8-O-Acetylharpagide is a nonsteroidal ecdysteroid agonist

Vol. 26, No. 6. pp. S 19-523, 1996 Copyright 0 1996 Elsevier Science Ltd Printed in Great Britain. All rights reserved 096% I748/96 $15.00 + 0.00

insect Biochem. M&c.

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Rapid Communication 8GAcetylharpagide is a Nonsteroidal Ecdysteroid Agonist ALEX ELBRECHT,*§ YULI CHEN,* TANNIS JURGENS,? OTTO D. HENSENS,? DEBRA L. ZINK,? HANS T. BECK,$ MICHAEL J. BALICK,J ROBERT BORRISi Received 24 October 1995; revised and accepted 27 November 1995

We have identified a novel nonsteroidal ecdysteroid agonist. This compound was isolated from a methanol extract of Ajugu reptuns L. (Lamiaceae) and the structure was identified by spectroscopic methods as 8-0-acetylharpagide. We have characterised this compound as an ecdysteroid agonist in a transactivation assay using P-galactosidase as the reporter gene regulated by ecdysteroid response elements. In khis assay, 8-Gacetylharpagide has an EC,, of 22 ~.LM. The compound also competes with tritiated-ponasterone A for binding to the Drosophila ecdysteroid receptor. Finally, it induces differentiation of Drosophila Kc cells as would be expected of an ecdysteroid agonist. This iridoid glycoside is common to several plant species and may play a role in the natural defense mechanisms of plants. Copyright 0 1996 Elsevier Science Ltd Ecdysteroid Receptor Insecticide Drosophila

INTRODUCTION

of action of ecdysone. As with other steroid hormones, virtually all of the effects exerted by ecdysone occur by regulating gene transcription (Beato, 1989). This regulation is mediated through interactions with specific receptor proteins. The ecdysteroid receptor has been cloned from lepidopteran and dipteran species including Drosophila and sequence analysis has shown that this protein belongs to a relatively large family of nuclear receptor proteins which includes other steroid receptors, thyroid hormone receptors, retinoic acid and retinoic acid-related receptors, as well as numerous orphan receptors (Koelle et al., 1991). Even within this family there is a clear preference for any particular steroid or nonsteroidal agonist or antagonist. In particular, nonsteroidal agonists have been rare finds but examples include 1,2dibenzoyl- 1-rert-butylhydrazine for the ecdysteroid receptor and diethylstilbestrol for the estrogen receptor. With 8-0-acetylharpagide we have identified a new class of nonsteroidal ecydsteroid agonists. Phytoecdysteroids are common constituents of plants suggesting that they might act as feeding deterrents for phytophagous insects (Lafont and Horn, 1989). We had speculated that plants might also contain nonsteroidal ecdysteroid agonists which would serve a similar purpose and would not be metabolized as quickly. 8-O-Acetylharpagide could be an example of such a compound.

Insect growth is marked by a series of discontinuous events called molts. Molting is a complicated process involving numerous biological systems and initiated by the steroid hormone ecdysone. Much of the molting process is not understood but disruption can be lethal and nonsteroidal ecdysteroid agonists are being developed for use as insecticides (Wing, 1988; Wing et al., 1988). Specifically, treatment of lepidopteran larvae with the compound 1,2-dibenzoyl- 1-tert-butylhydrazine cause the larvae to initiate a precocious, incomplete and lethal molt (Carlson and Long, 1989). Although death does not occur until 2-3 days post-treatment, the larve do not feed or grow. Thus, targeting molting for the development of agents useful in insect management provides an alternative to the more popular neurotoxic insecticides. The only nonsteroidal compounds identified to date appear to be safe for beneficial insects and mammals (Carlson and Long, 1989). This safety is due, in part, to the mechanism

*Department of Genetics and Molecular Biology. Merck Research Laboratories, P.O. Box 2000. Rahway, NJ 07065, U.S.A. tNatural Product Chemistry, Merck Research Laboratories, P.O. Box 2000, Rahway, NJ 07065, U.S.A. *Institute for Economic Botany, New York Botanical Garden. Bronx. NY 10458, U.S.A. (iAuthor for correspondence. 519

ALEXELBRECHT

520 MATERIALS

AND METHODS

Plant material, comprising entire plants of Ajuga reptans L. (Lamiaceae), was collected from a population in cultivation on the grounds of the New York Botanical Garden, Bronx, NY, in October 1990. Voucher specimens (Beck 1358a) are deposited in the herbarium of that institution. Following air drying, the plant sample (838 g, dry weight) was milled to a coarse powder and extracted twice with methanol. Removal of solvent in vacua afforded 116 g of dark green tarry residue, which was dissolved in 95% (aqueous) methanol and defatted by repeated extraction with n-hexane. The defatted alcoholic phase was concentrated to dryness in vacua and resulted in a residue weight of 101 g. A 3 g portion of this residue was chromatographed on a column of silica gel-60 (230400 Mesh, EM Science), using a dichloromethane: ethanol step gradient, starting with dichloromethane and continuing with mixtures of increasing polarity. Biological activity was monitored in a cMK7 cell transactivation assay described below. Active fractions eluting with dichloromethane:ethanol (5:7) were pooled and evaporated to dryness in vacua to afford 465 mg of brown residue. Preparative thin layer chromatography of a 100 mg portion of this residue on silica gel plates (EM Science) developed in dichloromethane:methanol (5: 1) and visualization under UV light at 254 nm allowed delineation of a band corresponding to a major component of this fraction. This component was then removed from the plate, eluted and evaporated to dryness in vacua to afford 26.5 mg of an off-white amorphous solid with pronounced activity in the cMK7 cell transactivation assay. Comparison of proton NMR spectra (300 MHz), proton homonuclear correlation spectra (COSY),” C-NMR spectra (75 MHz), DEPT spectra and high resolution mass spectra (FAB and electron impact modes) with literature values (Lichti and von Wartburg, 1966; Scarpati et al., 1965; Bianco et al., 1981; Takeda et al., 1987) allowed identification of the compound as the known iridoid glucoside, 8-0-acetylharpagide. cMK7 cells were obtained from Dr D. Hogness (Stanford University, Palo Alto, CA). These cells were prepared by transfection of Drosophila S2 cells with a Drosophila ecdysteroid receptor expression vector and an ecdysteroid responsive B-galactosidase reporter construction (Koelle et al., 199 1). We have adapted these cells for an assay in a microtiter format. Cells were maintained in culture as described (Koelle et al., 1991) and 2 x 10” cells in 250 ~1 of medium were transferred to each well of a microtiter plate. The cells were incubated at 25°C for 24 h with test compound dissolved in 5 ~1 of dimethyl sulfoxide. The following day, the cells were centrifuged for 10 min at 500 g (2000 rpm) in an IEC Centra-7R, and the supernatant was decanted. The cells were lysed by addition of 50 I_L~ of 1 x cell lysis buffer (Promega) and incubation at room temperature for 15 min with gentle rocking. 5 ~1 of lysate were transferred to 100 ~1 of assay

etd

buffer (40 mM NaH,PO,, 60 mM Na,HPO,, 5 mM KCl, pH 8.0) and incubated for 30 min at 37°C. After addition of 100 ~1 of stop buffer (300 mM glycine, 15 mM EDTA, pH 11.2), B-galactosidase enzyme activity was determined by measuring fluorescence in a 7620 Microplate Fluorometer (Cambridge Technology, Inc.). Ponasterone A binding activity was determined using extracts of cMK9 cells obtained from Dr D. Hogness (Stanford University, Palo Alto, CA). The cell line was developed by transfection of Drosophila S2 cells with a plasmid containing the Drosophila EcR cDNA and a Drosophila metallothionein promoter (Koelle et al., 1991). The cells were grown in culture as described and were induced with 700 pM copper sulphate for 24 h before they were harvested. Cell extracts were prepared as described by Koelle et al. (1991). Hormone-binding assays were run as described by Koelle et al. (1991) with the exception that 24, 25,26, 27-3H(N)-ponasterone A (195.2 Ci/mmol, NEN) was used at a final concentration of 0.5 nM, instead of iodinated ponasterone A. Also, instead of filtration through glass fiber discs, dextrancoated charcoal was used to separate and remove unbound ligand. Kc0 cells obtained from Dr T. Benyajati (University of Rochester, NY) were used for cell differentiation experiments. Cells were maintained in D22 cell culture medium (Sigma). On day 0, 1 x 10h cells were placed in 6-well culture dishes in 2 ml of medium. Compounds were added in either 5 ~1 ethanol or 5 pl 50% ethanol: 50% dimethyl sulfoxide, and the cells were incubated at 25°C. Total cell number and the number of cells with ecdysone induced processes were determined 24 h later. RESULTS AND DISCUSSION In an effort to identify natural nonsteroidal ecdysteroid agonists we have screened extracts from a variety of plant sources. Induction of B-galactosidase enzyme activity in a transactivation assay was used to identify ecdysteroid agonists and monitor isolation. The genus Ajuga (Lamiaceae) is a well known producer of phytoecdysteroids and potential insect antifeedants of the clerodane diterpene structural class. These compounds have recently been reviewed (Camps and Coll, 1993). While not recognized as potential insecticides, a number of iridoid glycosides have also been isolated from this genus including 8-0-acetylharpagide (Assaad and Lahloub, 1988; Takeda et al., 1987; Shimomura et al., 1987, inter alia) which was isolated in the present study. Thus, the isolation of 8-0-acetylharpagide from A. reptans was not surprising. The bicyclic iridoid skeleton is usually elaborated with various arrays of functional groups, often including glycosides and esters, resulting in a diverse group of structurally related compounds. The extent of this diversity can best be appreciated by noting that 569 new iridoids were described in the scientific literature from 1980 through 1989 (for recent reviews, see Boros and Sterm-

S-O-ACETYLHARPAGIDE

itz, 1990, 1991). Iridoids are widely distributed in plants belonging to portions of the Class Magnoliopsida (dicotyledons) especially in the Subclass Asteridae. The structural diversity of these compounds suggests that their study may be a fertile area for the discovery of other nonsteroidal ecdysteroid agonists, with potential for use as insecticides. Although S-0-acetylharpagide does not exhibit the tetracyclic configuration of steroids, it behaves as an ecdysteroid agonist (Fig. 1). In the transactivation assay both 20-hydroxyecdysone and 8-0-acetylharpagide induce P-galactosidase enzyme activity to the same extent (Fig. 2). Background in this assay is low and the maximum fold induction of approximately 400 provides for a very broad sensitivity range. Although induction for both compounds was maximal, there was a considerable difference in the E(& values with 8-0-acetylharpagide being approximately lOOO-fold less potent than 20-hydroxyecdysone. The EC,, obtained for 20-hydroxyecdysone in the transactivation assay is within a factor of two to values obtained by others in cell-based assays (Wing, 1988). The potency of 8-0-acetylharpagide also compares favourably with the nonsteroidal ecdysteroid agonist 1, 2-dibenzoyl- 1-tert-butylhydrazine, 22 PM (Fig. 2) vs 4.8 PM (Wing, 1988), respectively.

A OH

HO HO

20-hydroxyecdysone

B

HO

OH

8-acetylharpagide FIGURE 1. Structures of 20-hydroxyecdysone and S-O-acetylharpagide. The molecular structures 20.hydroxyecdysone and S-O-acetylharpagide are shown in panels A and B, respectively. The structure for S-0-acetylharpagide was identified by spectroscopic means.

521

100

IO’

102

103

Concentration

104

105

106

(nM)

FIGURE 2. Dose-response curves for 20-hydroxyecdysone and S-Oacetylharpagide. P-Galactosidase enzyme activity in cMK7 cells was determined as described in the Materials and Methods section. Enzyme activity was measured in fluorescence units and is shown as a percentage of maximum 20-hydroxyecdysone induced activity. The maximum fold induction over background for 20-hydroxyecdysone was approximately 400. Values for 20-hydroxyecdysone (open circles) and for S0-acetylharpagide (solid circles) are expressed as the mean+/-standard deviation with n = 3. The EC,,s for 20-hydroxyecdysone and 8-O-acetylharpagide are 35 nM and 22 PM, respectively.

Although the transactivation assay is a very specific assay, if does not prove an interaction at the level of the ecdysteroid receptor. Radiolabelled ponasterone A has been used to characterize ecdysteroid receptors from different arthropod species (Cherbas et al., 1988; Mao and Kaufman, 1994). The relative ability of 20-hydroxyecdysone and 8-0-acetylharpagide to displace ponasterone A in cytosol extracts of Drosophila cells is shown in Fig. 3. E&s of approximately 70 nM and 100 PM were obtained for 20-hydroxyecdysone and 8-O-acetylharpagide respectively. Again, the value for 20-hydroxyecdysone agrees well with one published by Wing (1988) using Drosophila Kc cell extracts. At the highest concentration tested (100 FM) there was no competition with labelled testosterone for the human androgen receptor (Dr J. Toney, personal communication). This indicates the specificity of 8-0-acetylharpagide for the ecdysteroid receptor when compared with another steroid receptor, although the interaction is weak relative to 20-hydroxyecdysone and requires a concentration of approximately 100 /.LMto displace 50% of the ponasterone A. However, these values are consistent with the results obtained for 20-hydroxyecdysone and 8-0-acetylharpagide in the transactivation assay. It has been shown that Drosophila Kc cells respond to treatment with ecdysone by extending long processes and subsequently aggregating (Courgeon, 1972; Cherbas et al., 1980). This response has been used to characterize the nonsteroidal ecdysteroid agonist 1, 2-dibenzoyl- lterr-butylhydrazine (Wing, 1988). Drosophila Kc0 cells treated with the ethanol vehicle alone are not signifi-

522

ALEX ELBRECHT

‘*OL

0-l

10-3

# 10-Z

....

,...1

lO_’

100

IO’

Concentration

102

103

104

105

106

(nM)

FIGURE 3. Displacement of “H-ponasterone A with 20-hydroxyecdysane and 80-acetylharpagide. Different concentrations of 20-hydroxyecdysone (open circles) or 80acetylharpagide (solid circles) were used to displace Wponasterone A at a final concentration of 0.5 nM. Binding activity was determined in cMK9 cell extracts as described in the Materials and Methods section. Ponasterone A binding activity is expressed as a percentage of maximum. The values for maximum amount of ‘H-ponasterone A binding and nonspecific binding are approximately 4000 dpm and 600 dpm, respectively. Nonspecific binding was determined in the presence of 10 OOO-fold molar excess of 20hydroxyecdysone. The results are expressed as the mean+/-of duplicate determinations. The E&s for 20.hydroxyecdysone and 8-O-acetylharpagide are 70 nM and 100 PM, respectively.

cantly different from untreated cells (Fig. 4, Panel A). These cells have a round morphology with very few elaborating processes. Treatment for 24 h with 0.5 PM 20hydroxyecdysone (Fig. 4, Panel B) causes the cells to flatten and to develop the long processes described by others (Courgeon, 1972; Cherbas et al., 1980). This morphology is also exhibited by cells treated with 50 PM 80-acetlyharpagide, as would be expected for an ecdys-

et al.

teroid agonist, and demonstrates that 8-O-acetylharpagide can regulate the different ecdysteroid responsive genes involved in cell differentiation. Quantitation of this effect is shown in Table 1. We have shown that a natural constituent of A. reptuns L. acts as an ecdysteroid agonist. It activates transcription of an ecdysteroid inducible promoter in a transactivation assay, it competes with the ecdysteroid ponasterone A for binding to Drosophila ecdysteroid receptor preparations, and it causes differentiation of ecdysteroid responsive cells in culture. This class of compounds is common to many different plant species and since it has been shown that other ecdysteroid agonists function as insect growth regulators, it may serve as a natural feeding deterrent for phytophagous insects. Interestingly, 8-0acetylharpagide has insect antifeedant properties (Kubo, 1993), although we do not know if the antifeedant effects result from ecdysteroid receptor mediated actions. We are currently determining the insecticidal activity of 80-acetylharpagide and related compounds. TABLE

1. Quantitation

of Kc cell differentiation ylharpagide

Treatment

Percentage Experiment

Untreated 0.25% ethanol 0.5 PM 20.hydroxyecdysone 50 PM 8-0-acetylharpagide

2 0 39 13

induced

by 8-O-acet-

of Cells Differentiated

I

Experiment

2

3 4 47 27

The results were obtained 24 h after treatment and represent the average of duplicate measurements from two independent experiments. All cells with processes were considered as differentiated regardless of the length of the processes. Although measurements were not taken, processes were included when, by microscopic examination, the length was equal to or greater than the diameter of the cell

FIGURE 4. Differentiation of Kc cells induced by 20.hydroxyecdysone or 8-0-acetylharpagide. Kc0 cells were cultured for 24 h in 6-well dishes (Falcon) and photographed under phase contrast of an inverted microscope at 320 x magnification. Panel A: cells cultured in the presence of 0.25% ethanol (vehicle). Panel B: cells cultured in the presence of 0.5 PM 20-hydroxyecdysone. Panel C: cells cultured in the presence of 50 PM 8-0-acetylharpagide.

8-O-ACETYLHARPAGIDE

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would like to thank L. Van der Ploeg for discussions and for reading the manuscript.

Acknowledgements-AE