Potent and Selective Partial Ecdysone Agonist Activity of Chromafenozide in Sf9 Cells

Biochemical and Biophysical Research Communications 292, 1087–1091 (2002) doi:10.1006/bbrc.2002.6771, available online at http://www.idealibrary.com on

Potent and Selective Partial Ecdysone Agonist Activity of Chromafenozide in Sf9 Cells Tetsuya Toya,* Hiroshi Fukasawa,† Akio Masui,* and Yasuyuki Endo† ,1 *Research & Development Laboratories, Nippon Kayaku Company, Ltd., 225-1 Koshikiya, Ageo, Saitama 362-0064, Japan; and †Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

Received March 7, 2002

Chromafenozide (ANS-118) is a non-steroidal ecdysone mimic and its insecticidal effect is highly specific to lepidoptera. In order to evaluate the transcription-inducing activity via nuclear ecdysone receptor (EcR) and the mode of action of chromafenozide, ecdysone-responsive reporter gene assay systems were developed in Sf 9 and Kc cells. Ponasterone A, a full EcR agonist, induced reporter transcription in a dose-dependent manner in both Sf 9 and Kc cells. In contrast, chromafenozide activated reporter transcription with comparable potency to ponasterone A only in Sf 9 cells, although its maximum activity was 4-fold lower than that of ponasterone A. When chromafenozide was applied together with ponasterone A to Sf 9 cells, it antagonized ponasterone A at nanomolar concentrations. These results suggest that chromafenozide is a potent partial EcR agonist specific to lepidoptera; it appears to bind lepidopteran EcR with comparable affinity to ponasterone A, but may activate the EcR in a different manner. © 2002 Elsevier Science (USA) Key Words: chromafenozide; ANS-118; insect hormone; ecdysone; ponasterone A; luciferase; agonist; assay; Sf 9 cells; Kc cells.

Two different types of hormones, juvenile hormones (JHs, Fig. 1. 1) and 20-hydroxyecdysone (20-HE, Fig. 1. 2), are known to regulate molting and metamorphosis in insect development (1, 2). JHs are mainly secreted during larval development to preserve the larval stage. On the other hand, 20-HE acts at every stage of the insect’s growth. When last instar larvae stop secreting JHs, development to pupa, followed by adult, occurs under the regulation of 20-HE. As these hormones play crucial roles in the insect’s endocrine systems, it is considered that both agonists and antagonists could be To whom correspondence should be addressed. Fax: ⫹81-3-58414768. E-mail: [email protected]. 1

developed as new insect growth regulators (IGRs) that might act specifically in arthropods. The actions of 20-HE are mediated by a liganddependent transcription factor, nuclear ecdysone receptor (EcR), which was first isolated in Drosophila melanogaster in 1991 as a member of the steroid/ thyroid hormone receptor superfamily (3, 4, 5). Since then, its homologues have been identified in over 10 arthropods, including Chironomus tentans, Bombyx mori, Manduca sexta, and Aedes aegypti (6). The EcRs show high homology in their DNA-binding domains and ligand-binding domains (LBD), ⬎90% and ⬎60% respectively, in these species. To form the functional ecdysone receptor, EcR heterodimerizes with another nuclear receptor, USP (ultraspiracle), which is an insect homologue of RXR (9-cis retinoic acid receptor) (7). In the presence of 20-HE, the EcR-USP heterodimer is highly stabilized and binds to EcRE through its DNA-binding domain to activate transcription of the ecdysone-responsive early genes (e.g., E74A and BR-C), which in turn induce early-late genes (e.g., DHR3 and E78), followed by induction of many late genes. Furthermore, these ecdysoneresponsive genes regulate each other so that their inter-relationships construct an ecdysone-inducible signal network. 20-HE plays a crucial role in controlling this network as an agonist of EcR (6, 8). 1-tert-Butyl-1,2-dibenzoyl hydrazine (RH-5849, Fig. 1. 4) was reported as the first nonsteroidal ecdysone agonist in 1988 (9, 10). Although there is no structural similarity between 20-HE and RH-5849, RH-5849 shows molting hormonal activity in the intact insect, and at the tissue, cellular, and molecular levels. As a result of optimization studies of RH-5849, RH-5992 (tebufenozide, Fig. 1. 5) and chromafenozide (ANS-118, Fig. 1. 6) have been developed as highly potent ecdysone agonists (11, 12). Chromafenozide has a basic dibenzoylhydrazine structure, like RH-5849, and shows hormonal and insecticidal activities specifically on lepidopteran insects, although RH-5849 shows ac-

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FIG. 1. Structures of JHs (1), 20-hydroxyecdysone (2), ponasterone A (3), RH-5849 (4), RH-5992 (5), and chromafenozide (6).

tivities on dipteran, lepidopteran and coleopteran insects. In this report, in order to examine the mode of action of chromafenozide, we developed ecdysteroid responsive reporter gene assay systems using the ecdysone response element and luciferase gene in Sf 9 (Spodoptera frugiperda) and Kc (Drosophila melanogaster) cells, and evaluated its EcR-activating activity and its specificity for lepidopteran EcR. MATERIALS AND METHODS Chemicals. 20-HE and ponasterone A was purchased from Invitrogen (USA). Chromafenozide was synthesized by the condensation of 5-methylchroman-6-carbonyl chloride and tert-butylhydrazine followed by the reaction of 3,5-dimethylbenzoyl chloride as previously described (13). Each compound was dissolved in ethanol and stored at ⫺20°C. Construction of reporter plasmid. The ecdysone responsive reporter plasmid, pEcR-LUC, was constructed by inserting three copies of ecdysone response element (gagacaag-GGTTCA-A-TGCACTtgt) and a truncated Drosophila melanogaster heat shock protein 70 promoter upstream of the firefly luciferase gene in a pGL3-basic vector (Promega, USA). Reporter gene assay. Sf 9 cells (Invitrogen) were maintained in Sf 900 II medium (Invitrogen) as a suspension culture in a spinner flask. Kc cells, kindly provided by Dr. Kumiko Ui-Tei (Japan Medical College), were maintained at 26°C as an adherent culture in Schneider’s Drosophila Medium (Invitrogen) supplemented with 10% FBS. Before transfection, cells were seeded in 24- or 96-well plates (2.4 ⫻ 10 5 cells/well for 24-well plate) in Sf 900 II or Schneider’s Drosophila Medium containing 10% FBS. After 24 h culture at 27°C, the reporter plasmid, pEcR-LUC (0.2 ␮g/well for 24-well plate), was transiently introduced into cells by cationic lipid transfection reagent Tfx-20 (Promega) according to the manufacturer’s instructions. After an additional 24 h of culture at 27°C, the medium was replaced with fresh medium and test compounds were added to the wells. After 24 h, the cells were harvested and lysed in reporter lysis buffer (Promega), then the lysates were assayed for luciferase, using luciferase assay reagent (Promega). Luciferase data were normalized to protein concentrations determined by BCA assay reagent (Pierce) and represent the mean (⫾ standard deviation) of triplicate assays.

RESULTS AND DISCUSSION Partial EcR Agonist Activity of Chromafenozide in Sf 9 Cells To evaluate the mode of ANS-118’s action, an ecdysone responsive reporter plasmid was constructed and transiently introduced into Sf 9 cells. When the transfected cells were cultured with 20-HE or the wellknown high-affinity ecdysone analogue ponasterone A, reporter luciferase was induced in a dose-dependent manner (Fig. 2a). Ponasterone A showed over 10-fold higher potency than 20-HE. This is consistent with their reported relative binding affinities to EcR (13, 14, 15). So, this reporter system can reveal functional ecdysone receptor-mediated transcriptional activation with adequate sensitivity. When chromafenozide was added to the reportertransfected Sf 9 cells, it induced luciferase with a similar dose-response relation to ponasterone A from 1 nM to 1 ␮M, although the maximum induction by chromafenozide at 1 ␮M was 5-fold lower than that by ponasterone A (Fig. 2a). Interestingly, at a higher concentration range (⬎1 ␮M), the inductive activity of chromafenozide decreased dose-dependently. So, the overall dose-response curve of chromafenozide was bell-shaped. No obvious cell toxicity of chromafenozide was observed at these concentrations. Selective Activity of Chromafenozide between Species Because chromafenozide shows molting hormone activity that is highly specific to lepidopteran larvae, its activity on ecdysone receptor-mediated transcription in Kc cells, derived from the dipteran Drosophila melanogaster, was examined by using the same reporter plasmid. Ponasterone A and 20-HE induced luciferase in a dose-dependent manner in Kc cells as well as in Sf 9 cells (Fig. 2b). Kc cells were more sensitive to these compounds than Sf 9 cells, but the relative relation of

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FIG. 2. EcR-mediated transcription-inducing activities of 20-HE, ponasterone A and chromafenozide in Sf 9 cells (a) and Kc cells (b). Each bar represents the mean ⫾ standard deviation (n ⫽ 3).

the potencies of the two compounds was unchanged. Chromafenozide only slightly induced luciferase even at 1 ␮M (Fig. 2b), confirming that the compound is highly specific to the lepidopteran. Antagonistic Activity of Chromafenozide against Ponasterone A As chromafenozide seems to act as a partial agonist at lower concentrations, its antagonistic activity against the full agonist ponasterone A was next evaluated. When reporter-transfected Sf 9 cells were treated with various doses of chromafenozide in the presence of 0.1 ␮M ponasterone A, the induction of

luciferase activity by ponasterone A was reduced with increasing concentration of chromafenozide (10 nM– 100 ␮M) (Fig. 3). When the added concentration of chromafenozide was the same as that of ponasterone A, the luciferase activity decreased to about half of the full induction by ponasterone A (Fig. 3). Antagonistic activities of chromafenozide against 20-HE in Sf 9 and Kc cells were also tested (Fig. 4). In Sf 9 cells, the induction of luciferase activity by 1 ␮M 20-HE was reduced to about half by adding 0.01– 0.1 nM chromafenozide. In contrast, 1–10 ␮M of chromafenazole was necessary to lesson the luciferase activity to about half of that by 0.1 ␮M 20-HE in Kc cells. So, the

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FIG. 3. Antagonistic activity of chromofenozide against ponasterone A (0.1 ␮M) in Sf 9 cells. Each bar represents the mean ⫾ standard deviation (n ⫽ 3).

specificity of chromafenazole to the lepidopteran was confirmed in these antagonist tests again. DISCUSSION Chromafenozide has been shown to exhibit the potent hormonal and insecticidal activities in lepidopteran larvae, but its mode of function at the molecular level has not been well elucidated (12). In this report,

EcR-responsive reporter gene assay systems were developed and used for evaluation of the EcR-activating activity of chromafenozide. Mikitani reported an EcR-responsive reporter gene assay using Kc cells and suggested that nonsteroidal ecdysone agonists have high specificity among insects, so that assay systems to be developed for evaluation of their activities should use cells from appropriate species of insects (16). As chromafenozide is specific to lepidopteran insects, both lepidopteran Sf-9 cells and dipteran Kc cells were used for comparison here. The well known synthetic ecdysteroid ponasterone A showed strong transcription-inducing activities in both reporter assays using Sf 9 and Kc cells. This is consistent with the reported result that ponasterone A has a strong affinity with EcR, and there is only a slight difference between dipteran and lepidopteran insect cells (13, 14, 15, 17). In contrast, chromafenozide showed potent transcription-inducing activity only in Sf 9 cells. Although the potency of chromafenozide is comparable with that of ponasterone A judging from their submicromolar ED 50 values in Sf 9 cells, its maximal activity at 1 ␮M was 5-fold lower than that of ponasterone A. This result indicates that chromafenozide may be a partial agonist of EcR. Furthermore, chromafenozide alone showed antagonistic activity at above micromolar concentrations, so its overall doseresponse curve is bell-shaped. Although the reason for the biphasic response to chromafenozide is not clear, some mechanism not involving EcR might operate in the higher concentration range. In dipteran Kc cells, chromafenozide showed only weak activity even at the highest concentration exam-

FIG. 4. Antagonistic activities of chromofenozide against 20-HE in Sf 9 cells (a) and Kc cells (b). Cells were treated with increased concentrations of chromafenozide as shown in addition with 1 ␮M (in Sf 9 cells) or 0.1 ␮M (in Kc cells) 20-HE. Each bar represents the mean ⫾ standard deviation (n ⫽ 3). 1090

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ined, 1 ␮M. Although LBDs of EcRs show high homology (⬎60%) among various insects (6), there are presumably slight structural differences between lepidopteran EcR and dipteran EcR that are recognized by chromafenozide, resulting in its high specificity to lepidoptera. Similar results were observed in the experiments on RH-5992, a non-steroidal ecdysone agonist (18). It also causes larvae mortality against lepidopteran insects and its binding affinity to dipteran EcR was only about 1/100 of that to lepidopteran EcR. Supporting the idea that chromafenozide is a partial EcR agonist, co-treatment with chromafenozide and ponasterone A decreased the reporter transcription induced by ponasterone A in a manner dependent upon the dose of chromafenozide. In this co-treatment assay, the potency of chromafenozide was comparable to that of ponasterone A. Recently, Wurtz et al. have constructed homology models of the Chironomus tentans EcR LBD and proposed novel superpositions of 20-HE and RH-5849 complexed with EcR (19). In these models, RH-5849 cannot fill the ecdysteroid-binding cavity and does not form hydrogen bonds with amino acid residues with which ecdysteroids bind. As in these models, the structural difference between ponasterone A and chromafenozide may result in differences in the binding mode with EcR, so that chromafenozide may not induce the full conformational alteration which is needed for maximal activation of EcR. Chromafenazole also antagonized against 20-HE, and its antagonistic activity was selective to Sf 9 cells. So the potent hormonal and insecticidal activities of chromafenazole in lepidopteran larvae are ascribable to its selective partial agonist or antagonist activity against endogeneous ecdysteroid. In conclusion, considering the transcription-inducing activity of chromafenozide in EcR-mediated reporter assay and the structural similarity to RHcompounds, we suggest that chromafenozide bind to the ecdysone-binding site in EcR and modulates the transcription of the genes that regulate the molt as a potent partial EcR agonist, resulting in disruption of the normal ecdysone-inducible signal network. Thus, the biological responses at the intact insect level, cellular level, and also at the molecular level indicate that chromafenozide is a potent and selective ecdysteroid agonist. ACKNOWLEDGMENT We express our thanks to Dr. Kumiko Ui Tei of Japan Medical College for providing the Kc cell line.

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