Cucurbitacins Are Insect Steroid Hormone Antagonists Acting at The

Biochem. J. (1997) 327, 643–650 (Printed in Great Britain) 643 Cucurbitacins are insect steroid hormone antagonists acting at the ecdysteroid receptor ! Laurence DINAN*1, Pensri WHITING*, Jean-Pierre GIRAULT†, Rene LAFONT‡, Tarlochan S. DHADIALLA§, Dean E. CRESS§, Bruno MUGATs, Christophe ANTONIEWSKIs and Jean-Antoine LEPESANTs *Department of Biological Sciences, University of Exeter, Washington Singer Laboratories, Perry Road, Exeter, Devon EX4 4QG, U.K., †Universite! Rene! Descartes, Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, CNRS-URA 400, 45 rue des Saints-Pe' res, 75270 Paris cedex 06, France, ‡E; cole Normale Supe! rieure, De! partement de Biologie, Laboratoire de Biochimie, CNRS-EP 119, 46 rue d’Ulm, 75230 Paris cedex 05, France, §Rohm & Haas Co., Research Laboratories, 727 Norristown Road, P.O. Box 904, Spring House, PA 19477-0904, U.S.A., and sInstitut Jacques Monod, 2 Place Jussieu, F-75251 Paris cedex 05, France Two triterpenoids, cucurbitacins B and D, have been isolated B (cucB) prevents stimulation by 20E of an ecdysteroid-re- from seeds of Iberis umbellata (Cruciferae) and shown to be sponsive reporter gene in a transfection assay. CucB also prevents responsible for the antagonistic activity of a methanolic extract the formation of the Drosophila ecdysteroid receptor}Ultra- of this species in preventing the 20-hydroxyecdysone (20E)- spiracle}20E complex with the hsp27 ecdysteroid response induced morphological changes in the Drosophila melanogaster element as demonstrated by gel-shift assay. This is therefore the BII permanent cell line. With a 20E concentration of 50 nM, first definitive evidence for the existence of antagonists acting at cucurbitacins B and D give 50% responses at 1.5 and 10 µM the ecdysteroid receptor. Preliminary structure}activity studies #$ respectively. Both cucurbitacins are able to displace specifically indicate the importance of the ∆ -22-oxo functional grouping in bound radiolabelled 25-deoxy-20-hydroxyecdysone (ponasterone the side chain for antagonistic activity. Hexanorcucurbitacin D, A) from a cell-free preparation of the BII cells containing which lacks carbon atoms C-22 to C-27, is found to be a weak ecdysteroid receptors. The Kd values for cucurbitacins B and D agonist rather than an antagonist. Moreover, the side chain (5 and 50 µM respectively) are similar to the concentrations analogue 5-methylhex-3-en-2-one possesses weak antagonistic required to antagonize 20E activity with whole cells. Cucurbitacin activity. INTRODUCTION being developed as a commercial insecticide. To identify ecdy- Insect development is strictly regulated by the interplay between steroid antagonists among natural products we have initiated a a number of chemically different classes of hormone. The steroid screening programme with a sensitive, robust and convenient hormones of insects are the ecdysteroids, which are involved at microplate-based bioassay on an ecdysteroid-responsive cell line each stage of the insect’s life cycle and in the regulation of many [14,15]. This has identified several plant extracts with potent developmental, biochemical and physiological processes (re- ecdysteroid antagonist activities. Here we report the bioassay- viewed in [1]). Thus, in the search for new agents to control insect guided purification and identification of the active principles pest species, interference with ecdysteroid action is an attractive, from one of these extracts and the initial characterization of their but as yet little exploited, target. Not only are the hormonal molecular mode of action. Cucurbitacins are thus demonstrated actions of ecdysteroids specific to invertebrates, but the ecdy- to be the first definitive insect steroid hormone antagonists acting steroids are chemically distinct from vertebrate steroid hormones, at the level of the ecdysteroid receptor. suggesting that agents specifically disrupting ecdysteroid meta- bolism}mode of action should not affect vertebrate steroid hormone systems. MATERIALS AND METHODS Hormone agonists and antagonists are powerful tools for the molecular dissection of hormone action. Steroid hormone Chromatography antagonists have been identified for the oestrogens, androgens, General HPLC conditions have been described previously [16]. progestogens and glucocorticoids, and compounds such as tam- Analytical and semi-preparative C"), silica and DIOL columns oxifen have found medical application [2,3]. No unequivocal were purchased from Jones Chromatography. Solid-phase ex- antagonists have yet been identified in vertebrate systems for traction (SPE) columns (C") and silica SEP-PAK) were obtained vitamin D [4] or in invertebrates for ecdysteroids. The recent from the Waters Division of Millipore. Chromatography sep- rapid advances in the characterization of the ecdysteroid recep- aration conditions are described below as appropriate. tors from Drosophila melanogaster [5,6] and other insect species [7–10] have made insect systems excellent general models for the study of steroid hormone action [11]. Steroidal ecdysteroid Bioassay agonists exist in the form of phytoecdysteroids – more than 150 analogues – which have been isolated from various species of The microplate-based bioassay for ecdysteroid agonists and plant [12]. The first non-steroidal ecdysteroid agonists antagonists based on the ecdysteroid-specific response of the have been identified [13] and at least one of these (RH5992) is Drosophila melanogaster BII cell line was performed as described Abbreviations used: cucB, cucurbitacin B; cucD, cucurbitacin D; EcRE, ecdysteroid response element; 20E, 20-hydroxyecdysone; RP, reverse-phase; SPE, solid-phase extraction. 1 To whom correspondence should be addressed. 644 L. Dinan and others previously [15]. The concentration of 20-hydroxyecdysone (20E) Molecular modelling used in the antagonist assay was 50 nM. Molecular modelling was performed with Alchemy III software from Tripos (St. Louis, MO, U.S.A.). Energy minimization was Plant material performed in acuo without electrostatic charges. Superimpo- Seeds for screening purposes were purchased from commercial sition of 20E and cucurbitacin D (cucD) was performed with the seed companies, mainly Chiltern Seeds, Ulverston, Cumbria, fitting procedure of the software and by fitting the atoms of the U.K. Seeds of Iberis umbellata (candytuft) were donated by C- and D-rings of the two compounds. Suttons Seeds (Torquay, Devon, U.K.). Plants of I. umbellata were grown in a domestic garden and harvested when mature Mode of action of the antagonists (with flowers and fruits). Cell-free receptor binding assay Reference cucurbitacins BII cells were homogenized by sonication and centrifuged (16000 g for 20 min at 4 mC) as described previously [18]. Aliquots Cucurbitacins E and I were purchased from Extrasynthese SA, (100 µl) of the supernatant were incubated in a final volume of $ Genay, France. Other cucurbitacins were gifts from Professor H. 200 µl with [ H]ponasterone A (180 Ci}mmol; 0.2 nM final Achenbach (Department of Pharmaceutical Chemistry, Uni- concentration) in the presence of known concentrations of A1 or versity of Erlangen, Erlangen, Germany) and Professor T. A2 (see below) or a 250-fold excess of unlabelled ponasterone A. Konoshima (Kyoto Pharmaceutical University, Kyoto, Japan). Kd values were calculated from IC&! values with the LIGAND program [19]. Alkenyl ketones Hept-3-en-2-one, hex-4-en-3-one, 5-methylhex-3-en-2-one and Gel-retardation assays 6-methylhepta-3,5-dien-2-one were purchased from Aldrich Gel-shift assays were performed as described by Antoniewski et (Gillingham, Dorset, U.K.) or Lancaster Synthesis (Morecambe, al. [20] with D. melanogaster BII cell nuclear extract, except that Lancs., U.K.). incubations and gel separations were performed at 20 mC. The probe was a 27-mer corresponding to the hsp27 ecdysteroid response element (EcRE) sequence [20,21] labelled at the 5« end Extraction and analysis of plant material $# with P. Gels were vacuum-dried and then substances were Seed samples were ground in a pestle and mortar; plant material detected and quantified by means of a PhosphorImager. was freeze-dried for 4 days. Samples (approx. 25 mg) were extracted three-times with methanol (1 ml) at 55 mC. The pooled extracts were mixed with 1.3 ml of water and 2 ml of hexane. The Transfection assays hexane phase (containing non-polar lipids and}or pigments) was D. melanogaster S2 cells were transfected in the presence discarded. Portions of each aqueous methanol phase were of DOTAP ²N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl- analysed for their agonist and antagonist activities by means of ammonium methylsulphate´ with a construct consisting of ten bioassay, and for the presence of ecdysteroids by radioimmuno- copies of the hsp27 EcRE coupled to the Fbp1 minimal promoter assay [17]. The antisera do not cross-react with cucurbitacins B (bp ®69 to 80) and the lacZ reporter gene [22]. After 24 h the and D (T. V. Savchenko and L. Dinan, unpublished work). cells were exposed to 20E and}or cucurbitacins; after a further Preliminary chromatographic characterization of the antagonist 24 h the cells were extracted and the β-galactosidase activity was activities was obtained by monitoring normal-phase (silica, 1 g) measured by the method of [22]. and reverse-phase (RP) (C"), 0.5 g) SPE and HPLC separations with the bioassay in antagonist mode. Coupled transcription and translation of Drosophila ecdysteroid receptor (DmEcR) and Ultraspiracle (DmUSP) proteins Isolation of antagonists from candytuft

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