Diacylhydrazine Research Articles

Ecdysteroid-mimicking compounds act as both agonists and antagonists to the crustacean ecdysone receptor

To characterize the potential endocrine-disrupting chemicals (EDCs) in the environment that interact with the crustacean ecdysone receptor (EcR), we established a method involving in silico modeling/molecular docking and in vitro reporter gene assay. Cherry shrimp (Neocaridina davidi) EcR (NdEcR) and retinoid X receptor (NdRxR) were identified and cloned for use in this method. A theoretical 3D model of NdEcR ligand-binding domain (LBD) was built in silico based on sequence homology with the established X-ray structure of insect EcR. The interaction of the NdEcR LBD with ecdysteroids, diacylhydrazine (DAH) pesticides, and other potential EDCs was evaluated using molecular docking programs. The results revealed that the ligand-binding pocket in the NdEcR LBD was flexible and adaptive for accommodating ligands of different shapes. The agonistic and antagonistic activities of the candidate compounds were further assessed by in vitro reporter gene assay using human cell lines transiently transfected with NdEcR and NdRxR expression plasmids and a reporter plasmid containing synthesized ecdysone response element. The assay was validated by the dose-dependent responses of EcR-mediated gene transcription after treating the transfected cell lines with ecdysteroids, 20-hydroxyecdysone, and ponasterone A. Examination of the candidate compounds using the reporter gene assay revealed restricted functional specificity to ecdysteroids and DAHs. Three of the tested DAH pesticides originally targeting the insect EcR were found to be weak agonists and strong antagonists of NdEcR. These results suggest that DAHs are potential EDCs for crustaceans that disrupt their ecdysteroid signals by functioning as EcR agonists or antagonists.

Quantitative evaluation of the molting hormone activity in coleopteran cells established from the Colorado potato beetle, Leptinotarsa decemlineata

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 1μM (pEC50=5.99), 68nM (pEC50=7.17) and 1.3nM (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.

Selectivity of diacylhydrazine insecticides to the predatory bug Orius laevigatus: in vivo and modelling/docking experiments

Knowledge of pesticide selectivity to natural enemies is necessary for a successful implementation of biological and chemical control methods in integrated pest management (IPM) programmes. Diacylhydrazine (DAH)-based ecdysone agonists, also known as moulting-accelerating compounds (MACs), are considered to be a selective group of insecticides, and their compatibility with predatory Heteroptera, which are used as biological control agents, is known. However, their molecular mode of action has not been explored in beneficial insects such as Orius laevigatus (Fieber) (Hemiptera: Anthocoridae). In this project, in vivo toxicity assays demonstrated that the DAH-based RH-5849, tebufenozide and methoxyfenozide have no toxic effect against O. laevigatus. The ligand-binding domain (LBD) of the ecdysone receptor (EcR) of O. laevigatus was sequenced, and a homology protein model was constructed that confirmed a cavity structure with 12 α-helices, harbouring the natural insect moulting hormone 20-hydroxyecdysone. However, docking studies showed that a steric clash occurred for the DAH-based insecticides owing to a restricted extent of the ligand-binding cavity of the EcR of O. laevigatus. The insect toxicity assays demonstrated that MACs are selective for O. laevigatus. The modelling/docking experiments are indications that these pesticides do not bind with the LBD-EcR of O. laevigatus and support the supposition that they show no biological effects in the predatory bug. These data help in explaining the compatible use of MACs together with predatory bugs in IPM programmes.

Comparison of the activity of non‐steroidal ecdysone agonists between dipteran and lepidopteran insects, using cell‐based EcR reporter assays

Diacylhydrazine (DAH) analogues have been developed successfully as a new group of insect growth regulators, called ecdysone agonists or moulting accelerating compounds. These DAHs have been shown to manifest their toxicity via interaction with the ecdysone receptor (EcR) in susceptible insects, as does the natural insect moulting hormone 20-hydroxyecdysone (20E). A notable feature is their high activity and specificity, particularly against lepidopteran insects, raising the question as to whether non-lepidopteran-specific analogues can be isolated. However, for the discovery of ecdysone agonists that target other important insect groups such as Diptera, efficient screening systems that are based on the activation of the EcR are needed. In this study, a dipteran-specific reporter-based screening system with transfected S2 cells of Drosophila melanogaster Meig. was developed in order to discover and evaluate compounds that have ecdysone agonistic or antagonistic activity. A library of non-steroidal ecdysone agonists containing different mother structures with DAH and other related analogues such as acylaminoketone (AAK) and tetrahydroquinoline (THQ) was tested. None of the compounds tested was as active as 20E. This is in contrast to the very high activity of several DAH and AAK congeners in lepidopteran cells (Bombyx mori L.-derived Bm5 cells). The latter agrees with a successful docking of a DAH, tebufenozide, in the binding pocket of the lepidopteran EcR (B. mori), while this was not the case with the dipteran EcR (D. melanogaster). Of note was the identification of two THQ compounds with activity in S2 but not in Bm5 cells. Although marked differences in activity exist with respect to the activation of EcR between dipterans and lepidopterans, there exists a positive correlation (R = 0.724) between the pLC(50) values in S2 and Bm5 cells. In addition, it was found through protein modelling that a second lobe was present in the ligand-binding pocket of lepidopteran BmEcR but was lacking in the dipteran DmEcR protein, suggesting that this difference in structure of the binding pocket is a major factor for preferential activation of the lepidopteran over the dipteran receptors by DAH ligands. The present study confirmed the marked specificity of DAH and AAK analogues towards EcRs from lepidopteran insects. THQ compounds did not show this specificity, indicating that dipteran-specific ecdysone-agonist-based insecticides based on the THQ mother structure can be developed. The differences in activity of ecdysone agonists in dipteran and lepidopteran ecdysone-reporter-based screening systems are discussed.

Stereostructural behaviour of N–N atropisomers: Two conglomerate crystallisations and a crystallisation-induced deracemisation

The solid state behaviour of a number of compounds which show hindered rotation around an N–N bond, in some cases leading to axial chirality is described. A diacyl hydrazine, bisanthranoly hydrazine, 1 crystallises in the chiral space group P212121, presenting an example of conglomerate crystallisation. A tetra-acyl hydrazine derived from lactic acid, 2, shows kinetic resolution by crystallisation, as of the two isomers observed in the solution NMR, only one crystallises, again in the space group P212121. Two cyclic acyl hydrazines in the form of biquinazolinones are studied: 2,2′diphenyl-3,3-biquinazolinone, 3 crystallises in the achiral space group Pbca, while 3,3′-dimethyl-2,2′-biquinazoline-4′-thio-4-one, 4 crystallises in the chiral space group P21 giving another example of a conglomerate crystallisation. The single crystal structures of each of the species have been compared to powder XRD data to confirm that the single crystal structures are representative of the bulk material.

Molecular Docking of Cathepsin L Inhibitors in the Binding Site of Papain

The papain/CLIK-148 coordinate system was employed as a model to study the interactions of a nonpeptide thiocarbazate inhibitor of cathepsin L ( 1). This small molecule inhibitor, a thiol ester containing a diacyl hydrazine functionality and one stereogenic center, was most active as the S-enantiomer, with an IC 50 of 56 nM; the R-enantiomer ( 2) displayed only weak activity (33 microM). Correspondingly, molecular docking studies with Extra Precision Glide revealed a correlation between score and biological activity for the two thiocarbazate enantiomers when a structural water was preserved. The molecular interactions between 1 and papain were very similar to the interactions observed for CLIK-148 ( 3a and 3b) with papain, especially with regard to the hydrogen-bonding and lipophilic interactions of the ligands with conserved residues in the catalytic binding site. Subsequent docking of virtual compounds in the binding site led to the identification of a more potent inhibitor ( 5), with an IC 50 of 7.0 nM. These docking studies revealed that favorable energy scores and correspondingly favorable biological activities could be realized when the virtual compound design included occupation of the S2, S3, and S1' subsites by hydrophobic and aromatic functionalities of the ligand, and at least three hydrogen bonding contacts between the ligand and the conserved binding site residues of the protein.

Effects of the structures of ecdysone receptor (EcR) and ultraspiracle (USP) on the ligand-binding activity of the EcR/USP heterodimer

N-tert-Butyl-N,N '-diacylhydrazine (DAH) analogs are nonsteroidal ecdysone agonists. The binding activity of DAH analogs to the heterodimer of the ecdysone receptor (EcR) and ultraspiracle (USP) is diverse among insect species, which is probably the main factor causing their selective toxicity. We prepared EcR and USP proteins from lepidopteran Chilo suppressalis, dipteran Drosophila melanogaster and coleopteran Leptinotarsa decemlineata, and measured the binding activity of ecdysone agonists against various hybrid EcR/USP heterodimers. There was a linear relationship between binding activities (pIC50 values) before and after replacing native USP with that derived from other insects, suggesting that the selective toxicity of DAH analogs is mainly dependent on the EcR structure and not the USP structure.

Interpretation of Scoring Functions Using 3D Molecular Fields. Mapping the Diacyl-Hydrazine-Binding Pocket of an Insect Ecdysone Receptor

A method is presented for the interpretation of receptor docking score values (rough measures of binding affinities) of ligands in terms of 3D molecular field interaction contributions. The FlexX and FlexX-Pharm methods were used to dock the structures of designed sets of ligands into the ligand-binding pocket of a selected receptor. In the next step the relationship was investigated between the FlexX and CScore scores and 3D molecular fields obtained for the docked conformations of the ligands, using the CoMFA (Comparative Molecular Field Analysis) and CoMSIA (Comparative Molecular Similarity Indices Analysis) methods. The approach yielded highly significant CoMFA and CoMSIA models demonstrating that a high portion of the variance in the docking score values of the ligands can be explained by steric, electrostatic, hydrophobic, and hydrogen bond donor and acceptor molecular field interaction contributions. The approach was exemplified by using the crystal structure of the ligand-binding domain of the ecdysone receptor (EcR) of the moth Heliotis virescens as well as virtual molecule libraries of analogues of known diacyl-hydrazine (DAH) type ecdysteroid agonists. By docking appropriately designed virtual compound libraries into the DAH binding pocket of EcR followed by CoMFA and CoMSIA of the docked conformations, hitherto unexplored regions of the receptor cavity could be mapped. By mapping the significant molecular field interaction contributions onto the model of the receptor-ligand complex, important receptor-ligand interactions could be highlighted that may help the design of novel highly scored receptor ligands. An advantage of the method is that no experimental biological activity data are required to exhaustively map the receptor-binding site.

Mode of Action of Nonsteroidal Ecdysone Agonists, Diacylhydrazine Analogs

The binding affinity of insect molting hormone agonists, N-tert-butyl-N,N′-diacylhydrazine (DAH) analogs, against the molting hormone receptor proteins (EcR/USP) was measured. For the lepidopteran Chilo suppressalis, the receptor-binding activity of DAH analogs correlated linearly with their molting hormonal activity (in vitro) and insecticidal activity (in vivo). The binding activity of DAH analogs of C. suppressalis EcR/USP was higher than that of the dipteran Drosophila melanogaster EcR/USP. The binding activity of EcR/USP changed little when USP was exchanged between C. suppressalis and D. melanogaster. These results suggest that the selective toxicity of DAH analogs toward Lepidoptera is caused by the difference in the structure of EcR, not by that of USP. © Pesticide Science Society of Japan

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.

Biochemical and biological mode of action of ecdysone agonists on the spruce budworm

The mode of action of RH-5992 (tebufenozide), a non-steroidal ecdysone agonist, on the eastern spruce budworm, Choristoneura fumiferana, was investigated. This diacyl hydrazine compound, upon ingestion, initiates a precocious incomplete molt that is lethal in most lepidopteran larvae including the spruce budworm. This was found to be induced when the larvae ingested the compound early in the stadium prior to the appearance of the ecdysone peak in the hemolymph. The larvae stopped feeding within 8h post feeding (PF) and remained quiescent just as they do in preparation for a normal molt. Head capsule slippage started at 12h PF, became pronounced by 24h, and by 48h an untanned new head capsule was visible behind the old one. The lack of tanning of the new cuticle was due to the failure of dopadecarboxylase gene expression. Although the old cuticle was loose around the entire body, indicating that apolysis had occurred, there was no evidence of ecdysis of the old cuticle, suggesting that eclosion hormone was probably not released. The transcription factor, Choristoneura hormone receptor 3 (CHR3), which is normally expressed at the onset of the hemolymph ecdysone peak, was expressed in the epidermis 1h PF of RH-5992 confirming that this analogue acts through the ecdysone receptor system. This unique mode of action at the molecular level of this ecdysone agonist and its effectiveness as an environmentally benign control agent for the spruce budworm are described.

Synthesis, structure and reactivity of N,O-metallacyclic (dicarbonyldiazene) platinum complexes

Reaction of cis-Pt(PR13)2Cl2 (R1=Me, Ph) with diacyl hydrazine R2OCNHNHCOR2 (R2=Me, Ph) in refluxing ethanol and NaHCO3 afforded platinum diazene diacyl complexes Pt(PR13)2(R2OCNNCOR2) (1, R1=R2=Ph; 3, R1=Me, R2=Ph; 4, R1=R2=Me). Reaction of Pt(PPh3)2(C2H4) with diazene dicarboxylates R2OCNNCOR2 (R2=OEt, OPri) resulted in the formation of Pt(PPh3)2(R2OCNNCOR2) (2, R2=OEt; 5, R2=OPri). Multinuclear NMR spectroscopy on 1–5 revealed that the dicarbonyl-substituted diazene ligand is coordinated asymmetrically, consistent with a five membered Pt–N–N–C–O ring. Compound 5 crystallises from THF–benzene as the solvate Pt(PPh3)2(PriO2CNNCO2Pri)·(C6H6)2; its X-ray crystal structure shows that the coordination sphere of platinum is essentially square planar and coplanar with the five membered Pt1–O1–C5–N2–N1 ring. The Pt1–O1 distance is 2.027(4) Åand the Pt1–N1 distance is 2.050(5) Å. The N1–N2 distance is 1.421(6) Åwhile the N2–C5 and C5–O1 distances are 1.273(7) and 1.314(7) Å, respectively. Complexes 1–5 show sensitivity towards chlorinated solvents (CH2Cl2, CHCl3) under photolysis conditions, forming the corresponding cis-platinum bis(phosphine) dichloride complexes; the same products are formed in a slower thermal reaction with 2 and 5. Complexes 2 and 5 react photochemically with ethylene in [2H8]THF yielding Pt(PPh3)2(C2H4) but 1, 3 and 4 are inert. Addition of an excess of dppe ligand to 2 and 5 resulted in the displacement of PPh3 and the formation of Pt(dppe)(EtO2CNNCO2Et) and Pt(dppe)(PriO2CNNCO2Pri) [dppe=1,2-bis(diphenylphosphino)ethane].