Characterization and transcriptomic analyses of the toxicity induced by toosendanin in Spodoptera frugipreda.

The fall armyworm, Spodoptera frugiperda, is a destructive agricultural pest that seriously threatens global food security. Insecticide resistance of this pest has gradually formed in recent years due to improper usage, and alternative methods are badly needed. Toosendanin (TSN) is a botanical compound with broad-spectrum insecticidal activities against many pests. However, the effects of TSN on S. frugiperda are still unclear. In this study, the growth inhibition phenomenon, including weight loss and prolonged developmental duration, in the larvae with TSN exposure was clearly observed. Compared to the control group, a total of 450 and 3314 differentially expressed genes (DEGs) were identified by RNA-Seq in the larvae groups treated with 10 and 20 mg/kg TSN, respectively. Furthermore, the DEGs involved in the juvenile hormone and ecdysone signal pathways and downstream processes, including detoxifying enzyme genes, chitin synthesis and metabolism genes, and cuticular protein genes, were found. Our findings suggest that TSN regulates the expression of key genes in juvenile hormone and ecdysone signal pathways and a series of downstream processes to alter the hormone balance and cuticle formation and eventually inhibit larval growth, which laid the foundation for the molecular toxicological mechanism research of TSN on S. frugiperda larvae.

Diallyl trisulfide reduced the reproductive capacity of male Sitotroga cerealella via the regulation of juvenile and ecdysone hormones.

Environmental pollution and resistance in animals are major concerns for the application of synthetic pesticides. Diallyl trisulfide (DAT), an active compound in garlic essential oil, is a novel tool for active and safe control of agricultural insect pests. In this study, we analysed the effects of DAT (0.01 µL/L) on the protein content in male reproductive tissues (accessory glands, ejaculatory ducts, and testis), and juvenile hormone (JH) and ecdysone titres in a highly detrimental pest of stored products, Sitotroga cerealella. Evaluation of the expression profile of JH and ecdysone pathway-related genes in various tissues indicated that the accessory gland protein and ecdysone titres were markedly decreased after DAT fumigation, whereas the testis protein content and JH titre were increased. However, the protein content of the ejaculatory ducts remained unchanged between the treated and control groups. Further investigation revealed that DAT disrupted the mRNA expression of key enzymes involved in JH and ecdysone pathways. While increased mRNA levels of juvenile hormone acid O-methyltransferase (JHMAT) and Kruppel homologue 1 (Kr-h1) were observed after 4 and 7 h of DAT fumigation, the levels of juvenile hormone epoxide hydrolase (JHEH) were substantially reduced 3 h post-fumigation. mRNA levels of the ecdysone-responsive gene, FTZF1, and cytochrome P450 enzyme, CYP315A1, were notably decreased at 7 h and 4 h, respectively, post-fumigation, whereas CYP314A1 and CYP302A1 mRNA levels decreased after 3 h and 4 h, respectively. While DAT fumigation disrupted sperm number in the testis, ejaculatory ducts, and seminal vesicles, topical application of the 20-hydroxyecdysone (20E) analogue also lowered sperm number in the ejaculatory ducts. Topical application of methoprene, a JH analogue, increased the protein content in the testes, but not in the accessory glands or ejaculatory ducts. However, the survival rate was not affected by the topical application of methoprene or 20E. These data suggest that DAT regulates JH and ecdysone via its molecular pathway genes and modulates endocrine secretion during the male reproductive process.

The plant-derived triterpenoid, cucurbitacin B, but not cucurbitacin E, inhibits the developmental transition associated with ecdysone biosynthesis in Drosophila melanogaster.

In insects, some sterols are essential not only for cell membrane homeostasis, but for biosynthesis of the steroid hormone ecdysone. Dietary sterols are required for insect development because insects cannot synthesize sterols de novo. Therefore, sterol-like compounds that can compete with essential sterols are good candidates for insect growth regulators. In this study, we investigated the effects of the plant-derived triterpenoids, cucurbitacin B and E (CucB and CucE) on the development of the fruit fly, Drosophila melanogaster. To reduce the effects of supply with an excess of sterols contained in food, we reared D. melanogaster larvae on low sterol food (LSF) with or without cucurbitacins. Most larvae raised on LSF without supplementation or with CucE died at the second or third larval instar (L2 or L3) stages, whereas CucB-administered larvae mostly died without molting. The developmental arrest caused by CucB was partially rescued by ecdysone supplementation. Furthermore, we examined the effects of CucB on larval-prepupal transition by transferring larvae from LSF supplemented with cholesterol to that with CucB just after the L2/L3 molt. L3 larvae raised on LSF with CucB failed to pupariate, with a remarkable developmental delay. Ecdysone supplementation rescued the developmental delay but did not rescue the pupariation defect. Furthermore, we cultured the steroidogenic organ, the prothoracic gland (PG) of the silkworm Bombyx mori, with or without cucurbitacin. Ecdysone production in the PG was reduced by incubation with CucB, but not with CucE. These results suggest that CucB acts not only as an antagonist of the ecdysone receptor as previously reported, but also acts as an inhibitor of ecdysone biosynthesis.

Developing new drugs that activate the protective arm of the renin-angiotensin system as a potential treatment for respiratory failure in COVID-19 patients.

COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has reached pandemic proportions with negative impacts on global health, the world economy and human society. The clinical picture of COVID-19, and the fact that Angiotensin converting enzyme 2 (ACE2) is a receptor of SARS-CoV-2, suggests that SARS-CoV-2 infection induces an imbalance in the renin-angiotensin system (RAS). We review clinical strategies that are attempting to rebalance the RAS in COVID-19 patients by using ACE inhibitors, angiotensin receptor blockers, or agonists of angiotensin-II receptor type 2 or Mas receptor (MasR). We also propose that the new MasR activator BIO101, a pharmaceutical grade formulation of 20-hydroxyecdysone that has anti-inflammatory, anti-fibrotic and cardioprotective properties, could restore RAS balance and improve the health of COVID-19 patients who have severe pneumonia.

Comparison of pharmacokinetics of phytoecdysones and triterpenoid saponins of monomer, crude and processed Radix Achyranthis Bidentatae by UHPLC-MS/MS.

1. The aim of this study was to develop a selective, rapid, accurate and sensitive ultrahigh performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for pharmacokinetic (PK) studies of phytoecdysones and triterpenoid saponins after oral administration of five monomers, crude, wine-processed and salt-processed Radix Achyranthis bidentatae (RAB).2. A Thermo Hypersil GOLD C18 column (100 mm × 2.1 mm, 1.9 µm) coupled with a mobile phase of (A) acetonitrile and (B) water (both containing 0.3% acetic acid) was used for sample separation. The mass analysis was performed in a triple quadruple mass spectrometer using selected reaction monitoring (SRM) with negative scan mode.3. The results showed that this method exhibited desirable sensitivity, precision, stability and repeatability. The extraction recoveries of the compounds ranged from 94.2 to 99.8% and the matrix effects ranged from 93.3 to 100.5%. Comparing the Cmax and AUC of five analytes in those groups showed this tendency: salt-processed RAB > wine-processed RAB > crude RAB > monomer group. The results confirmed the feasibility of TCM theory to enhance the efficacy of processed RAB.

Ecdysteroid-containing food supplements from Cyanotis arachnoidea on the European market: evidence for spinach product counterfeiting.

Phytoecdysteroids like 20-hydroxyecdysone ("ecdysterone") can exert a mild, non-hormonal anabolic/adaptogenic activity in mammals, and as such, are frequently used in food supplements. Spinach is well-known for its relatively low ecdysteroid content. Cyanotis arachnoidea, a plant native in China, is among the richest sources of phytoecdysteroids, and extracts of this plant are marketed in tons per year amounts via the internet at highly competitive prices. Here we report the investigation of a series of food supplements produced in Germany and claimed to contain spinach extracts. Twelve ecdysteroids including two new compounds were isolated and utilized as marker compounds. A comparative analysis of the products with Cyanotis and spinach extracts provides evidence that they were manufactured from Cyanotis extracts instead of spinach as stated. Based on the chromatographic fingerprints, 20-hydroxyecdysone 2- and 3-acetate are suggested as diagnostic markers for related quality control. This case appears to represent an unusual type of dietary supplement counterfeiting: undeclared extracts from alternative plants would supposedly 'guarantee' product efficacy.

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

BACKGROUND: A 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 FINDINGS: Administration 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. CONCLUSIONS: The 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.

Seasonal and geographical impact on the morphology and 20-hydroxyecdysone content in different tissue types of wild Ajuga bracteosa Wall. ex Benth.

Ajuga bracteosa is an endangered medicinal herb which contains several natural products of therapeutic importance like 20-hydroxyecdysone (20-HE). As geography and habitat play a crucial role in the metabolism and morphology of a plant, the present study was aimed at evaluating the impact of phytogeography, season and tissue type on morphology and 20-HE content of A. bracteosa. The results revealed large morphological variations in various ecotypes of A. bracteosa. However, plants from the same altitude, regardless of their phytogeography, represented similar morphology. Effect of habitat on 20-HE content remained non-significant except for Karot (1608µg/g) and Kahuta (728µg/g). Effect of tissue types was significant (p value <0.016) for 20-HE content and followed ascending order: rootspring (1071µg/g)>summer (617µg/g). The aerial tissue types contained more 20-HE content in all seasons; especially during winter its amount radically rose in flowers (µ=2814µg/g). The aerial portion of Karot ecotype harvested in winter offers a valuable source of 20-HE. To confirm the effect of low temperature on 20-HE content, profiling of A. bracteosa raised in vitro at different temperature regime was carried out. On the basis of these results we hypothesize that chilling cold hampers vegetative growth and triggers stress induced 20-HE accumulation as a defense response.

20-OH-ecdysone prevents hot flushes in ovariectomized rats.

Hot flushes are due to the lack of estrogens and are the most characteristic climacteric complaints. Hormone replacement therapy was the standard treatment but now its use is limited because of side effects. Need therefore arises to search for non-estrogenic alternatives. The molting hormone 20-beta-hydroxyecdysone (Ecd) is produced by several plants including spinach and has no estrogenic or androgenic properties but enhances GABAergic effects in neurons. Since GABAergic compounds can ameliorate hot flushes, we investigated the effects of Ecd on subcutaneous body temperature of intact and ovariectomized (ovx) rats. The subcutaneous body temperature was recorded at 5-min intervals over a period of 3 hours. Rats were then ovx, and skin temperatures were recorded after an acute intravenous (5 mg) and during subchronic and chronic oral application of Ecd (73 mg/animal/day). For additional control purposes, a group of ovx rats received food containing estradiol-17 ß (E2). Skin temperature in individual ovx animals fluctuated largely with peaks (hot flushes) occurring every 20-40 minutes. Following the i.v. treatment with Ecd, skin temperature dropped by more than 1 °C, an effect much larger than in the controls. One and two weeks later, hot flushes were only seen in ovx controls but not in intact, E2-, or Ecd-treated animals. As a consequence, E2 and Ecd intake significantly (p < 0.05) reduced the mean temperature in ovx rats during the various time points of the study. These results suggest that Ecd is efficient to prevent hot flushes in ovx rats.

[Studies on the chemical constituents of Paris bashanensis].

OBJECTIVE: Study the chemical constituents of the rhizomes of Paris bashanensis to search after the alternative resourc for the Chinese medicinal material Rhizoma Paridis. METHODS: The n-BuOH extracts of P. bashanensis was applied to silica gel column and eluted with EtOAc-EtOH,then the gained fractions were further purified by chromatography on Sephadex LH-20 column and PreRP-HPLC to give pure compounds whose structures were elucidated mainly on the basis of analyzing the spectral data of MS,1H-NMR, 13C-NMR,2D-NMR. RESULTS: Five compounds were isolated and identified as P-ecdysone (1), pinnatasterone(2), pennogenin-3-0-alpha-L-rhamnopyranosyl (1-->2)-[alpha-L-arabinofuranosyl ( 1- -4) ] -pf-D-glycopyranoside (3), diosgenin-3-O-alpha-L-rhamnopyranosyl (1-->2) - [ a-L-arabinofuranosyl (1-->4)]-beta-D-glycopyranoside(4), pennogenin-3-O-alpha-L-rhamnopyranosyl (1-->4) -a-L-rhamnopyranosyl (1-4)-[alpha-L-rhamnopyranosyl (1-->2)]-beta-D-glycopyranoside (5). CONCLUSION: Compound 1-5 are obtained from this plant for the first time.

Virtual screening for ligands of the insect molting hormone receptor.

Insect growth is regulated by the orchestrated event of ecdysteroids and their receptor proteins. Agonists/antagonists of ecdysteroid receptor are predicted to disrupt normal growth, providing good candidates of new insecticides. A database of over 2 million compounds was subjected to a shape-based virtual screening cascade to identify novel nonsteroidal hits similar to the known EcR ligand ponasterone A. Testing revealed micromolar hits against two strains of insect cells. Docking experiments against EcR were used to support the predicted binding mode of these ligands based on their overlay to ponasterone A.

Two minor phytoecdysteroids of the plant Silene viridiflora.

Chemical investigations of Silene viridiflora (L.) yielded a new ecdysteroid, 20-hydroxyecdysone 20,22-monoacetonide-25-acetate (1), and a known ecdysteroid, 2-deoxypolypodine B-3-beta-D-glucoside (2). The elucidation of the chemical structures was established by 1D and 2D NMR experiments.

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.

Selenoprotein P regulation by the glucocorticoid receptor.

Maintenance of the antioxidant activity of selenoproteins is one potential mechanism of the beneficial health effects of selenium. Selenoprotein P is the primary selenium distribution protein of the body as well as the major selenium containing protein in serum. The transcriptional regulation of selenoprotein P is of interest since the extrahepatic expression of this gene has demonstrated differentiation-dependent expression in development as well as under different disease states. SEPP1 displays patterned expression in numerous tissues during development and the loss of SEPP1 expression has been observed in malignancy. In addition, factors that influence inflammatory processes like cytokines and their regulators have been implicated in selenoprotein P transcriptional control. Herein, we identify a retinoid responsive element and describe a mechanism where the glucocorticoid receptor negatively regulates expression of selenoprotein P. Luciferase reporter assays and quantitative PCR were used to measure selenoprotein P transcription in engineered HEK-293 cells. When stimulated with ecdysone analogs, selenoprotein P expression was increased with the use of a fusion transcription factor that contains the glucocorticoid receptor DNA binding domain, an ecdysone ligand-binding domain, and a strong transactivation domain as well as the retinoid X receptor. The native glucocorticoid receptor inhibited selenoprotein P transactivation, and selenoprotein P was further attenuated in the presence of dexamethasone. Our results may provide insight into a potential mechanism by which selenium is redistributed during development, differentiation or under conditions of critical illness, where glucocorticoid levels are typically increased.

Furostanol saponins and ecdysones with cytotoxic activity from Helleborus bocconei ssp. intermedius.

Two furostanol saponins helleboroside A (1) and helleboroside B (2) were isolated from the methanol extract of Helleborus bocconei Ten. subsp. intermedius (Guss.) Greuter and Burdet, along with the furospirostanol saponin 4 and two ecdysones: ecdysterone (5) and polypodyne B (6). Compound 2 was enzymatically hydrolysed to give product 3. The biological activity of all compounds was tested against rat C6 glioma cells showing a significant cytotoxicity for compounds 3, 4 and 6.

Simultaneous determination of three phytoecdysteroids in the roots of four medicinal plants from the genus Asparagus by HPLC.

INTRODUCTION: The genus Asparagus is known to contain phytoecdysteroids that have been shown to exhibit many beneficial pharmacological properties such as improving lipid metabolism, modulating immunological responses, etc. Currently, knowledge about the contents of phytoecdysteroids in the roots of Asparagus species is limited and HPLC methods for their analyses are unsatisfactory. OBJECTIVE: To develop an HPLC method for the simultaneous determination of three phytoecdysteroids, 20-hydroxyecdysone, ecdysone and ajugasterone C, in the roots of four Asparagus species. METHODOLOGY: Reference standards of phytoecdysteroids were isolated from the roots of Asparagus filicinus by open column chromatography. HPLC analysis was performed on an Alltima C(18) column with gradient elution using aqueous 0.2% formic acid solution containing 0.2% isopropanol and acetonitrile. RESULTS: All calibration curves showed good linear correlation coefficients (r(2) > 0.9994) within the tested ranges. Limits of detection (S/N = 3) and quantification (S/N = 10) for the three analytes were less than 2.7 and 9.9 ng, respectively. Intra- and inter-day RSDs of retention times and peak areas were less than 2.61%. The recoveries were between 93.2 and 107.5%, and the RSDs were less than 3.83% for the root samples of A. filicinus. CONCLUSION: The HPLC method established is appropriate for the efficient quantitative and qualitative analyses of important phytoecdysteroids in Asparagus species. This study showed that A. filicinus is rich in phytoecdysteroids, especially 20-hydroxyecdysone. However the three studied phytoecdysteroids were not detected in A. cochinchinensis, A. officinalis and A. setaceus.

Molecular cloning of the ecdysone receptor and the retinoid X receptor from the scorpion Liocheles australasiae.

cDNAs of the ecdysone receptor and the retinoid X receptor were cloned from the Japanese scorpion Liocheles australasiae, and the amino acid sequences were deduced. The full-length cDNA sequences of the L. australasiae ecdysone receptor and the L. australasiae retinoid X receptor were 2881 and 1977 bp in length, respectively, and the open reading frames encoded proteins of 560 and 414 amino acids. The amino acid sequence of the L. australasiae ecdysone receptor was similar to that of the ecdysone receptor-A of the soft tick, Ornithodoros moubata (68%) and to that of the ecdysone receptor-A1 of the lone star tick, Amblyomma americanum (66%), but showed lower similarity to the ecdysone receptors of Orthoptera and Coleoptera (53-57%). The primary sequence of the ligand-binding region of the L. australasiae ecdysone receptor was highly homologous to that of ticks (85-86%). The amino acid sequence of the L. australasiae retinoid X receptor was also homologous to the amino acid sequence of ultraspiracles of ticks (63%) and insects belonging to the orders Orthoptera and Coleoptera (60-64%). The identity of both the L. australasiae ecdysone receptor and the L. australasiae retinoid X receptor to their lepidopteran and dipteran orthologs was less than 50%. The cDNAs of both the L. australasiae ecdysone receptor (L. australasiae ecdysone receptor-A) and the L. australasiae retinoid X receptor were successfully translated in vitro using a rabbit reticulocyte lysate system. An ecdysone analog, ponasterone A, bound to L. australasiae ecdysone receptor-A (K(D) = 4.2 nM), but not to L. australasiae retinoid X receptor. The L. australasiae retinoid X receptor did not enhance the binding of ponasterone A to L. australasiae ecdysone receptor-A, although L. australasiae retinoid X receptor was necessary for the binding of L. australasiae ecdysone receptor-A to ecdysone response elements.

Simultaneous qualitation and quantification of four phytoecdysones in Radix Achyranthis Bidentatae by high-performance liquid chromatography with diode array detection.

A high-performance liquid chromatographic method with diode array detection was developed and validated to simultaneously identify and quantify four phytoecdysones,i.e., polypodine B (1), ecdysterone (2), 25-R inokosterone (3) and 25-S inokosterone (4), in Radix Achyranthis Bidentatae. The analysis was performed using a C(18) column with 6% tetrahydrofuran aqueous and acetonitrile isocratic elution, and the detection was carried out by DAD at 242 nm. The identities of the analytes were determined by comparing the retention time and UV spectrum with those of reference compounds. The validation of the method included linearity, sensitivity, recovery and stability. All calibration curves of the four phytoecdysones showed good linear regression (r(2) >or= 0.9993). The limit of detection (S/N = 3) and limit of quantification (S/N = 10) were less than 7.5 and 12.3 ng, respectively. The method provided desirable repeatability with overall intra- and inter-day variations of less than 4.67%. The obtained recoveries varied between 95.1 and 104.4% while the relative standard deviations were below 4.85% (n = 3). The analysis results indicated that the contents of the investigated phytoecdysones in Radix Achyranthis Bidentatae from different locations were highly variant, and the genuine crude drug indigenous to Henan province did not possess the highest concentration of phytoecdysones.

Detection of saponins in extracts from the rhizomes of Paris species and prepared Chinese medicines by high performance liquid chromatography-electrospray ionization mass spectrometry.

A highly sensitive and specific method, based on high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS), was developed for simultaneous detection of eleven steroidal saponins and one phytoecdysone in extracts from the rhizomes of thirteen PARIS species and four prepared Chinese medicines (PCMs). The HPLC experiments were performed by means of a reversed-phase C18 column (4.6 mm x 150 mm, 5 microm) and mobile phase system consisting of 0.1 % aqueous formic acid and acetonitrile under gradient elution conditions. The most intensive electrospray ionization signals were found in the negative ion spectra due to HCOO- adducts. The limits of detection (LODs) for the saponins were lower than 40 ng/mL in selected ion monitoring (SIM) mode. The identification of the saponins in the extracts of PARIS species and PCMs was confirmed using their retention times and mass spectral comparisons to standard compounds. The validated method was successfully applied for simultaneous detection of twelve standard compounds in the analytes so that it provided a new means for quality evaluation of Rhizoma Paridis and Chinese multiherb remedies that contain Rhizoma Paridis. The results showed that most species of PARIS contain steroidal saponins, which provided evidence for expanding the botanical origin of Rhizoma Paridis.

High-throughput screening of ecdysone agonists using a reporter gene assay followed by 3-D QSAR analysis of the molting hormonal activity.

In this study, 172 diacylhydrazine analogs were examined for their ability to activate an ecdysone (molting hormone)-dependent reporter gene in a silkworm (Bombyx mori) cell-based high-throughput screening assay. The measured EC(50) values (concentration required to cause an effect in 50% of the cells) were used to construct a 3-D QSAR model that describes the ecdysone agonist activities of the diacylhydrazine analogs. Of these compounds, 14 exhibited no activity and were excluded from the 3-D QSAR analysis. The resulting equation described approximately 74% of the activity for 158 compounds. The final equation consisted of 42% electrostatic and 58% steric effects (r(2) = 0.74 and q(2) = 0.45). Comparative molecular field analysis (CoMFA) was used to visualize the steric and electrostatic potential fields that were favorable and unfavorable for biological activity. Of particular interest was the observation that the hydrophobic parameter (logP) was not necessary for describing the observed activities, although previous studies have cited the importance of hydrophobic parameters in both classical and 3-D QSAR analyses of these compounds. Modeling studies of the B. mori ecdysone receptor supported the observed physicochemical parameters required for activity reported by the CoMFA models. Comparison of the present analysis with those performed using other lepidopteran assay systems evidenced a high degree of correlation (r(2) = 0.81 for a Sf-9 cell-based assay and r(2) = 0.89 for a Chilo suppressalis integument-based assay), indicating that it is valid to compare the results generated with the B. mori cell-based system to those generated with previous lepidopteran assays. This novel assay system is amendable to a high-throughput screening format and should greatly increase our ability to discover novel agonists of molting hormone (ecdysone) activity.