nM. Chromatographic analysis under native conditions showed that EcR, when expressed alone, migrated as a variety of complexes, mostly coming out in the void volume as denatured, insoluble, aggregate. In contrast, purified extracts of coexpressed EcR and USP eluted as a single peak with a mobility indicating a heterodimer. The majority of the coexpressed fusion receptors, following purification, formed functional steroid binding sites. A detailed scheme is provided for the expression and isolation of milligram quantities of highly purified receptor dimer.     

Nicotinic acetylcholine receptors: targets for commercially important insecticides

Nicotinic acetylcholine receptors (nAChRs) are major excitatory neurotransmitter receptors in both vertebrates and invertebrates. In insects, nAChRs are the target site for several naturally occurring and synthetic compounds that exhibit potent insecticidal activity. Several compounds isolated from plants are potent agonists or antagonists of nAChRs, suggesting that these may have evolved as a defence mechanism against insects and other herbivores. Nicotine, isolated from the tobacco plant, has insecticidal activity and has been used extensively as a commercial insecticide. Spinosad, a naturally occurring mixture of two macrocyclic lactones isolated from the microorganism Saccharopolyspora spinosa, acts upon nAChRs and has been developed as a commercial insecticide. Since the early 1990s, one of the most widely used and rapidly growing classes of insecticides has been the neonicotinoids. Neonicotinoid insecticides are potent selective agonists of insect nAChRs and are used extensively in both crop protection and animal health applications. As with other classes of insecticides, there is growing evidence for the evolution of resistance to insecticides that act on nAChRs.     

Exploration of the binding affinities between ecdysone agonists and EcR/USP by docking and MM-PB/GBSA approaches

Ecdysone receptor (EcR) is a significant target in the identification of new environmentally friendly pesticides. There are two types of ecdysone agonists: steroidal ecdysone agonists and dibenzoylhydrazines (DBHs). In this study, various modeling methods (homology modeling, molecular docking, MD simulation, binding free energy calculation, and per-residue binding free energy decomposition) were utilized to study the different binding mechanisms of two types of ecdysone agonists. Our theoretical results indicated that the relative binding potencies of DBHs can be ranked sufficiently accurately using the MOE docking method. However, MM/PBSA calculations more accurately predicted the binding affinities between steroidal ecdysone agonists and EcR-LBD. To identify the key residues involved in ecdysone agonist binding, the binding free energy (ΔG _Bind) was decomposed into the energy contributions of individual residues. The results revealed that nine residues—Ile339, Thr343, Met380, Met381, Tyr403, Tyr408, Asp419, Gln503, and Asn504—determined the binding affinities of the DBHs. Glu309, Met342, Arg383, Arg387, and Leu396 were important influences on the binding affinities of the steroidal ecdysone agonists.

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Graphical abstract The ecdysone receptor (EcR) is related to insect growth and has been shown to be a useful target for insecticides

     

The Drosophila EcR gene encodes an ecdysone receptor, a new member of the steroid receptor superfamily

The steroid hormone ecdysone triggers coordinate changes in Drosophila tissue development that result in metamorphosis. To advance our understanding of the genetic regulatory hierarchies controlling this tissue response, we have isolated and characterized a gene, EcR, for a new steroid receptor homolog and have shown that it encodes an ecdysone receptor. First, EcR protein binds active ecdysteroids and is antigenically indistinguishable from the ecdysone-binding protein previously observed in extracts of Drosophila cell lines and tissues. Second, EcR protein binds DNA with high specificity at ecdysone response elements. Third, ecdysone-responsive cultured cells express EcR, whereas ecdysone-resistant cells derived from them are deficient in EcR. Expression of EcR in such resistant cells by transfection restores their ability to respond to the hormone. As expected, EcR is nuclear and found in all ecdysone target tissues examined. Furthermore, the EcR gene is expressed at each developmental stage marked by a pulse of ecdysone.     

Nucleocytoplasmic shuttling of the ecdysteroid receptor (EcR) and of ultraspiracle (Usp) from Drosophila melanogaster in mammalian cells: energy requirement and interaction with exportin

The small G protein Ran, which is important for nucleocytoplasmic shuttling of proteins is present, but does not interact with EcR, Usp, and EcR/Usp. As shown by oligomycin treatment, EcR, Usp, and EcR/Usp import is energy dependent. Export of EcR and EcR/Usp is mediated by exportin-1 (CRM-1) as shown by the inhibiting effect of leptomycin B (LMB). Usp remains in the nucleus for more than 24 h. Nuclear retainment of EcR and Usp is energy dependent as shown by treatment with oligomycin. No export signal could be identified for Usp. The data confirm that EcR and Usp can enter the nucleus independently and that intracellular localization is regulated individually for each receptor. It is also demonstrated that the export signal of EcR is inaccessible after heterodimerization with Usp.     

Spatial patterns of ecdysteroid receptor activation during the onset of Drosophila metamorphosis

Ecdysteroid signaling in insects is transduced by a heterodimer of the EcR and USP nuclear receptors. In order to monitor the temporal and spatial patterns of ecdysteroid signaling in vivo we established transgenic animals that express a fusion of the GAL4 DNA binding domain and the ligand binding domain (LBD) of EcR or USP, combined with a GAL4-dependent lacZ reporter gene. The patterns of beta-galactosidase expression in these animals indicate where and when the GAL4-LBD fusion protein has been activated by its ligand in vivo. We show that the patterns of GAL4-EcR and GAL4-USP activation at the onset of metamorphosis reflect what would be predicted for ecdysteroid activation of the EcR/USP heterodimer. No activation is seen in mid-third instar larvae when the ecdysteroid titer is low, and strong widespread activation is observed at the end of the instar when the ecdysteroid titer is high. In addition, both GAL4-EcR and GAL4-USP are activated in larval organs cultured with 20-hydroxyecdysone (20E), consistent with EcR/USP acting as a 20E receptor. We also show that GAL4-USP activation depends on EcR, suggesting that USP requires its heterodimer partner to function as an activator in vivo. Interestingly, we observe no GAL4-LBD activation in the imaginal discs and ring glands of late third instar larvae. Addition of 20E to cultured mid-third instar imaginal discs results in GAL4-USP activation, but this response is not seen in imaginal discs cultured from late third instar larvae, suggesting that EcR/USP loses its ability to function as an efficient activator in this tissue. We conclude that EcR/USP activation by the systemic ecdysteroid signal may be spatially restricted in vivo. Finally, we show that GAL4-EcR functions as a potent and specific dominant negative at the onset of metamorphosis, providing a new tool for characterizing ecdysteroid signaling pathways during development.     

Functional characterization of ecdysone receptor gene switches in mammalian cells

Regulated expression of transgene is essential in basic research as well as for many therapeutic applications. The main purpose of the present study is to understand the functioning of the ecdysone receptor (EcR)-based gene switch in mammalian cells and to develop improved versions of EcR gene switches. We utilized EcR mutants to develop new EcR gene switches that showed higher ligand sensitivity and higher magnitude of induction of reporter gene expression in the presence of ligand. We also developed monopartite versions of EcR gene switches with reduced size of the components that are accommodated into viral vectors. Ligand binding assays revealed that EcR alone could not bind to the nonsteroidal ligand, RH-2485. The EcR's heterodimeric partner, ultraspiracle, is required for efficient binding of EcR to the ligand. The essential role of retinoid X receptor (RXR) or its insect homolog, ultraspiracle, in EcR function is shown by RXR knockdown experiments using RNAi. Chromatin immunoprecipitation assays demonstrated that VP16 (activation domain, AD):GAL4(DNA binding domain, DBD):EcR(ligand binding domain, LBD) or GAL4(DBD):EcR(LBD) fusion proteins can bind to GAL4 response elements in the absence of ligand. The VP16(AD) fusion protein of a chimera between human and locust RXR could heterodimerize with GAL4(DBD):EcR(LBD) in the absence of ligand but the VP16(AD) fusion protein of Homo sapiens RXR requires ligand for its heterodimerization with GAL4(DBD):EcR(LBD).     

斜纹夜蛾蜕皮激素受体与超气门蛋白的原核表达及活性检测

双酰基肼类杀虫剂模拟天然蜕皮激素作用影响幼虫蜕皮。昆虫蜕皮激素受体的高度敏感性和专一性要求必须建立新的杀虫活性检测技术, 以适应快速准确和大批量筛选的要求。本研究采用RT-PCR技术, 获取斜纹夜蛾Spodoptera litura蜕皮激素受体（EcR）与超气门蛋白（USP）功能域目的基因, 构建EcR、 USP功能区基因原核表达载体（pEHISEGFPTEV-EcR_cde和pEHISEGFPTEV-USP_cde）。载体经诱导表达和蛋白纯化, 获得EcR和USP功能区纯化蛋白。在蛋白浓度l mg/mL, ³H-PonA终浓度8 nmol/L的条件下, 采用放射性配基受体结合分析测定了4种药剂（虫酰肼、 呋喃虫酰肼、 抑食肼和灭幼脲）不同浓度下的放射性比活的变化。结果显示： 随着药剂浓度的逐渐增大, 前3种药剂的放射性比活都有不同程度的降低, 其中虫酰肼的放射性比活降低程度最大, 其次是呋喃虫酰肼和抑食肼, 灭幼脲的放射性比活基本无变化。这些结果表明相同条件下虫酰肼比呋喃虫酰肼和抑食肼有更高的杀虫活力, 本研究的方法可对双酰基肼类杀虫剂或者先导化合物进行初步筛选。     

Spiroindolines identify the vesicular acetylcholine transporter as a novel target for insecticide action

The efficacy of all major insecticide classes continues to be eroded by the development of resistance mediated, in part, by selection of alleles encoding insecticide insensitive target proteins. The discovery of new insecticide classes acting at novel protein binding sites is therefore important for the continued protection of the food supply from insect predators, and of human and animal health from insect borne disease. Here we describe a novel class of insecticides (Spiroindolines) encompassing molecules that combine excellent activity against major agricultural pest species with low mammalian toxicity. We confidently assign the vesicular acetylcholine transporter as the molecular target of Spiroindolines through the combination of molecular genetics in model organisms with a pharmacological approach in insect tissues. The vesicular acetylcholine transporter can now be added to the list of validated insecticide targets in the acetylcholine signalling pathway and we anticipate that this will lead to the discovery of novel molecules useful in sustaining agriculture. In addition to their potential as insecticides and nematocides, Spiroindolines represent the only other class of chemical ligands for the vesicular acetylcholine transporter since those based on the discovery of vesamicol over 40 years ago, and as such, have potential to provide more selective tools for PET imaging in the diagnosis of neurodegenerative disease. They also provide novel biochemical tools for studies of the function of this protein family.