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Insect Biochemistry and Molecular Biology 37 (2007) 865–875

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A photoaffinity, non-steroidal, ecdysone agonist, bisacylhydrazinecompound, RH-131039: Characterization of binding and

functional activity

Tarlochan S. Dhadiallaa,�, Dat Leb, Subha R. Pallic, Alexander Raikheld, Glenn R. Carlsone

aDow AgroSciences, LLC, Indianapolis, IN 46268, USAbPolySciences, Warrington, PA 18976, USA

cDepartment of Entomology, University of Kentucky, Lexington, KY 40546, USAdDepartment of Entomology, University of California Riverside, CA 92521, USA

e305 Britt Road, North Wales, PA 19454, USA

Received 5 March 2007; received in revised form 15 May 2007; accepted 16 May 2007

Abstract

In this paper we describe the synthesis, ligand-binding and functional activity characteristics of the photoaffinity, non-steroidal,

ecdysone agonist, bisacylhydrazine compound, 3-benzoyl-benzoic acid N-tert-butyl-N0-(2-ethyl-3-methoxy-benzoyl)-hydrazide (RH-131039).

Tritiated RH-131039 is the first non-steroidal photoaffinity compound that was shown to bind specifically to ecdysone receptors (EcRs)

from insects belonging to the orders Diptera and Lepidoptera. The spruce budworm (Choristoneura fumiferana) ecdysone receptor

(CfEcR) bound with high affinity (Kd ¼ 2.2370.27 nM) to this compound. When irradiated with UV light (l ¼ 350 nm) under

equilibrium ligand-binding conditions, RH-131039 attached specifically and covalently to the CfEcR ligand-binding domain (LBD).

RH-131039 also bound to cloned ecdysone receptor proteins from three dipteran insects, Drosophila melanogaster, Aedes aegypti

and Chironomous tentans. This paper also describes and invokes caution in interpretation of ligand-binding results obtained

using crude cellular extracts containing target receptors, as illustrated with the use of Drosophila Kc cells that have functional EcR

and L57 cells (derivatives of Kc cells in which EcR-B isoforms have been knocked out by ‘‘parahomologous’’ recombination). Tritiated

RH-131039 is a useful tool to dissect ligand-binding and functional differences for EcRs from different arthropod species.

r 2007 Elsevier Ltd. All rights reserved.

Keywords: Photoaffinity labeling; Limited proteolysis; Ecdysteroid receptor; Non-steroidal ecdysone agonist; Photoaffinity ecdysone agonist; Dipteran

EcR; Lepidopteran EcR

1. Introduction

With the discovery of the first non-steroidal ecdysoneagonist, unsubstituted bisacylhydrazine (RH-5849) andcloning of the ecdysone receptor (EcR) from Drosophila

melanogaster (Koelle et al., 1991) and many otherarthropod species (Henrich, 2005; Palli et al., 2005),significant research interest was generated in the use ofthe non-steroidal ecdysone agonists in understanding theirbiological effects in vivo and in vitro (Oberlander et al.,1995; Dhadialla et al., 1998, 2005; Dhadialla and Ross, 2007)

e front matter r 2007 Elsevier Ltd. All rights reserved.

mb.2007.05.009

ing author. Tel.: +1317 337 3755.

ess: tdhadialla@dow.com (T.S. Dhadialla).

Since the discovery of the first insect active bisacylhydra-zine, RH-5849 (Wing, 1988), four bisacylhydrazine com-pounds (tebufenozide, methoxyfenozide, halofenozide, andchromafenazide) have been commercialized (Hsu, 1991;Heller et al., 1992; RohMid, 1996; Yanagi et al., 2000;Carlson et al., 2001).During insect development, growth and morphogenesis

are regulated by the steroid hormone 20-hydroxyecdysone(20E) and the sesquiterpene juvenile hormones. Amongstthe insects studied, the activity of 20E is manifested viainteraction with nuclear receptor heterodimers consistingof the EcR (Koelle et al., 1991) and ultraspiracle proteins(USP, Oro et al., 1992; Henrich et al., 1990), whichrecognize specific ecdysone receptor response elements

ARTICLE IN PRESST.S. Dhadialla et al. / Insect Biochemistry and Molecular Biology 37 (2007) 865–875866

(EcRE) present in the promoter regions of 20E regulatedgenes (Yao et al., 1992; Yao et al., 1993). It is this stableinteraction of the quaternary complex (20E, EcR/USPand EcRE) which brings about the transcription of20E-responsive genes. Although all the data from ligand-binding experiments indicated that 20E binds to aminoacid residues in the EcR ligand-binding domain (LBD)(Koelle et al., 1991; Yao et al., 1992, 1993; Dhadialla et al.,1998, 2006; Hu, 1998), the visual proof for this came fromcrystal structures of liganded and unliganded EcR LBDsfrom cotton bollworm, Heliothis virescens, and the sweetpotato whitefly, Bemesia tabacci (Billas et al., 2003;Carmichael et al., 2005). While in both studies, the potentphytoecdysteroid, ponasterone A was used as the steroidalligand, Billas et al. (2003) also co-crystallized theH. virescens EcR (HvEcR) LBD with a non-steroidal bisa-cylhydrazine compound. The results of this study con-firmed the earlier hypothesis (Kumar et al., 2002, 2004)that 20E and active bisacylhydrazines occupy different butoverlapping spaces in the ligand-binding pocket of EcRs.

Before the EcR crystal structures were published, therewas a great deal of interest in understanding the 3Dinteraction of 20E or ponasterone A with EcR LBDs. Thisinterest was further escalated after the molecular basis ofthe action of the non-steroidal bisacylhydrazine insecticidalcompounds was understood (reviewed by Dhadialla et al.,1998, 2005; Dhadialla and Ross, 2007). Due to the novelmode of action of these insecticidal compounds, and inparticular, the selective toxicity of tebufenozide, methox-yfenozide, and chromafenozide to predominantly lepidop-teran insects, it was felt that by understanding theinteraction of these compounds with specific amino acidresidues in EcR LBDs, it would be possible to design anddiscover new ecdysone agonists using molecular modelingapproaches for activity against pests from other insectorders such as Homoptera, Hemiptera, Orthoptera etc.Different labs adopted different approaches that includedhomology modeling of EcR LBDs based on crystal stru-ctures of vertebrate steroid receptors and ligand docking(Kumar et al., 2002, 2004) and using photoaffinity radio-labeled bisacylhydrazines (briefly discussed in Dhadiallaet al., 2005) as well as ecdysteroids (Bourne et al., 2002) Wepursued both these approaches. Here, we describe thesynthesis of a photoaffinity radiolabeled bisacylhydrazineand characterization of its ligand-binding properties toseveral EcRs.

2. Materials and methods

Chemical synthesis and radio-labeling of the RH-131039is described in the on-line supplementary material.

2.1. EcR and USP proteins

EcR protein from the spruce budworm, Choristoneura

fumiferana (CfEcR) lacking the N-terminal A/B transacti-vation domain and full-length ultraspiracle protein

(CfUSP) were expressed as GST fusions in the expressionvector, pGEX-3X (Pharmacia, Bai d’Urfe, Quebec, Canada,Palli S.R., unpublished) for use in ligand-binding studiesand photoaffinity labeling experiments. Truncated CfEcRproteins were produced as described previously (Perera etal., 1999a, b). D. melanogaster EcR and USP (DmEcR andDmUSP) proteins were produced in E. coli as His-Tagfusions from constructs provided by Drs Peter and LucyCherbas, Indiana University, IN. EcR and USP from theyellow fever mosquito, Aedes aegypti (AaEcR andAaUSP), were produced by in vitro transcription andtranslation of cloned AaEcR and AaUSP as described(Kapitskaya et al., 1996). Chironomus tentans EcR andUSP (CtEcR, CtUSP) bacterial GST-fusion proteins wereproduced from cloned DNA encoding the two proteins,kindly provided by Professors Klaus and MargaretSpindler, University of Ulm, Germany.

2.2. Radiolabelling of EcR proteins and limited proteolysis

experiments

Ligand-induced conformational changes in EcR wereinvestigated using an ecdysteroid, muristerone A, and non-steroidal ecdysone agonists, RH-5992 and RH-131039, and35S-labelled DmEcR. DmEcR-GST fusion proteins weremetabolically labeled by including 35S-methionine (specificactivity 41000Ci/mmol; Perkin-Elmer, Shelton, CT) inin vitro transcription-and-translation reaction mixture(Promega, Madison, WI) using DmEcR-GST LBD cDNAcloned in a pGEM vector following manufacturer’sinstructions. From the resulting translation mixture,unincorporated 35S-methionine was removed by centrifu-ging the reaction mixture in a micro-centricon tube(MWCO 10,000). In each experiment, bacterial fusionproteins of 35S-labelled DmEcR and unlabeled DmUSP(1:10 ratio v/v) were incubated in the absence or presenceof muristerone A, RH-5992 or RH-131039. Followingequilibrium ligand–receptor binding, the reaction mixturewas subjected to limited proteolysis with increasingamounts of proteinase K at room temperature. Proteolysiswas stopped after 20min by adding 2� SDS-PAGEsample buffer to the reaction mixture followed by heatingthe sample to 95 1C for 5min. The samples were analyzedusing SDS-PAGE and fluorography.

2.3. Radiometric ligand-binding assays

Ligand-binding assays using tritiated ponasterone Awere performed as described earlier (Kapitskaya et al.,1996; Pereira et al., 1999a, b). Briefly, 1 ml each of bacterialextracts containing EcR or USP (tagged or not) were mixedin a final reaction volume of 100 ml buffer (10mMTris–HCl, pH 7.2, 1mM DTT) and 2.39� 10�9M [3H]ponasterone A (specific activity of 170Ci/mmol; NEN,Boston, MA, now Perkin-Elmer, Shelton, CT), andincubated for 2 h at 4 1C in the absence (total binding) orpresence of either 20E (10�4M; non-specific binding) or

ARTICLE IN PRESST.S. Dhadialla et al. / Insect Biochemistry and Molecular Biology 37 (2007) 865–875 867

different concentrations of competitor ligands. Specificbinding of tritiated ponasterone A was determined bysubtracting non-specific binding from total binding. TheIC50 values (concentration of competitor that gives 50%displacement of specific binding of tritiated ponasterone A)were calculated from competitive dose-response curvesgenerated for each of the compounds tested. Each reactionwas done in triplicate.

The same procedure as for tritiated ponasterone Abinding was followed in experiments that required the useof tritiated RH-131039 (tritiated PAL; specific activity of84.5Ci/mmol). For radioligand-binding assays, a total of40,000 dpm (0.22 pmol) per reaction vial were used. Non-specific binding was determined using 10�5M non-radio-active RH-131039 or RH-5992, or 10�4M 20E. Bothtritiated ponasterone A and PAL were routinely purified insmall batches by reverse phase-HPLC (RP-HPLC).

2.4. Cell lines

Drosophila Kc167/M3 and L57 cell lines were obtainedfrom Drs. Peter and Lucy Cherbas, Indiana University, IN,and maintained in culture as described (Cherbas et al.,1989). L57 cells are Kc167/M3 cells that have been madedeficient in functional copy of EcR by ‘‘parahomologous’’gene targeting (Cherbas and Cherbas, 1997). L57 cells haveproven to be very useful for transfection with DNAsencoding EcRs from different insects to investigate theirresponsiveness to non-steroidal ecdysone agonists (Kumaret al., 2002). A cell line, IAL-PID2, derived from theimaginal discs of the Indian meal moth, Plodia interpunc-

tella, was obtained from Dr. Herbert Oberlander, USDA-ARS, Gainsville, FL, and the cells maintained in culture asdescribed earlier (Lynn et al., 1982).

2.5. Cell transfection, reporter gene activation, and cytosolic

extracts

L57 cells, grown in CCM3 medium (Hyclone) supple-mented with 10% FBS (Invitrogen), were transfected withCfEcR (CfEcR CDEF domains were cloned under thecontrol of baculovirus, Autographa californica multicapsidnucleopolyhedrosis virus IE1 promoter) and reporter(b-galactosidase reporter placed under 6X EcRE fromhsp27 gene and a minimal promoter as previouslydescribed by Koelle et al. (1991). For transfection,100,000 cells in 0.5ml of medium were seeded into eachwell of the 24-well plates. On the following day, the cellswere transfected with Lipofectamine 2000 Reagent (Invi-trogen) for 4 h. The transfected cells were cultured in themedium containing compounds. The reporter activity wasmeasured at 48 h after adding ligands using TROPIXchemiluminescent b-galactosidase assay kit. Cytosolicextracts of the two cell lines for ligand-binding studieswere prepared as described previously (Cherbas et al.,1988; Wing, 1988).

2.6. Photoaffinity labeling with 3H-RH-131039

Equilibrium binding reactions were set up using 3H-RH-131039 and CfEcR/CfUSP proteins in the absence orpresence of 10�4M cold RH-131039, RH-5992, and 20Eas non-steroidal and steroidal competitors. The differencein these reactions and regular ligand-binding reactionswas that 11.4� 106 dpm of RH-131039 were used ineach photoaffinity labeling reaction mix of 100 ml finalvolume. For each reaction mixture type (i.e. in absence orpresence of a competitor) 10� 100 ml reaction mixtureswere usually prepared. After equilibrium ligand-bindingconditions were achieved (90min at 4 1C), the 10 reactionssolutions of a kind were mixed. Fifty micro-liters fromeach such pooled reaction mixture type was processed toseparate bound from unbound radioactivity and determinespecifically bound radioactivity. The remainder of thereaction mixture (950 ml) was transferred into a UVtransparent glass tube and the contents irradiatedwith equidistance-fixed eight surrounding UV lights(l ¼ 350 nm) at 4 1C for 20min. The irradiated reactionmixes were then passed through Centricon (Millipore,Billerica, MA) micro-concentrators (MWCO 10,000) toseparate photoaffinity-labeled proteins from unboundtritiated PAL, as well as to concentrate the reactionmixtures down to �300 ml volumes. To each of theconcentrated reaction mix in the centricon was added2� SDS-PAGE sample buffer. The mixtures in the micro-concentrators were centrifuged to reduce the retentatevolume to �50 ml, which was collected in 1.5ml micro-centrifuge tubes. The volume was brought to 100 mlwith 1� SDS-PAGE buffer. The mixtures were heated at95 1C for 5min, and centrifuged at 12,000g for 1min. Fivemicro-liters of the supernatant was used to measureradioactivity. Thirty micro-liters of each sample wasapplied onto 8–16% Tris–glycine, SDS-PAGE pre-castgels for electrophoretic separation. The gels were stainedwith Coomassie brilliant blue solution and destainedto visualize proteins bands, after which the gels weresoaked in EnhanceTM (DuPont, DE) to enhance the signal.The gels were dried and exposed to X-ray films for 2–4 daysfor visualization of the peptide(s) covalently linked totritiated PAL.

3. Results

3.1. Synthesis and selection of photoaffinity ligand

Of several benzophenone and azido derivatives ofRH-2485, RH-131039 (Fig. 1) was selected based on itsaffinity for lepidopteran and dipteran EcRs and ease of itstritiation. The methyl position in the methoxy group wassubstituted with tritiated methyl at NEN laboratories(Waltham, Massachusetts). The synthesis route for theprecursor for tritiation is shown in supplementary Fig. 1and described in supplementary Materials and Methodssection available on line.

ARTICLE IN PRESST.S. Dhadialla et al. / Insect Biochemistry and Molecular Biology 37 (2007) 865–875868

3.2. RH-131039 induces EcRE-regulated reporter gene

As shown in Fig. 2, both 20E and RH-131039 causeddose-dependent induction of reporter gene activity in cellstransfected with DmEcR or CfEcR. The cells transfectedwith reporter construct alone did not show induction ofreporter gene activity after ligand administration. BothDmEcR and CfEcR transfected cells showed two orders ofmagnitude greater sensitivity to 20E than to RH-131039.These data show that photoaffinity-labeled RH-131109 isan active ligand capable of transactivating genes afterbinding to EcR.

30

40

50

3H-Pon. A

3H-RH-131039

PM

(10

3)

BO

UN

D

3.3. Ligand-binding characteristics of RH-131039

Cytosolic extracts, containing EcR complexes, from bothKc and L57 cells were used for binding of tritiatedponasterone A or PAL. Both tritiated ponasterone A andtritiated PAL bound to the Kc cell extracts (Fig. 3).However, as shown in Fig. 3, for the same amount ofradioactivity for each ligand and cellular extract proteinsused, there was �20 fold higher amount of tritiated PALbound than tritiated ponasterone A, a natural ligand forEcR complexes. Interestingly, when His-tagged DmEcRLBD bacterial fusion proteins were added to Kc cellextracts to assess effects on ligand binding, extremely highand significant amount of specific binding was obtained for

NH

N O

O

O

O

RH-131039

Fig. 1. Structure of RH-131039. The methyl of the methoxy group on the

benzoyl ring was tritiated to convert RH-131039 into a radioligand.

10-4 10-3 10-2 10-1 100 101

0

25

50

75

100

125

150

20E

RH-131039

Reporter

FO

LD

IN

DU

CT

ION

LIGAND CONCENTRATION (µM)

Fig. 2. Transactivation of luciferase reporter gene by 20E and RH-131039 in L5

(DmEcR) or (B) Choristoneura fumiferana (CfEcR). Each point is a mean7S

tritiated ponasterone A, and only a moderate increase inthe amount of specifically bound tritiated PAL, suggestingthat specific ponasterone A binding in the Kc extracts maybe limited by the amount of EcR present.We then investigated the relative ability of steroidal

(ponasterone A and 20E) and non-steroidal bisacylhydra-zine ecdysone agonists (RH-5992 and RH-131039) forreciprocal competition for binding to Kc cellular extractusing tritiated ponasterone A or tritiated RH-131039as radioligands. Binding of tritiated ponasterone A toDmEcR/DmUSP in Kc cellular extracts could be competedto non-specific binding levels with excess of both steroidaland non-steroidal ecdysone compounds, the reciprocalwas not true when tritiated RH-131039 was used as aradioligand (results not shown). In other words, binding oftritiated RH-131039 could not be competed with excessunlabeled ecdysteroids, 20E and ponasterone A.In order to explore this further, we made use of L57 cell

extracts for testing binding of both tritiated ponasterone A

10-5 10-4 10-3 10-2 10-1

0

10

20

30

40

50

60

20E

RH-131039

LIGAND CONCENTRATION (µM)

7 cells transfected with either EcR from either (A) Drosophila melanogaster

EM of three replicated experiments.

- +DmEcR - +DmEcR

0

10

20

Kc Cell Extract

SP

EC

IFIC

D

L-57 Cell Extract

Fig. 3. Binding of tritiated ponasterone A or tritiated RH-131039 to

cytosolic extracts of Drosophila Kc and L57 cells in the absence (�) or

presence (+) of DmEcR-His-tagged bacterially produced fusion protein.

For each radioligand 40,000 cpm were included in equal volumes of

binding reaction mixtures containing the same concentration of cellular

extract7bacterially expressed DmEcR-LBD proteins. Each bar is the

mean7SEM of three replicates.

ARTICLE IN PRESS

AaEcR/AaUSP

0

5

10

15

20

3H-Pon A

3H-RH-131039

SP

EC

IFIC

CP

M (

10

3)

BO

UN

D

DmEcR/DmUSP

Fig. 5. Tritiated ponasterone A and tritiated RH-131039 binding to

bacterial fusion proteins of DmEcR/DmUSP (His tagged)—LBDs and

full-length AaEcR/AaUSP. The amounts of radioactivity for each ligand

were kept constant at 40, 000 cpm in the reaction mixtures containing

equal amounts of bacterial extracts containing the EcR/USP proteins.

Each bar represents the mean7SEM of three replicated results.

T.S. Dhadialla et al. / Insect Biochemistry and Molecular Biology 37 (2007) 865–875 869

and RH-131039. As would be expected, extracts of L57cells, which have been made deficient of EcR isoforms byparahomologous recombination, did not show specificponasterone A binding. Surprisingly, significant amountsof specifically bound tritiated PAL was observed (Fig. 3).Like for experiments with Kc cell extracts, when we addedHis-tagged bacterial fusion protein of DmEcR LBD to L57extracts, high amounts of specific tritiated ponasterone Abinding was obtained as one would expect with formationof EcR/USP heterodimers. Unlike for Kc cell extracts,binding of tritiated PAL to L57 cell extracts under thesame conditions produced reduced specific binding. Therelevance of the fluctuations in tritiated PAL binding toproteins in Kc or L57 cell extracts following addition ofexogenous heterologously produced DmEcR is not wellunderstood. It is, however, clear that in both cases there isenough USP protein to heterodimerise with exogenouslyadded DmEcR protein based on ponasterone A bindingdata. The results with tritiated PAL for binding to L57extracts suggest that tritiated PAL may be binding to lowlevel of DmEcR-A isoforms present in the L57 cells

3.4. Limited proteolysis fragment protection assay

To determine the conformation of EcR proteins uponbinding to ligands, we performed limited proteolysisfragment protection assay. As shown in Fig. 4A, murister-one A induced a conformational change in EcR to producea 36 kDa peptide fragment resistant to further proteolyticdigestion. RH-5992 at the tested concentration (1 mM)did not induce a similar conformational change in EcR.On the contrary to RH-5992, 1 mM RH-131039 induceda conformational change in EcR similar to that inducedby muristerone A represented by a 36 kDa proteinaseK-resistant peptide fragment (Fig. 4B).

Fig. 4. Fluorographs of electrophoresis gels on which 35S-methionine-labeled D

or presence of unlabeled DmUSP and absence or presence of 1 mM muristero

reach equilibrium ligand-binding conditions. The reaction mixtures were then s

of proteinase K (lanes a–e; same concentration of enzyme was used for same am

without any proteinase K treatment, but incubated under similar time and tem

mix subjected to elctrophoretic separation under denaturing conditions by SDS

A was able to afford proteolytic protection to DmEcR peptide fragment of abo

DmEcR for limited proteinase K protection. In a similar experiment (B), both m

peptides following limited proteolysis with proteinase K of the indicated react

Binding characteristics of tritiated ponasterone Aand tritiated PAL to EcR/USP bacterial fusion proteinsfrom dipteran insects, D. melanogaster, A. aegypti, andC. tentans): we tested the relative binding of equal amountsof tritiated ponasterone A and tritiated PAL to bacteriallyproduced fusion proteins of full-length AaEcR/AaUSPand LBD of DmEcR/DmUSP. As shown in Fig. 5, tritiatedponasterone A bound well to DmEcR/DmUSP LBDsand AaEcR/AaUSP. Tritiated PAL also bound specificallyto AaEcR/AaUSP. However, we were unable to detectspecific binding of tritiated PAL to DmEcR/DmUSP LBDfusion proteins.To determine if the USP protein present in AaEcR and

DmEcR heterodimer complex makes a difference in the

mEcR-His-tagged LBD proteins (solid arrows at an angle) in the absence

ne A or RH-5992 (A) or 1 mM unlabeled RH-131039 (B) were allowed to

ubjected to limited proteolysis in the presence of increasing concentrations

ount of sample in lanes labeled with similar letters; lane a is control sample

perature conditions for samples in other lanes) and the resulting reaction

-PAGE. Comparison of lane d in (A) demonstrates that only muristerone

ut 36 kDa. RH-5992 was ineffective in providing a conformation change in

uristerone A and RH-131039 (1mM; lanes d and e) resulted in 36 kDa size

ion mixtures (dotted arrows).

ARTICLE IN PRESST.S. Dhadialla et al. / Insect Biochemistry and Molecular Biology 37 (2007) 865–875870

binding of tritiated PAL, we performed EcR and USP mix-n-match experiments. As shown in Fig. 6, while tritiatedPAL bound to AaEcR, we could not detect its binding toDmEcR irrespective of the use of either AaUSP orDmUSP. Interestingly, the choice of DmUSP with eitherof the EcR proteins resulted in significantly reducedbinding of tritiated ponasterone A. Similarly, use ofDmUSP as a partner for AaEcR resulted in lower bindingto PAL when compared to the binding achieved by AaEcRand AaUSP heterodimer.

We also investigated the relative binding of tritiatedponasterone A to GST fusion proteins of C. tentans EcRand USP (CtEcR/CtUSP) in the absence or presence of20E, RH-5992, or RH-131039. As shown in Fig. 7A, thebinding of radiolabeled ponasterone A could be competedto similar non-specific binding levels with 10�4M 20E, aswell as 10�5M RH-5992. However, the presence of 10�4

and 10�5M RH-131039 only partially (�44% and 22%,respectively) reduced binding of tritiated ponasterone A. Inreciprocal experiments (Fig. 7B), where tritiated PAL wasused as a radioligand, a relatively insignificant amount oftritiated PAL bound to CtEcR/CtUSP in the absence ofa competitor. This binding was �18 fold less comparedto binding of tritiated ponasterone A. Neither 10�4 nor10�5M 20E or RH-5992, respectively, competed for thislow level of tritiated PAL binding. Slightly reduced, butnot significantly different from total, binding of tritiatedPAL was obtained in the presence of excess unlabeledPAL. From these results, it appears that tritiated PALbinds predominantly in a non-specific manner to CtEcR/CtUSP.

AaEcR/AaUSP AaEcR/DmUSP

Specific

cpm

(10

3)

Bound

15.0

12.5

10.0

7.5

5.0

2.5

0.0

EcR / USP

Fig. 6. In order to test the effect of DmEcR or AaEcR heterodimerizing wit

mixtures were prepared to contain bacterially produced proteins as indicated on

(40,000 cpm per reaction mixture) were incubated with receptor proteins und

unlabeled 20E (in the case of ponasterone A as the radioligand) or 10�4M un

bound radioligand7SEM of three replicates.

3.5. Binding characteristics of tritiated PAL to full-length

and truncated CfEcR

In several previous publications, the binding of tritiatedponasterone A to CfEcR/CfUSP-GST fusion proteins hasbeen shown to be specific and saturable (Kothapalli et al.,1995; Perera et al., 1999a, b). To determine if the binding oftritiated PAL to CfEcR/CfUSP-GST fusion proteins is alsospecific and saturable, we carried out experiments togenerate saturation isotherms. Under equilibrium bindingconditions for tritiated PAL to these proteins, the bindingwas found to be specific and saturable (Fig. 8). TritiatedPAL bound CfEcR/CfUSP with high affinity (Kd ¼

2.2.370.027 nM as calculated by non-linear regressionanalysis of the data).

3.6. Demonstration of photo-activated covalent attachment

of RH-131039 to CfEcR(CDEF)/CfUSP

To visualize covalent binding of tritiated PAL to CfEcR,we carried out equilibrium binding reactions using CfEcR/CfUSP proteins and tritiated RH-131039 in the absence orpresence of excess amounts of unlabeled PAL, RH-5992,and 20E. The reaction mixture was then photo-reacted atl350 nm at 4 1C for 20min. Previous experiments had shownthat under these conditions the integrity of EcR proteinsremains intact (data not shown). After exposure to UVlight, the proteins covalently bound to tritiated RH-131039were visualized by SDS-PAGE followed by fluorographyof the dried gel. The results presented in Fig. 9, clearlyshowed that tritiated RH-131039, in the absence of a

DmEcR/AaUSP DmEcR/DmUSP

3H-Pon A

3H-RH-131039

Combinations

h homologous or heterologous USP proteins of the two species, reaction

the X-axis of the bar diagram. Equal amounts of the two tritiated ligands

er equilibrium binding conditions in the absence or presence of 10�4M

labeled RH-131039. Each bar represents the mean amount of specifically

ARTICLE IN PRESS

0 1 2 3 4 5 6

pm

ole

3H

-RH

-131039 B

OU

ND

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RE

E

Non-specific

Specific

pmole 3H-RH-131039

0.10

0.05

0.00

R2 = 0.6948

0.5

0.4

0.3

0.2

0.1

0.0

0.120.100.080.060.040.020.00

Fig. 8. Saturation isotherm for the binding of tritiated RH-131039 to CfEcR/CfUSP-GST fusion proteins. The binding reactions were carried out as

described under Materials and Methods. The receptor proteins were incubated with increasing concentrations of tritiated RH-131039 in the absence or

presence of 10�4M unlabeled RH-131039 and the binding reactions allowed to proceed under equilibrium binding conditions determined earlier. Specific

binding of tritiated RH-131039 was determined by subtracting non-specific binding in the presence of cold competitor from total binding (no competitor).

Each point in the saturation isotherms is a replicate of three binding reactions7SEM. The data were used to generate a Scatchard plot (inset), which also

gives the 95% confidence limits (dotted lines) for the data. The calculated values for the dissociation constant (Kd) for RH-131039 binding to CfEcR/

CfUSP proteins was 2.2370.27 nM. The Bmax value was 1.0770.03 with an R2¼ 0.98. GraphPad PRIZMs software was used to calculate the Kd and

Bmax values.

DP

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(10-4M)

RH-5992

(10-5M)

Cold PAL

(10-4M)

Cold PAL

(10-5M)

COMPETITORS COMPETITORS

1.8

1.6

1.2

1.0

0.8

0.6

0.4

0.2

0.0

Fig. 7. Competitive binding of tritiated ponasterone A (A) and tritiated RH-131039 (B) were tested using GST-fusion proteins of C. tentans EcR and USP

(CtEcR/CtUSP). Equal amounts of radioactivity (40,000 cpm per reaction) of each of the two radioligands and receptor protein mixtures were used for the

binding reactions. The radioligands were incubated with the receptor proteins in the absence (ethanol control) or in the presence of indicated

concentrations of unlabeled 20E, RH-5992, or RH-131039. Each bar is a mean of three replicated binding reactions7SEM.

T.S. Dhadialla et al. / Insect Biochemistry and Molecular Biology 37 (2007) 865–875 871

competitor, binds to a protein with an expected sizeof CfEcR (DEF domain)–GST fusion protein. TritiatedRH-131039 binding to this single, labeled protein bandwas competed with excess amounts of cold non-steroidal

ecdysone agonists, RH-131039, RH-5992, and steroid, 20E.In a similar experiment, covalent binding of tritiated PALcould also be shown to 50 end truncated CfEcR (withoutthe A/B 50 domain; Fig. 10). The fluorographs shown in

ARTICLE IN PRESS

215

120

84

60

39.2

28

16

CfEcR-GST

(CDEF)

O

O

N

H

N

O

O

A C

O

N

H

N

O

H

OH

O

HO

HO OH

OH

RH-5992

20E

Competitors

MWM

(kDa)B D

RH-131039

Fig. 9. Covalent attachment of tritiated RH-131039 to CfEcR (CDEF domain)/CfUSP heterodimer proteins after equilibrium binding in the absence or

presence of cold competitors (10�5M RH-131039, 10�5M RH-5992, and 10�4M 20E; structures shown at the bottom of the fluorograph) followed by

irradiation of the reaction mixtures with UV350nm as described under Materials and Methods. Following the irradiation step, contents of the binding

reactions were subjected to SDS-PAGE followed by fluorography of the gel to visualize specific binding of the radioactive RH-131039. This is a

representation of a typical result from one of several such experiments demonstrating the covalent binding of tritiated RH-131039 to CfEcR (CDEF)-GST

fusion protein.

MWM

(kDa)

250

98

64

50

36

30

16

RH-5992 10-5M - + - - - + - -

RH-131039 10-5M

Pon. A 10-5M

- + - - - + -

- - - -

CfEcR(CDEF)

CfEcR-A

-

- + - +

Fig. 10. The experiment described in Fig. 9 was repeated using either full-length CfEcR-A isoforms or truncated CfEcR containing only CDEF domains.

Both proteins were produced using Promega in vitro transcription and translation kit. The lysate containing the translated CfEcR (CDEF)/CfUSP

proteins was incubated with tritiated RH-131039 in absence and presence of excess cold competitors (RH-5992, RH-131039 or ponasterone A) and the

irradiated reaction contents subjected to SDS-PAGE as described under Fig. 9. The results clearly show covalent specific binding of tritiated RH-131030 to

both full-length and truncated forms of CfEcR proteins.

T.S. Dhadialla et al. / Insect Biochemistry and Molecular Biology 37 (2007) 865–875872

ARTICLE IN PRESST.S. Dhadialla et al. / Insect Biochemistry and Molecular Biology 37 (2007) 865–875 873

Figs. 9 and 10 clearly show the specificity of binding oftritiated PAL to CfEcR.

4. Discussion

The major contribution of this work is the synthesis andcharacterization of a benzophenone substituent for photo-active covalent attachment to EcR LBD. The use of thistritiated photoaffinity compound, RH-131039, provided avisual demonstration of its specific attachment to CfEcRand differences in its binding activity to DmEcR, AaEcR,and CtEcR. RH-131039 behaved similar to earlier well-characterized bisacylhydrazine insecticide compounds,tebufenozide (RH-5992) and methoxyfenozide (RH-2485)in its binding affinity (Kd ¼ 2.2370.27 nM; Dhadialla etal., 1998, 2005) and its ability to transactivate reportergenes in L57 cells transfected with CfEcR (Figs. 8 and 2,respectively). Tritiated RH-131039 bound specifically andcovalently (upon photo activation) to CfEcRLBD and thisbinding could be competed with excess cold RH-131039,20E, or RH-5992 before photo-activation steps. Moreover,the specific binding of both tritiated ponasterone A andRH-131039 to CfEcR/CfUSP–GST fusion proteins couldbe competed reciprocally to non-specific ligand-bindinglevels with excess unlabeled ecdysteroidal (ponasterone Aand 20E) and non-steroidal ecdysone agonist (RH-5992and RH-131039) compounds.

Besides synthesis and characterization of the photoaffi-nity stable ecdysteroidal ligand, RH-131039, this paperalso presents new data on its ligand-binding properties toother EcR/USP heterodimers. Surprising results wereobserved when the binding of RH-131039 was tested withproteins prepared from Drosophila Kc cells and L57 cells aswell as DmEcR and DmUSP fusion proteins expressed inbacteria. When relative binding of tritiated RH-131039 andponasterone A to cytosolic extracts of Kc cells was tested,significantly higher binding of RH-131039 than ponaster-one A binding was observed. When bacterially producedHis-tagged DmEcR protein was added to the cytosolicextracts, the binding of ponasterone A increased byabout 25-fold when compared to the binding with cytosolicextracts alone, and the activity of RH-131039 increasedby about 30%. These data suggest that the binding ofponasterone A to Kc cell cytosolic extracts is not limitedby the amount of DmUSP protein but by the amountof DmEcR present in these extracts. Results fromexperiments involving binding of tritiated ponasterone Aand RH-131039 to Kc cellular extracts and reciprocalcompetitive displacement of these radioligands with excessunlabeled ecdysteroidal (ponasterone A and 20E) and non-steroidal bisacylhydrazine ecdysone agonist (RH-5992 andRH-131039) compounds, suggested that RH-131039 (andperhaps RH-5992) may preferentially bind to some otherprotein at low concentrations (pM to nM range) used as aradioligand, but then shift to EcR/USP binding at highconcentrations (mM to mM range) used for competitivedisplacement.

In L57 cell extracts specific binding of ponasterone Awas not detected, as would be expected, since the cells aredeficient in EcR-B1 and -B2 isoforms. However, thesurprising result was that tritiated RH-131039 yieldedsignificant specific binding to L57 extracts. The resultsobtained from binding of tritiated RH-131039 andponasterone A to bacterially expressed LBDs of DmEcR-and DmUSP-His tagged fusion proteins demonstratedundetectable binding of RH-131039 and significant specificbinding of tritiated ponasterone A. In transactivationexperiments, L57 cells transfected with DmEcR-induced25-fold increase in reporter gene activity at 1 mM concen-tration of RH-131039 compared to about 100-fold induc-tion with 1 mM 20E (Fig. 2).In limited protease protection experiments, binding of

1 mM RH-131039 to 35S-methionine-labeled DmEcR andunlabeled DmUSP afforded a proteinase K resistantpeptide fragment of DmEcR, which was similar inmolecular weight to the one obtained using 1 mMmuristerone A (Fig. 4B). These results are similar to thoseobtained by Yao et al. (1993) in protease protectionexperiments using DmEcR/DmUSP and another bisacyl-hydrazine ecdysone agonist, RH-5849, or the ecdysteroid,muristerone A, as ligands.Taken together, these data suggest that while RH-131039

binds and interacts functionally with DmEcR, it does soonly at higher concentrations. In regular ligand-bindingexperiments, concentrations in the range of pico molartritiated RH-131039 may not have been sufficient to detectspecifically bound radioactivity to His-tagged DmEcR/DmUSP proteins. Hu (1998) tested RH-5992 (tebufeno-zide) for reporter gene transactivation experiments in L57cells transfected with DmEcR. The results in this studyshowed that in L57 cells the 100% induced state of thereporter gene with 20E could be competed down withincreasing concentrations of RH-5992, suggesting anantagonist role for RH-5992. However, continued increasein RH-5992 concentrations resulted in a maximum of 75%competition of 20E-induced reporter gene activation, afterwhich there was a sustained 25% activation of the reportergene. Based on these results the authors suggested that thenon-steroidal ecdysone agonist, RH-5992, interacts withDmEcR as an antagonist at lower concentrations andchanges to an agonist at higher concentrations. It ispossible that RH-131039 may be acting the same way whenbinding to DmEcR.Tebufenozide (RH-5992) binds with EcR/USP from the

midge, C. tentans, with extremely high-affinity (Kd ¼ �3nM;reviewed in Dhadialla et al. (2005) We expected thatRH-131039 would also bind with very high affinity toCtEcR/CtUSP proteins. On the contrary, we observed lowbinding of RH-131039, compared to that with ponasteroneA, to CtEcR/CtUSP proteins. In light of its high affinitybinding to CfEcR/CfUSP and AaEcR/AaUSP proteins,this is a surprising result. It does, however, reinforce thatslight modifications of a ligand can affect its interactionwith a given receptor, a phenomenon that is normally

ARTICLE IN PRESST.S. Dhadialla et al. / Insect Biochemistry and Molecular Biology 37 (2007) 865–875874

observed during the chemical synthesis and structure–activity relationship of chemotypes within a chemical series.

The data presented in this paper demonstrated the use ofa photoaffinity non-steroidal bisacylhydrazine compoundthat interacts in a similar manner to previously well-characterized bisacylhydrazine insecticidal compounds,tebufenozide, and methoxyfenozide, in its specific bindingto lepidopteran EcR/USP LBD proteins. For the first timevisual demonstration of covalent attachment of anecdysteroidal photoaffinity compound, RH-131039, to anEcR protein has been demonstrated. The use of thiscompound in experiments also generated data that showedcomplexities involved in using crude cellular extracts forligand-binding assays. However, with appropriate well-controlled experiments, there is utility for photoaffinityradiolabeled compounds such a RH-131039 for character-ization of their target sites.

Acknowledgments

We would like to acknowledge the technical assistance ofseveral summer interns from the University of Arizona,Tucson, AZ and Mr. Jianzhong Zhang for experimentsdone at Rohm and Haas Company, Spring House, PA.TSD acknowledges Ronald Ross for helping review thechemical synthesis of RH-131039 section, and the criticalreview of the manuscript by Drs. Kent Steele, DavidYoung and Mark Hertlein.

Appendix A. Supplementary materials

Supplementary data associated with this article can befound in the online version at doi:10.1016/j.ibmb.2007.05.009.

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