A luciferase reporter assay for ecdysone agonists using HEK293T cells.

Ecdysone agonists are a class of insecticides that activate the ecdysone receptor (EcR) heterodimerized with the ultraspiracle (USP). Here, we report a new luciferase reporter assay for ecdysone agonists. The assay employs mammalian HEK293T cells transiently transfected with the EcR and USP genes of Chilo suppressalis, along with the taiman (Tai) gene of Drosophila melanogaster that encodes a steroid receptor coactivator. This assay system gave results consistent with those of radioligand binding assays and showed sensitivity superior to that of the existing in vitro methods. In addition, use of the heterologous host cells precludes perturbation from intrinsic players of the ecdysone signaling, which is a potential drawback of insect cell-based methods. This reporter system is suitable for detailed structure-activity analysis of ecdysone agonists and will serve as a valuable tool for the rational design of novel insect growth regulators.

A Commercial Extract of Cyanotis arachnoidea Roots as a Source of Unusual Ecdysteroid Derivatives with Insect Hormone Receptor Binding Activity.

Ecdysteroids act as molting hormones in insects and as nonhormonal anabolic agents and adaptogens in mammals. A wide range of ecdysteroid-containing herbal extracts are available worldwide as food supplements. The aim of this work was to study such an extract as a possible industrial source of new bioactive ecdysteroids. A large-scale chromatographic isolation was performed from an extract of Cyanotis arachnoidea roots. Ten ecdysteroids (1-10) including eight new compounds were isolated and characterized by extensive nuclear magnetic resonance studies. Highly unusual structures were identified, including a H-14ß (1, 2, 4, and 10) moiety, among which a 14ß(H)17ß(H) phytosteroid (1) is reported for the first time. Compounds with an intact side chain (4-10) and 11 other natural or semisynthetic ecdysteroids (11-21) were tested for insect ecdysteroid receptor (EcR) binding activity. Two new compounds, i.e., 14-deoxydacryhainansterone (5) and 22-oxodacryhainansterone (6), showed strong EcR binding activity (IC50 = 41.7 and 380 nM, respectively). Six compounds were identified as EcR agonists and another two as antagonists using a transgenic ecdysteroid reporter gene assay. The present results demonstrate that commercial C. arachnoidea extracts are rich in new, unusual bioactive ecdysteroids. Because of the lack of an authentic plant material, the truly biosynthetic or artifactual nature of these compounds cannot be confirmed.

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.

Quantitative structure-activity relationship of substituted imidazothiadiazoles for their binding against the ecdysone receptor of Sf-9 cells.

Imidazothiadiazoles (ITDs) are a class of potent nonsteroidal ecdysone agonists with larvicidal activity. Previously, we performed the Hansch-Fujita type of quantitative structure-activity relationship (QSAR) analysis for ITD analogs (Yokoi et al., Pestic. Biochem. Physiol.2015, 120, 40-50). The activity was reasonably explained by hydrophobicity and electronegativity of substituents on the imidazothiadiazole ring system. However, the limited data points (n = 8) hampered the examination of other physicochemical parameters. In the present study, we expanded the library of ITD congeners and evaluated their receptor-binding affinity using intact Sf-9 cells. The QSAR analysis for the expanded set revealed the significance of the third physicochemical parameter, the negative steric effect for long substituents. We also evaluated the larvicidal activity of the synthesized compounds against Spodoptera litura; however, it was not correlated to the binding affinity. The results obtained here suggests that the pharmacokinetic properties must be improved to enhance the larvicidal activity of ITDs.

Brassinolide-like activity of castasterone analogs with varied side chains against rice lamina inclination.

Brassinolide (BL) and castasterone (CS) are the representative members of brassinosteroid class of plant steroid hormone having plant growth promoting activity. In this study, eleven CS analogs bearing a variety of side chains were synthesized to determine the effect of the side chain structures on the BL-like activity. The plant hormonal activity was evaluated in a dwarf rice lamina inclination assay, and the potency was determined as the reciprocal logarithm of the 50% effective dose (ED50) from each dose-response curve. The reciprocal logarithm of ED50 (pED50) was decreased dramatically upon deletion of the C-28 methyl group of CS. The introduction of oxygen-containing groups such as hydroxy, methoxy, and ethoxycarbonyl was also unfavorable to the activity. The pED50 was influenced by the geometry of carbon-carbon double bond between C-24 and C-25 (cis and trans), but the introduction of a fluorine atom at the C-25 position of the double bond did not significantly change the activity. The binding free energy (ΔG) was calculated for all ligand-receptor binding interactions using molecular dynamics, resulting that ΔG is linearly correlated with the pED50.

Structure-activity relationship of imidazothiadiazole analogs for the binding to the ecdysone receptor of insect cells.

Diacylhydrazines are the first non-steroidal ecdysone agonists, and five compounds are used as insecticides in agriculture. After the discovery of diacylhydrazine-type compounds, numerous non-steroidal structures were reported as ecdysone agonists. Among various ecdysone agonists, imidazothiadiazoles are reported to be very potent in vitro; however, the experimental detail for the structure identification and bioassays are not stated in the paper (Holmwood and Schindler, Bioorg. Med. Chem. 17, 4064-4070, 2009). In our present study, we synthesized 18 imidazothiadiazole-type compounds and confirmed the chemical structures by spectrometric analyses. The binding activity of the synthesized compounds to the ecdysone receptor was evaluated in terms of the concentration required for 50% inhibition of [(3)H]ponasterone A incorporation [IC50 (M)] into lepidopteran (Sf-9), coleopteran (BCRL-Lepd-SL1), and dipteran (NIAS-AeAl2) cells. 6-(2-Chlorophenyl)-2-(trifluoromethyl)imidazo[2,1-b] [1,3,4]-thiadiazol-5-yl)acrylamide analogs with CONHR (secondary amide) were very potent against Sf-9 cells, but further alkylation (tertiary amide: CONR2) decreased the activity dramatically. Additionally, a primary amide analog (CONH2) was inactive. The activity also decreased 150-fold by the saturation of olefin region of the acrylamide moiety. In addition, various substituents were introduced at the 2-position of the imidazothiadiazole ring to disclose the physicochemical properties of the substituents which are important for receptor binding. The activity increased by 7500-fold with the introduction of the CF2CF2CF3 group compared to the unsubstituted compound against Sf-9 cells. Quantitative structure-activity relationship analysis for these substituents indicated that hydrophobic and electron-withdrawing groups were favorable for binding. Some of the compounds with strong receptor binding activity showed good larvicidal activity against Spodoptera litura. In contrast, the binding affinity of imidazothiadiazole analogs was low or not observed against dipteran and coleopteran cells.

Receptor-binding affinity and larvicidal activity of tetrahydroquinoline-type ecdysone agonists against Aedes albopictus.

Tetrahydroquinolines (THQs), a class of nonsteroidal ecdysone agonists, are good candidates for novel mosquito control agents because they specifically bind to mosquito ecdysone receptors (EcRs). We have recently performed quantitative structure-activity relationship (QSAR) analyses of THQs to elucidate the physicochemical properties important for the ligand-receptor interaction. Based on previous QSAR results, here, we newly synthesized 15 THQ analogs with a heteroaryl group at the acyl moiety and evaluated their binding affinity against Aedes albopictus EcRs. We also measured the larvicidal activity of the combined set of previously and newly synthesized compounds against A. albopictus to examine the contribution of receptor-binding to larvicidal activity. Multiple regression analyses showed that the binding affinity and the molecular hydrophobicity of THQs are the key determinants of their larvicidal activity.

Virtual screening identifies a novel piperazine-based insect juvenile hormone agonist.

Juvenile hormone (JH) agonists constitute a subclass of insect growth regulators and play important roles in insect pest management. In this work, a multi-step virtual screening program was executed to find novel JH agonists. A database of 5 million purchasable compounds was sequentially processed with three computational filters: (i) shape and chemical similarity as compared to known JH-active compounds; (ii) molecular docking simulations against a Drosophila JH receptor, methoprene-tolerant; and (iii) free energy calculation of ligand-receptor binding using a modified MM/PBSA (molecular mechanics/Poisson-Boltzmann surface area) protocol. The 11 candidates that passed the three filters were evaluated in a luciferase reporter assay, leading to the identification of a hit compound that contains a piperazine ring system (EC50=870 nM). This compound is structurally dissimilar to known JH agonists and synthetically easy to access; therefore, it is a promising starting point for further structure optimization.

Nonsteroidal ecdysone receptor agonists use a water channel for binding to the ecdysone receptor complex EcR/USP.

The ecdysone receptor (EcR) possesses the remarkable capacity to adapt structurally to different types of ligands. EcR binds ecdysteroids, including 20-hydroxyecdysone (20E), as well as nonsteroidal synthetic agonists such as insecticidal dibenzoylhydrazines (DBHs). Here, we report the crystal structures of the ligand-binding domains of Heliothis virescens EcR/USP bound to the DBH agonist BYI09181 and to the imidazole-type compound BYI08346. The region delineated by helices H7 and H10 opens up to tightly fit a phenyl ring of the ligands to an extent that depends on the bulkiness of ring substituent. In the structure of 20E-bound EcR, this part of the ligand-binding pocket (LBP) contains a channel filled by water molecules that form an intricate hydrogen bond network between 20E and LBP. The water channel present in the nuclear receptor bound to its natural hormone acts as a critical molecular adaptation spring used to accommodate synthetic agonists inside its binding cavity.

Transcription-inducing activity of natural and synthetic juvenile hormone agonists through the Drosophila Methoprene-tolerant protein.

BACKGROUND: Juvenile hormones (JHs) are a class of sesquiterpenoids that play a pivotal role in insect growth and reproduction. Synthetic JH agonists (JHAs), including pyriproxyfen, have been widely used as insecticides to control agricultural pests and disease vectors. The antimetamorphic action of JHAs is mediated by their intracellular receptor, the heterodimer of Methoprene-tolerant (Met) and Taiman (Tai) proteins. Although a range of bioassay systems has been developed to detect the activity of JHAs, each of these systems has its own drawback(s), such as poor reproducibility, the use of radioactive ligands or the effect of endogenous JH-signaling factors. RESULTS: To address these issues, we constructed a new luciferase reporter assay for JHAs in mammalian HEK293T cells transiently transfected with the Drosophila Met and Tai genes. This reporter system gave highly reproducible results and showed nanomolar sensitivity to natural JHs. We then applied this reporter system to a structure-activity relationship (SAR) analysis of 14 natural and synthetic JHAs, leading to identification of the ligand structural factors important for the transcription-inducing activity. CONCLUSION: Because this reporter system is not affected by the signaling cascade downstream of the JH receptors, it is suitable for evaluating the intrinsic activity of JHAs. The SAR results obtained in this study therefore provide invaluable information on the rational design of novel JHA insecticides.

Asymmetric synthesis of tetrahydroquinoline-type ecdysone agonists and QSAR for their binding affinity against Aedes albopictus ecdysone receptors.

BACKGROUND: Tetrahydroquinolines (THQs) are a class of non-steroidal ecdysone agonists that specifically bind to mosquito ecdysone receptors (EcR). The THQ scaffold contains two chiral centers at the C-2 and C-4 positions, resulting in four stereoisomers. We have previously shown that the (2R,4S)-isomers are the most biologically active; however, the lack of a practical synthetic method for these isomers has hampered further structure-activity studies. RESULTS: In this study, a chiral phosphoric acid-catalyzed Povarov reaction was employed to develop a facile asymmetric synthesis of THQs with a (2R,4S)-configuration, which allowed the preparation of a 40-compound library of enantiopure THQs. Evaluation of their binding affinity against Aedes albopictus EcR, followed by quantitative structure-activity relationship (QSAR) analyses, uncovered the physicochemical properties of THQs that are important for the ligand-receptor interaction. The most potent THQ derivative was twofold more active than the molting hormone, 20-hydroxyecdysone. CONCLUSION: The QSAR results provide valuable information for the rational design of novel mosquito-specific ecdysone agonists. © 2018 Society of Chemical Industry.