Proc. Nail. Acad. Sci. USA Vol. 85, pp. 2096-2100, April 1988 Biochemistry 26-4125I]Iodoponasterone A is a potent ecdysone and a sensitive radioligand for ecdysone receptors (steroid hormone/26-iodoponasterone A/Drosophila/Kc cells) PETER CHERBAS*t, Lucy CHERBAS*, SHOEI-SHENG LEEt§, AND Koji NAKANISHIt *Department of Biology and Program in Molecular, Cellular and Developmental Biology, Indiana University, Bloomington, IN 47405; and tDepartment of Chemistry, Columbia University, New York, NY 10027 Communicated by Carroll M. Williams, December 7, 1987 ABSTRACT The effects of ecdysone, the steroid molting introduction of [3H]ponasterone A (>100 Ci/mmol; 1 Ci = hormone of arthropods, are of considerable interest both to 37 GBq) has been critical to recent progress in the study of insect physiologists and to those studying steroid-regulated ecdysone receptors (31, 36, 38). Still, this material has not gene expression. Yet progress in understanding ecdysone been widely available and, given the small amounts of receptors has been inhibited by the lack of a suitable highly biological material that often must be assayed, is still mar- radioactive hormone analog with high affinity for the receptor. ginal in its specific activity. Here we report that the synthetic Here we report that the synthetic ecdysteroid 26-iodoponaste- analog 26-iodoponasterone A (called hereafter iodoponaste- rone A is one of the most active ecdysones known, inducing rone) is still more active biologically than ponasterone A, half-maximal morphological transformation in Drosophila that it can be synthesized readily with carrier-free 125I, and Kc167 cells when present at 0.5 nM. 26-[12'I5Iodoponasterone that the radiolabeled material (2175 Ci/mmol) functions well A can be prepared at a specific activity of 2175 Ci/mmol (1 Ci as a ligand for the detection of ecdysone receptors. = 37 GBq) by reaction of the precursor 26-mesylinokosterone with carrier-free Na'25I. The radiolabeled material binds to Kc167 cell ecdysone receptors specifically and with affinity (Kd MATERIALS AND METHODS ca. 3.8 x 10-10 M). Thus, 26-['2I]iodoponasterone A ap- Cells. Kc167 cells were a gift from M. Bourouis (43). We pears to be a superior radioligand for ecdysone receptors on have adapted them to growth in M3 medium (44) supple- grounds both of affinity and of specific activity. Its ready mented with 5% fetal calf serum. availability should greatly facilitate studies of these receptors. Bioassay. Ecdysones were bioassayed by their activity in inducing morphological transformation of Kc cells (42). The Steroid hormone receptors are generally detected by the use assay procedure was that described in ref. 42 save that of high-affinity radioligands. Because such receptors are Kc167 cells were used. Ecdysteroids to be tested were present at very low concentrations (typically 1-10 ppm of dissolved at known concentrations in ethanol [assuming 6242 cellular protein), a useful ligand must exhibit not only high (ethanol) = 12,400 (45)], serially diluted, and tested in 1-ml affinity for the receptor but also unusually high radiological cell cultures. 20-Hydroxyecdysone, 26-mesylinokosterone, specific activity. Indeed, the development and use of such and iodoponasterone were checked for purity by HPLC (see radioligands has played an important role in the advance- below) and assayed in parallel at concentrations ranging ment of our knowledge of the vertebrate steroid receptors from 10 ,uM to 10 pM. Bioassays of 125I-substituted steroid (1-7). were carried out similarly. Dilutions of [1251]iodoponaste- Ecdysone,$ the steroid molting hormone of arthropods, rone were prepared (assuming a counting efficiency of 50% regulates the timing of development and metamorphosis at for 125I in solution), and cells were treated in parallel with it least in part by its effects on gene expression, effects that are and with a series of concentrations of 20-hydroxyecdysone. under study in numerous laboratories (9-29). Nevertheless, The cultures were compared qualitatively under the micro- our knowledge of the molecular biology and physiology of ecdysone receptors is still fragmentary. Ecdysone-binding scope, the iodoponasterone cultures being assigned posi- proteins thought to be ecdysone receptors have been iden- tions in the series, and then the 20-hydroxyecdysone series tified in ecdysone-responsive Drosophila tissues and cells in was scored in the usual way. culture. These receptors are present at low abundance (ca. Steroids. 20-Hydroxyecdysone and muristerone were pur- 1-2 x 103 molecules per cell); they exhibit high-affinity chased from Simes (Milan). Ponasterone A was the purified binding to active ecdysteroids, the relative affinities being natural product (40). Inokosterone was a gift from T. Take- approximately in proportion to relative ecdysone activities moto (Tohoku University, Sendai, Japan). All were homo- (9, 30-38); and their concentration and/or activity is de- geneous by HPLC save the ponasterone A, which was pressed in ecdysone-resistant tissue culture cells (K. Wing, estimated to be ca. 85% pure. 26-Mesylinokosterone and personal communication). Similar receptors have been iden- iodoponasterone were synthesized from inokosterone. De- tified from hypodermal cells of the crayfish Orconectes (39). tails of the syntheses and physical data for these compounds Because the natural hormone, 20-hydroxyecdysone, is will be reported elsewhere (S.-S.L., K.N., and P.C., unpub- effective only at relatively high concentration (ca. 10 nM) lished data; inquiries concerning the availability of both and its receptor complex is kinetically unstable, success in compounds should be directed to S.-S.L.). studies of the receptor(s) has relied upon use of a more potent analog, the phytoecdysone ponasterone A (40, 41), tTo whom reprint requests should be addressed. which is 10-50 times as active in biological assays and which §Present address: School of Pharmacy, College of Medicine, Na- is comparably more active as a ligand (30-38, 42). Thus, the tional Taiwan University, Taipei 100, Taiwan, Republic of China. IEcdysteroid is the generic name for compounds structurally related to a-ecdysone (8). We use ecdysone as the generic name for The publication costs of this article were defrayed in part by page charge compounds with the appropriate biological activity in accordance payment. This article must therefore be hereby marked "advertisement" with common usage in the literature and in analogy with the terms in accordance with 18 U.S.C. §1734 solely to indicate this fact. estrogen or progestin (9). Downloaded by guest on September 24, 2021 20% Biochemistry: Cherbas et al. Proc. Natl. Acad. Sci. USA 85 (1988) 2097 ["'Il]Iodoponasterone Synthesis. Radiolabeled iodopo- Cell Extracts. Whole-cell extracts have been prepared by nasterone was prepared from the mesyl intermediate as procedures slightly modified from those of ref. 36. Data are follows. The label was purchased as carrier-free Na125I (2175 shown for two extracts similarly prepared. Kc167 cells were Ci/mmol in dilute base free of reducing agents; Amersham) harvested at a density of 2 x 107 cells per ml, washed in lots of 1 or 2 mCi. It was transferred from the shipping vial extensively in saline, and sonicated in an ice-water bath in to a 100-,ul conical glass reaction vial by using dry acetone, 1.25 ml of buffer A [40 mM Hepes, pH 7.0 at 230C/1 mM which subsequently was evaporated under a stream of dry dithiothreitol/5% (vol/vol) glycerol]. The sonicate was ad- nitrogen gas. (To prepare dry solvents, reagent grade ace- justed to 0.4 M KCI by addition of 2.0 M KCI and incubated tone and acetonitrile in lots of a few milliliters were perco- for 5 min at 0C. Finally the extract was centrifuged for 50 lated through 2-ml columns of silica gel immediately before min at 20,000 rpm in a Beckman JA 20 rotor. The superna- use.) 26-Mesylinokosterone (19 nmol/mCi of 1251I) was added, tant was immediately diluted 1:9 with cold buffer A to bring and residual water was partially eliminated by repeatedly the extract to 40 mM KCI in buffer A. Extract 1 (used in the dissolving the mixture in a small volume of dry acetonitrile experiment of Fig. 3) contained 210 gg of protein per 100 ,ul; and evaporating. Finally the mixture was dissolved in dry extract 2 (experiments of Figs. 4 and 5) contained 380 ,ug of acetone (20 tkl/mCi of 1251) as reaction solvent, and the protein per 100 jLd (Bio-Rad protein assay). Extracts were reaction vessel was capped tightly and heated to 800C for 8-12 immediately frozen (liquid nitrogen) in 2-ml aliquots and hr. At the conclusion the acetone was evaporated, and the stored at - 90'C. Receptor activity remained constant under reaction mixture was dissolved in ca. 10 jul of methanol and these conditions for a period of at least 2 months. purified by HPLC. The major radioactive product was eluted Ligand Binding by Extracts. Experimental tubes contained at ca. 7 ml and cochromatographed with authentic iodopo- 0.1 volume (typically 14 ILI) of [1251]iodoponasterone in nasterone both by HPLC and by thin-layer chromatography buffer A, 0.1 volume of additional buffer A or competitor on silica gel. Typical yields have been 12-15%, based on input A, and 1 volume of extract. As the 1251 (six syntheses). Three lots of [1251]iodoponasterone were steroid dissolved in buffer used in the experiments described here. Each was checked by competitor steroid, we used muristerone (42)-the most HPLC and found to be >98% radiochemically pure; residual potent ecdysone available to us in quantity-at a final 26-mesylinokosterone was undetectable by absorbance. concentration of 4.5 ,uM. Binding reactions were carried out Iodoponasterone solutions are quite stable both radio- at room temperature for 1 hr. chemically and chemically when stored over a period of at Aliquots (10 pll) of each tube were removed and assayed to least 4 months in ethanol at - 20'C.