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.     

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。     

A photoaffinity, non-steroidal, ecdysone agonist, bisacylhydrazine compound, RH-131039: characterization of binding and functional activity

In this paper we describe the synthesis, ligand-binding and functional activity characteristics of the photoaffinity, non-steroidal, ecdysone agonist, bisacylhydrazine compound, 3-benzoyl-benzoic acid N-tert-butyl-N'-(2-ethyl-3-methoxy-benzoyl)-hydrazide (RH-131039). Tritiated RH-131039 is the first non-steroidal photoaffinity compound that was shown to bind specifically to ecdysone receptors (EcRs) from insects belonging to the orders Diptera and Lepidoptera. The spruce budworm (Choristoneura fumiferana) ecdysone receptor (CfEcR) bound with high affinity (K(d)=2.23+/-0.27 nM) to this compound. When irradiated with UV light (lambda=350 nm) under equilibrium ligand-binding conditions, RH-131039 attached specifically and covalently to the CfEcR ligand-binding domain (LBD). RH-131039 also bound to cloned ecdysone receptor proteins from three dipteran insects, Drosophila melanogaster, Aedes aegypti and Chironomous tentans. This paper also describes and invokes caution in interpretation of ligand-binding results obtained using crude cellular extracts containing target receptors, as illustrated with the use of Drosophila Kc cells that have functional EcR and L57 cells (derivatives of Kc cells in which EcR-B isoforms have been knocked out by "parahomologous" recombination). Tritiated RH-131039 is a useful tool to dissect ligand-binding and functional differences for EcRs from different arthropod species.     

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.     

Molecular determinants of differential ligand sensitivities of insect ecdysteroid receptors

The functional receptor for insect ecdysteroid hormones is a heterodimer consisting of two nuclear hormone receptors, ecdysteroid receptor (EcR) and the retinoid X receptor homologue Ultraspiracle (USP). Although ecdysone is commonly thought to be a hormone precursor and 20-hydroxyecdysone (20E), the physiologically active steroid, little is known about the relative activity of ecdysteroids in various arthropods. As a step toward characterization of potential differential ligand recognition, we have analyzed the activities of various ecdysteroids using gel mobility shift assays and transfection assays in Schneider-2 (S2) cells. Ecdysone showed little activation of the Drosophila melanogaster receptor complex (DmEcR-USP). In contrast, this steroid functioned as a potent ligand for the mosquito Aedes aegypti receptor complex (AaEcR-USP), significantly enhancing DNA binding and transactivating a reporter gene in S2 cells. The mosquito receptor also displayed higher hormone-independent DNA binding activity than the Drosophila receptor. Subunit-swapping experiments indicated that the EcR protein, not the USP protein, was responsible for ligand specificity. Using domain-swapping techniques, we made a series of Aedes and Drosophila EcR chimeric constructs. Differential ligand responsiveness was mapped near the C terminus of the ligand binding domain, within the identity box previously implicated in the dimerization specificity of nuclear receptors. This region includes helices 9 and 10, as determined by comparison with available crystal structures obtained from other nuclear receptors. Site-directed mutagenesis revealed that Phe529 in Aedes EcR, corresponding to Tyr611 in Drosophila EcR, was most critical for ligand specificity and hormone-independent DNA binding activity. These results demonstrated that ecdysone could function as a bona fide ligand in a species-specific manner.     

Influence of hormone on intracellular localization of the Drosophila melanogaster ecdysteroid receptor (EcR)

In the absence of hormone the ecdysteroid receptor (EcR) is distributed between the cytoplasm and the nucleus. Addition of the hormone muristerone A increases nuclear localization of wild type EcR within 5–10 min. Mutation of M504 to alanine, an amino acid, which is essential for ligand binding and which is situated in helix 5 of the ligand binding domain, abolishes hormone binding but still allows nuclear localization at only slightly reduced levels in the absence of hormone, whereas nuclear localization of EcR^M504R is nearly abolished. Cotransfection with ultraspiracle (USP), the invertebrate ortholog of RXR, leads to exclusively nuclear localization of wild type EcR and EcR^M504A indicating that basal heterodimerization in the absence of hormone is still possible. In the presence of Usp, EcR^M504R is only partially localized in the nucleus. EMSA experiments show that the ligand muristerone A enhances binding of wild type EcR, but only slighthly of mutated EcRs, to the canonical hsp 27 ecdysone response element. This is confirmed by transactivation studies. The results indicate that the architecture of the E-domain of EcR is important for nuclear localization even in the absence of a ligand.     

Inhibition of [3H]ponasterone A binding by ecdysone agonists in the intact Kc cell line.

Inhibition of the binding of [3H]ponasterone A ([3H]PoA) by ecdysone agonists including diacylhydrazines such as RH-5849, tebufenozide (RH-5992) and methoxyfenozide (RH-2485) was examined in intact Drosophila Kc cells. The reciprocal logarithm of the concentration at which there is 50% inhibition of [3H]PoA binding, pIC(50) (M), was determined as the binding activity for all compounds from each concentration-response curve. The order of the activity was PoA>20-hydroxyecdysone>cyasterone>inokosterone>or=makisterone A>methoxyfenozide>or=tebufenozide>ecdysone>RH-5849. The ranking of steroidal ecdysone analogs is consistent with that obtained against Spodoptera Sf-9 cells. Furthermore, in terms of pIC(50), all binding activity for ecdysone analogs, except ecdysone, estimated in the Kc cell line system was significantly higher than that for the Sf-9 cell line system. However, the activity of ecdysone was comparable between Kc and Sf-9 cells. The activity of diacylhydrazine analogs against Kc cells was significantly low compared with that against Sf-9 cells. The potency of methoxyfenozide was 1/200 that of PoA, which showed the highest activity in the Kc cell line system among all compounds tested. The activity of tebufenozide analogs having an n-pentyl or n-hexyl group instead of a 4-ethylphenyl group was similar to that of RH-5849.     

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.     

Ecdysone induction of MsrA protects against oxidative stress in Drosophila

The methionine sulfoxide reductases MsrA and MsrB reduce Met(O) to Met in epimer-specific fashion. In Drosophila, the major ecdysone induced protein is MsrA, which is regulated by the EcR-USP complex. We tested Kc cells for induction of MsrA, MsrB, EcR, and CAT by ecdysone and found that MsrA and the EcR were induced by ecdysone, but MsrB and CAT were not. When we tested for resistance to 20mM H2O2 toxicity, viability of Kc cells was reduced 3-fold. Pretreatment with 0.2 microM ecdysone for 48 h prior to exposure to H2O2, increased viability to 77% of controls. The EcR-deficient L57-3-11 knockout line was not responsive to ecdysone, and H2O2 resistance of both control and ecdysone-treated L57-3-11 cells was similar to that of the ecdysone-untreated Kc cells. These results show that hormonal regulation of MsrA is implicated in conferring protection against oxidative stress in the Drosophila model.     

Crystal structure of the ligand-binding domain of the ultraspiracle protein USP, the ortholog of retinoid X receptors in insects

The major postembryonic developmental events happening in insect life, including molting and metamorphosis, are regulated and coordinated temporally by pulses of ecdysone. The biological activity of this steroid hormone is mediated by two nuclear receptors: the ecdysone receptor (EcR) and the Ultraspiracle protein (USP). The crystal structure of the ligand-binding domain from the lepidopteran Heliothis virescens USP reported here shows that the loop connecting helices H1 and H3 precludes the canonical agonist conformation. The key residues that stabilize this unique loop conformation are strictly conserved within the lepidopteran USP family. The presence of an unexpected bound ligand that drives an unusual antagonist conformation confirms the induced-fit mechanism accompanying the ligand binding. The ligand-binding pocket exhibits a retinoid X receptor-like anchoring part near a conserved arginine, which could interact with a USP ligand functional group. The structure of this receptor provides the template for designing inhibitors, which could be utilized as a novel type of environmentally safe insecticides.