Ecdysone Receptor Ligand-binding Domain Research Articles

Identification of novel agonists and antagonists of the ecdysone receptor by virtual screening

Insect growth is regulated by the steroid hormone 20-hydroxyecdysone (20E), which works via the ecdysone receptor (EcR). To identify biologically active and novel ecdysone agonists/antagonists, ligand/structure-based virtual screening combined with pharmacophore modeling and molecular docking was performed to identify novel nonsteroidal lead compounds. Nine molecules were screened and selected for an in vitro cell-based reporter bioassay. The results showed that VS-006 and VS-009 exhibited antagonistic activity in S2 cells, whereas only VS-006 exhibited antagonistic activity in Bm5 cells. Molecular dynamic simulation of VS-006 complexed with the ligand binding domain of EcR validated the binding stability of VS-006 and highlighted the key residues for further lead optimization.

Steered molecular dynamics for studying ligand unbinding of ecdysone receptor

Ecdysone receptor (EcR) is an important target for pesticide design. Ligand binding regulates EcR transcriptional activity similar to other nuclear receptors; however, the pathways by which ligands enter and leave the EcR remain poorly understood. Here, we performed computational studies to identify unbinding pathways of an ecdysone agonist [the selective ecdysone agonist, BYI06830] from the EcR ligand binding domain (EcR LBD). BYI06830 can dissociate from EcR LBD via four different pathways with little effect on receptor structure. By comparing the potential of mean force (PMF) of four pathways, path 2 was considered to be the most likely exit path for BYI06830, which was located in the cleft formed by the H3-H4 loop, H6-H7 loop, and the H11 C-terminus. Furthermore, structural features along path 2 were analyzed and the structural snapshots of the metastable and transition states were isolated to illustrate the unbinding mechanism of ecdysone agonist from EcR LBD.

Identification of Ecdysone Hormone Receptor Agonists as a Therapeutic Approach for Treating Filarial Infections.

BackgroundA homologue of the ecdysone receptor has previously been identified in human filarial parasites. As the ecdysone receptor is not found in vertebrates, it and the regulatory pathways it controls represent attractive potential chemotherapeutic targets.Methodology/ Principal FindingsAdministration of 20-hydroxyecdysone to gerbils infected with B. malayi infective larvae disrupted their development to adult stage parasites. A stable mammalian cell line was created incorporating the B. malayi ecdysone receptor ligand-binding domain, its heterodimer partner and a secreted luciferase reporter in HEK293 cells. This was employed to screen a series of ecdysone agonist, identifying seven agonists active at sub-micromolar concentrations. A B. malayi ecdysone receptor ligand-binding domain was developed and used to study the ligand-receptor interactions of these agonists. An excellent correlation between the virtual screening results and the screening assay was observed. Based on both of these approaches, steroidal ecdysone agonists and the diacylhydrazine family of compounds were identified as a fruitful source of potential receptor agonists. In further confirmation of the modeling and screening results, Ponasterone A and Muristerone A, two compounds predicted to be strong ecdysone agonists stimulated expulsion of microfilaria and immature stages from adult parasites.ConclusionsThe studies validate the potential of the B. malayi ecdysone receptor as a drug target and provide a means to rapidly evaluate compounds for development of a new class of drugs against the human filarial parasites.

In silico docking studies of non-azadirachtin limonoids against ecdysone receptor of Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae)

Although specific binding of 20-hydroxyecdysone (20E) and its analogs (ecdysteroids) to the ecdysone receptor ligand-binding domain (EcR-LBD) in insects has been well documented, information on the EcR-ligand binding in Helicoverpa armigera is limited. Hence, an attempt has been made to screen effective natural plant-based agonists from a library of 25 non-azadirachtin neem limonoids and was compared with the commercially available insecticide, tebufenozide, through in silico approach. Results indicated that six compounds, namely nimbolide, azadirone, nimolinone, meliacinol, nimbocinol, azadiradione, efficiently docked with the active site of H. armigera EcR-LBD. The binding energies of top-ranked six molecules ranged from −10.54 to −12.22 kcal/mol, which was superior to the third-generation insect growth regulator (IGR), tebufenozide RH5992. Two factors are especially important in binding: (1) the residues Cys 508 and Asn 504, which are the most common in hydrogen-bonding interactions and (2) hydrophobic pocket residues—Asn 504, Met 507, Val 416, Tyr 408 and Thr 343. We also recognized one aromatic ring, 3–7 vicinal acceptors and 1–3 distal hydrophobic groups as minimum pharmacophoric feature. A significant correlation coefficient of 0.6823 was observed supporting positively the docking studies. These data could help in the application of natural compounds as alternatives to chemicals in pest management.

Sequencing and structural homology modeling of the ecdysone receptor in two chrysopids used in biological control of pest insects

In insects, the process of molting and metamorphosis are mainly regulated by a steroidal hormone 20-hydroxyecdysone (20E) and its analogs (ecdysteroids) that specifically bind to the ecdysone receptor ligand-binding domain (EcR-LBD). Currently, several synthetic non-steroidal ecdysone agonists, including tebufenozide, are commercially available as insecticides. Tebufenozide exerts its activity by binding to the 20E-binding site and thus activating EcR permanently. It appears that subtle differences in the architecture among LBDs may underpin the differential binding affinity of tebufenozide across taxonomic orders. In brief, first we demonstrated the harmlessness of tebufenozide towards Chrysoperla externa (Ce). Then, a molecular analysis of EcR-LBD of two neuropteran insects Chrysoperla carnea and Ce was presented. Finally, we constructed a chrysopid in silico homology model docked ponasterone A (PonA) and tebufenozide into the binding pocket and analyzed the amino acids indentified as critical for binding to PonA and tebufenozide. Due to a restrict extent in the cavity at the bottom of the ecdysone-binding pocket a steric clash occurred upon docking of tebufenozide. The absence of harm biological effect and the docking results suggest that tebufenozide is prevented of any deleterious effects on chrysopids.

Cloning and characterization of the ecdysone receptor and ultraspiracle protein from the water flea Daphnia magna

cDNAs encoding the ecdysone receptor (EcR) and ultra spiracle (USP) protein were cloned from the water flea Daphnia magna (Crustacea: Cladocera). The deduced EcR and USP amino acid sequences showed a high degree of homology to those of other crustaceans as well as insects. We isolated three isoforms of EcR that differ in the A/B domain. Quantitative PCR analysis indicated differing temporal expression patterns of the EcR isoforms during the molting period and demonstrated that the expression of one subtype correlated well with the timing of molt. Using cDNAs encoding EcR and USP, we constructed a Daphnia EcR/USP reporter based on a two-hybrid system. The gene fusions encoded the EcR ligand-binding domain (LBD) fused to the Gal4 DNA-binding domain, and the USP-LBD fused to the Vp16 activation domain. These chimeric genes were transfected with a luciferase reporter gene. Dose-dependent activation of the reporter gene could be observed when transfectants were exposed to Ec and other chemicals known to have Ec-like activities. This two-hybrid system may represent a useful reporter system for further examination of hormonal and chemical effects on Daphnia at the molecular level.

501. RheoSwitch® Therapeutic System-Inducible Recombinant AAV Vectors for Tightly Regulated Transgene Expression

Recent advances in recombinant AAV (rAAV) vector technology have made rAAV a vector of choice for many gene therapy applications. rAAV vectors for delivery of the RheoSwitch|[reg]| Therapeutic System (RTS) are ideal systems for regulatory control of long-term therapeutic gene expression in vivo. RTS inducible gene expression technology allows the precise timing and dose of the therapeutic protein to be controlled through the systemic administration of a highly specific, pharmacologically inert Activator Drug. The controlled expression of the gene of interest is activated by the RheoSwitch|[reg]| receptor (a heterodimer of an engineered ecdysone receptor ligand binding domain fused to a GAL4 DNA binding domain (GAL4:EcR) and an RXR protein fused to a VP16 activation domain (VP16:RXR)) only in the presence of Activator Drug. In the present study, we have optimized rAAV-based vectors by using different promoters for the expression of RheoSwitch|[reg]| receptor and by cloning the inducible transgene and the RheoSwitch|[reg]| receptor-expressing cassettes in different orientations. We then used a highly active, modified CMV promoter for the expression of RheoSwitch|[reg]| receptor and cloned the receptor cassette and inducible Gaussia luciferase (GLuc) or GFP cassette in a single rAAV. To evaluate the regulation of GLuc or GFP transgene expression, HEK293 and mouse neural stem cells (NSCs) were transduced with packaged virus. Addition of Activator Drug to transduced HEK293 and NSCs in culture induced transgene expression in a dose dependent manner. For in vivo evaluation, the packaged virus was injected into the quadriceps of mice and subsequently induced with Activator Drug. Both the inducible GLuc and GFP vectors demonstrated little or no basal activity and inducible expression. To reduce the size of the construct further, we tested a format in which a p53 activation domain was fused to the GAL4:EcR domain. This gene was cloned with an inducible secretable alkaline phosphatase (SEAP) in a single rAAV vector. Activator Drug-dependent induction and low basal activity were also observed for this format in vitro and in vivo. Regulatable rAAV vectors based on these designs are ideal for therapeutic gene delivery.

The X-ray Structure of a Hemipteran Ecdysone Receptor Ligand-binding Domain: COMPARISON WITH A LEPIDOPTERAN ECDYSONE RECEPTOR LIGAND-BINDING DOMAIN AND IMPLICATIONS FOR INSECTICIDE DESIGN

The ecdysone receptor is a hormone-dependent transcription factor that plays a central role in regulating the expression of vast networks of genes during development and reproduction in the phylum Arthropoda. The functional receptor is a heterodimer of the two nuclear receptor proteins ecdysone receptor (EcR) and ultraspiracle protein. The receptor is the target of the environmentally friendly bisacylhydrazine insecticides, which are effective against Lepidoptera but not against Hemiptera or several other insect orders. Here we present evidence indicating that much of the selectivity of the bisacylhydrazine insecticides can be studied at the level of their binding to purified ecdysone receptor ligand-binding domain (LBD) heterodimers. We report the crystal structure of the ecdysone receptor LBD heterodimer of the hemipteran Bemisia tabaci (Bt, sweet potato whitefly) in complex with the ecdysone analogue ponasterone A. Although comparison with the corresponding known LBD structure from the lepidopteran Heliothis virescens (Hv) ecdysone receptor revealed the overall mode of ponasterone A binding to be very similar in the two cases, we observed that the BtEcR ecdysteroid-binding pocket is structured differently to that of HvEcR in those parts that are not in contact with ponasterone A. We suggest that these differences in the ligand-binding pocket may provide a molecular basis for the taxonomic order selectivity of bisacylhydrazine insecticides.

313. Ligand-Specific Multiplex Gene Switch Expression in a Mouse Model

The ecdysone receptor (EcR)-based gene switch is used to regulate gene expression in a ligand-dependent manner. Gene switches may have utility in human gene therapies that require a specifically regulated level of gene expression, where it is desirable to terminate the therapy when it is no longer needed, or where continuation of the therapy would be unsafe. In addition, multiplexed gene switches allow for the differential regulation of several genes simultaneously. These EcR-based switches are comprised of EcR fused to a GAL4 DNA binding domain, and a chimera of human and insect retinoid X receptor fused to a VP16 activation domain (pVP16-Hs-LmRXR), which regulates a reporter gene that is placed under the control of a 6GAL4 response element and a transthyretin minimal promoter (p6xGAL4RE-TTR-SEAP). Three multiplex gene switches have been developed by mutating the EcR ligand-binding domain in a site-specific manner. Each switch shows specificity for either diacylhydrazines (DAH), tetrahydroquinolines (THQ) or ecdysteroid (EcD) ligands, and are not cross-activated by the other chemotypes. In mouse electroporation studies, a switch based on a V96T/N119F double mutant of EcR showed high specificity for EcD (Ponasterone A, PonA) but was completely refractory toward both DAH (GSTM-E) and THQ (RG-120499) ligands. Likewise, another double mutant of EcR, V128F/A110P, showed specificity toward THQ, but was refractory toward DAH (GSTM-E) and EcD (PonA) ligands. A third gene switch that uses A110P mutant of EcR was sensitive to GSTM-E ligand but not to either EcD (PonA) or THQ (RG-120499) ligands. A dual switch containing V96T/N119F and V128F/A110P EcR double mutants were used to demonstrate the simultaneous regulation of two genes in vivo.

Juvenile hormone potentiates ecdysone receptor-dependent transcription in a mammalian cell culture system

Insect development is guided by the combined actions of ecdysteroids and juvenile hormones (JHs). The transcriptional effects of ecdysteroids are mediated by a protein complex consisting of the ecdysone receptor (EcR) and its heterodimeric partner, Ultraspiracle (USP), but a corresponding JH receptor has not been defined conclusively. Given that the EcR ligand binding domain (LBD) is similar to that of the JH-responsive rat farnesoid-X-activated receptor (FXR), we sought to define experimental conditions under which EcR-dependent transcription could be promoted by JH. Chinese hamster ovary (CHO) cells were transfected with a plasmid carrying an ecdysteroid-inducible reporter gene, a second plasmid expressing one of the three amino-terminal variants of Drosophila EcR or an EcR chimera, and a third plasmid expressing either the mouse retinoid X receptor (RXR), or its insect orthologue, USP. Each of the EcR variants responded to the synthetic ecdysteroid, muristerone A (murA), but a maximal response to 20-hydroxyecdysone (20E) was achieved only for specific EcR combinations with its heterodimeric partner. Notably, the Drosophila EcR isoforms were responsive to 20E only when paired with USP, and only EcRB2 activity was further potentiated by JHIII in the presence of 20E. EcR chimeras that fuse the activator domains from VP16 or the glucocorticoid receptor to the Drosophila EcR DNA-binding and ligand-binding domains were responsive to ecdysteroids. Again, the effects of JHIII and 20E were associated with specific partners of the chimeric EcRs. In all experiments, the LBD of EcR proved to be the prerequisite component for potentiation by JHIII, and in this conformation may resemble the FXR LBD. Our results indicate that EcR responsiveness is influenced by the heterodimeric partner and that both the N-terminal domain of EcR and the particular ecdysteroid affect JHIII potentiation.

A new model for 20-hydroxyecdysone and dibenzoylhydrazine binding: a homology modeling and docking approach.

The ecdysone receptor (ECR), a nuclear transcription factor controlling insect development, is a novel target for insecticides such as dibenzoylhydrazines with low environmental and toxicological impacts. To understand the high selectivity of such synthetic molecules toward ECR, two homology models of the Chironomus tentans ECR ligand-binding domain (LDB) have been constructed by taking as templates the known LBD crystal structures of the retinoic acid and vitamin D receptors. Docking of 20-hydroxyecdysone (20E) and dibenzoylhydrazines to the receptor suggests a novel superposition of the natural and synthetic molecules; the N-tert-butyl substituent of the dibenzoylhydrazines extends significantly beyond the 20E volume. Our ECR-LBD protein models rationalize how 20E and dibenzoylhydrazines interact with the ligand-binding pocket. The homology model complexes provide new insights that can be exploited in the rational design of new environmentally safe insecticides.