Towards Coleoptera-specific high-throughput screening systems for compounds
with ecdysone activity: development of EcR reporter assays using weevil
(Anthonomus grandis)-derived cell lines and in silico analysis of ligand
binding to A. grandis EcR ligand-binding pocket
Thomas Soina, Masatoshi Igaa, Luc Sweversb, Pierre Rouge ´c, Colin R. Janssend, Guy Smagghea,*
aLaboratory of Agrozoology, Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium
bInsect Molecular Genetics and Biotechnology, Institute of Biology, National Centre for Scientific Research ‘‘Demokritos’’, Aghia Paraskevi Attikis, Athens, Greece
cSurfaces Cellulaires et Signalisation chez les Ve ´ge ´taux, UMR Universite ´ Paul Sabatier CNRS 5546, Castanet Tolosan, France
dLaboratory of Environmental Toxicology and Aquatic Ecology, Department of Applied Ecology and Environmental Biology, Faculty of Bioscience Engineering,
Ghent University, Ghent, Belgium
a r t i c l e i n f o
Received 10 February 2009
Received in revised form
9 June 2009
Accepted 10 June 2009
a b s t r a c t
Molting in insects is regulated by ecdysteroids and juvenile hormones. Several synthetic non-steroidal
ecdysone agonists are on the market as insecticides. These ecdysone agonists are dibenzoylhydrazine
(DBH) analogue compounds that manifest their toxicity via interaction with the ecdysone receptor (EcR).
Of the four commercial available ecdysone agonists, three (tebufenozide, methoxyfenozide and chro-
mafenozide) are highly lepidopteran specific, one (halofenozide) is used to control coleopteran and
lepidopteran insects in turf and ornamentals. However, compared to the very high binding affinity of
these DBH analogues to lepidopteran EcRs, halofenozide has a low binding affinity for coleopteran EcRs.
For the discovery of ecdysone agonists that target non-lepidopteran insect groups, efficient screening
systems that are based on the activation of the EcR are needed. We report here the development and
evaluation of two coleopteran-specific reporter-based screening systems to discover and evaluate
ecdysone agonists. The screening systems are based on the cell lines BRL-AG-3A and BRL-AG-3C that are
derived from the weevil Anthonomus grandis, which can be efficiently transduced with an EcR reporter
cassette for evaluation of induction of reporter activity by ecdysone agonists. We also cloned the almost
full length coding sequence of EcR expressed in the cell line BRL-AG-3C and used it to make an initial in
silico 3D-model of its ligand-binding pocket docked with ponasterone A and tebufenozide.
? 2009 Elsevier Ltd. All rights reserved.
Molting in insects is regulated by ecdysteroids; in most insects
the active ecdysteroid is 20-hydroxyecdysone (20E). Several
synthetic non-steroidal ecdysone agonists are on the market as
insecticides (Dhadialla et al., 1998; Yanagi et al., 2006). These
dibenzoylhydrazine (DBH) compounds have been shown to mani-
susceptible insects. Like 20E, theytransactivate a succession of molt
a group of molt-related genes. As a result of the expression of these
up-regulated genes, the larva undergoes premature apolysis and
head capsule slippage and takes on the appearance of the pharate
larva. However, unlike 20E, which is cleared at this juncture,
agonists are not cleared easily. Therefore, all the down-regulated
events that occur as the titer of 20E decreases are repressed by the
presence of the ecdysone agonist. The result is that the insect
remains trapped in the molting process and dies slowly from
starvation and desiccation (Dhadialla et al., 1998).
Four DBH ecdysone agonists are currently available on the
market. Tebufenozide (RH-5992), methoxyfenozide (RH-2485) and
(Dhadialla et al.,1998; Yanagi et al., 2006). Halofenozide (RH-0345)
is used to control coleopteran (scarabid larvae) and lepidopteran
insects in turf and ornamentals (Dhadialla et al.,1998). The success
of these compounds in insect control programs validates EcR as
a valuable target for the development of environmentally friendly
biorational insecticides (Nakagawa, 2005). However, as mentioned
above, current available ecdysone agonists target mainly lepidop-
teran insects together with a limited number of coleopteran insects
* Corresponding author. Tel.: þ32 9 2646150; fax: þ32 9 2646239.
E-mail address: firstname.lastname@example.org (G. Smagghe).
Contents lists available at ScienceDirect
Insect Biochemistry and Molecular Biology
journal homepage: www.elsevier.com/locate/ibmb
0965-1748/$ – see front matter ? 2009 Elsevier Ltd. All rights reserved.
Insect Biochemistry and Molecular Biology 39 (2009) 523–534
(Dhadialla et al., 1998). For the discovery of ecdysone agonists that
target other insect groups, efficient screening systems that are
based on the activation of EcR are needed.
Important to note is that DBH non-steroidal ecdysone agonists
have an unusual high affinity for the EcR of lepidopteran insects.
Binding studies have indicated that the dissociation equilibrium
constant (Kd) of binding of DBH analogues can differ by 1–2 orders
of magnitude between coleopteran and lepidopteran insects (Ogura
et al., 2005). While this illustrates the specificity of DBH analogues
for lepidopteran insects, the mechanism by which the higher
binding affinity is achieved is unknown. Deeper insight into the
mechanism of activation of coleopteran and lepidopteran EcRs
would provide the basis for the rational design of new molting
hormone agonists with increased specificity and efficiency for
coleopteran insects. Important tools to achieve this goal include (1)
the availabilityof coleopteran-specific cell-based ecdysone reporter
assays to allow fast screening for activity of compounds and (2)
appropriate models of the EcR ligand-binding domain (LBD) to
allow in silico docking studies of candidate ligands.
In this article, we report the molecular cloning and analysis of
the EcR of an embryonic cell line (BRL-AG-3C) derived from the
cotton boll weevil Anthonomus grandis (Stiles et al., 1992b) and the
use of this cell line in two assays with an ecdysone responsive
reporter construct to screen for ecdysone agonistic activity. We
compared these two reporter assays: one where we transfected the
cells with a plasmid with the reporter construct, and the other
where we infected the cells with a recombinant baculovirus that
has incorporated the same reporter construct in its genome. Similar
assays are already reported with the use of lepidopteran (Swevers
et al., 2004) or dipteran cells (Soin et al., 2008), but to our knowl-
edge not with coleopteran cells. As is the case with binding assays,
activities of DBH analogue compounds show much lower activity in
coleopteran than in lepidopteran reporter assays (Swevers et al.,
2004; Wheelock et al., 2006). Finally, wepresent a model of the LBD
of AgEcR bound to ecdysteroid and DBH analogue. Specifically, it
was of interest to compare the binding mode of a DBH analogue to
the AgEcR ligand-binding pocket (LBP) with the interactions
observed in the lepidopteran EcR-LBP to indicate differences that
could explain the differences in activity of DBH analogues recorded
in coleopteran and lepidopteran binding and cell-based reporter
assays (Ogura et al., 2005; see further below).
The weevil A. grandis (Curculionidae) is a very important pest
insect in cotton, although almost eradicated in the USA by an
eradication program from which the trials started in 1978 (Smith
and Swink, 2003). However, this species represents the order of
Coleoptera comprising agricultural pests such as the Colorado
potato beetle Leptinotarsa decemlineata and the corn rootworm
Diabrotica virgifera, storage pests such as Sitophilus oryzae and
Tribolium castaneum, and many tree and forest pests.
2. Material and methods
A technical grade of the ecdysone agonists RH-5849 (w100%),
halofenozide(>90% pure), tebufenozide
methoxyfenozide (>95% pure) were a kind gift of Rohm and Haas
Co. (Spring House, PA). 20E (?95% pure) was purchased from
Sigma–Aldrich and ponasterone A (PonA) from Invitrogen. Serial
dilutions of these test compounds were prepared in ethanol.
2.2. Cell lines
The embryonic cell lines derived from the cotton boll weevil
A. grandis, BRL-AG-3A and BRL-AG-3C (Stiles et al., 1992b), and
a Colorado potato beetle cell line established from pupal tissue,
BCIRL-Lepd-SL1 (Long et al., 2002), were cultured in EX-CELL?420
(Sigma–Aldrich) supplemented with 5% FBS (Invitrogen). The cell
lines were obtained from the Biological Control of Insects Research
Laboratory, USDA-ARS, Columbia, MO.
To confirm the species origin of the cell line BRL-AG-3C, we
cloned a fragment of the mitochondrial 16S rRNA gene from this
coleopteran cell line using the following universal primers: 50-
as forward primer and 50-GGTCTGAACTCAGATCATGT-30as reverse
primer (Douris et al.,1998). DNA fromthe cell linewas isolatedwith
the Tissue DNA Kit (Omega Bio-Tek). PCR amplification conditions
were 20 s at 94?C, 1 min at 46?C, 40s at 72?C for 30 cycles. PCR
products were cloned into the pGEM?-T vector (Promega) and
sequenced by Agowa. The partial sequence (GenBank accession no.
FJ423738) that was obtained showed very high identity to the
partial 16S rRNA sequences of Anthonomus rubi (GenBank accession
no. AJ495539; 91% identity), Anthonomus pomorum (GenBank
accession no. AJ495540; 89% identity) and Furcipes rectirostris
(GenBank accession no. AJ495541; 88% identity). These results
confirm the A. grandis origin of the cell line.
2.3. Testing ecdysone agonists on transfected or infected
coleopteran cell lines
Stiles et al. (1992a) reported that Lipofectin? was very efficient
to transfect the BRL-AG-3C cell line. So we tried to transfect the
A. grandis cell lines BRL-AG-3A and BRL-AG-3C and the L. decemli-
neata cell line BCIRL-Lepd-SL1 with Lipofectin? (Invitrogen). Awell
of a 6-well plate was filled with 1 ? 106cells. After the cells were
attached the cells were washed twice with serum-free EX-CELL?
420 (Sigma–Aldrich). For one well or 1 ?106cells,15 ml Lipofectin?
was incubated together with 85 ml of the same medium for 45 min
at room temperature and then 15 min together with 1.5 mg of the
reporter construct ERE-b.act.luc (Soin et al., 2008) before adding to
the cells. The cells were incubated for 5 h with the transfection
medium. Transfected cells at a density of around 50,000 cells in
100ml wereincubated for 24h with 20E oran ecdysone agonist. The
Steady-Glo?Luciferase Assay System kit (Promega) was used for
measuring the luciferase expression and the luminescence was
measured with an Infinite M200 luminometer (Tecan). For every
concentration four replicates were used and each experiment was
repeated three times.
We also tried to infect the same coleopteran cell lines with
a genetically modified baculovirus, BmNPV/A.GFP/ERE-b.act.luc
(Swevers et al., 2008), and to use these infected cells to test the
ecdysone responsiveness of (potential) ecdysone agonists. Cells
were seeded in a 96-well plate at a density of 500,000 cells/ml.
After 1 h incubation to let the cells adhere to the substrate, 50 ml
medium of each well was aspirated and replaced by‘virus medium’.
This ‘virus medium’ is the supernatant of a Bm5 cell culture after
infectionwith the modified baculovirus (Swevers et al., 2008). Then
1 ml of a concentration range of ecdysone agonist or 20E was added
to the cell suspension in the wells. After 24 h of incubation the
luciferase expression was measured with the Steady-Glo Luciferase
Assay System kit (Promega). For everyconcentration four replicates
were used and each experiment was replicated three times.
Median effective concentrations, EC50s, were calculated with
GraphPad Prism Version 4.00 (GraphPad software) using sigmoid
dose–response (variable slope). One should be cautious when
comparing the activities of ecdysone agonists with only using these
EC50-values. An EC50-value is calculated by calculation of the point
of inflection of the sigmoid dose–response curve and it does not say
anything about the plateau of the curve (i.e. the maximal induc-
tion). It is possible when comparing two compounds that
T. Soin et al. / Insect Biochemistry and Molecular Biology 39 (2009) 523–534 524
compound one has a higher EC50-value but a higher maximal
induction capacity. We express the concept of potency of an
ecdysone agonist with an EC50-value and the concept of efficacy
with the maximal induction capacity.
Cytotoxicityof RH-5849, halofenozide,
methoxyfenozide was tested on the BRL-AG-3C cells to test if the
results of the reporterassayswere not influenced byacytotoxiceffect
of these non-steroidal ecdysone agonists. Cytotoxicity assays were
performed with the CellTiter 96?AQueousOne Solution Cell Prolifera-
tion Assay (Promega). This assay uses 3-(4,5-dimethylthiazol-2-yl)-5-
salt (MTS) and phenazine ethosulfate (PES), an electron coupling
reagent. The MTS tetrazolium compound is bioreduced by cells into
a colored formazan product that is soluble in tissue culture medium
(Cory et al., 1991). This conversion is presumably accomplished by
NADPH or NADH produced by dehydrogenase enzymes in metaboli-
cally active cells. The non-steroidal ecdysone agonists were tested at
For each concentration three replicates were used and each experi-
ment was replicated two times.
2.4. Cloning and molecular analysis of AgEcR
Total RNA was purified using the RNeasy kit (Qiagen) according
to the manufacturer’s protocol. This RNA was used as template for
cDNA synthesis with the Transcriptor First Strand cDNA Synthesis
Kit (Roche Applied Science) to clone a partial sequence of AgEcR
and for cDNA synthesis with the SMART? RACE cDNA Amplifica-
tionKit (Clontech). Thedegenerate
ATNGAYATGTAYATG-30(F) and 50-GCCATNCKRAACATCATNAC-30
(R), were designed based on the amino acid sequences, CEIDMYM
and VMMFRMA, which are completely conserved in the following
coleopteran insects: L. decemlineata (GenBank accession no.
BAD99297), Tribolium molitor (GenBank accession no. CAA72296)
and T. castaneum (GenBank accession no. NP_001107650) to clone
a partial sequence of the EcR. These amino acid sequences are
located in the DNA binding domain (DBD) and the LBD, respec-
tively. Amplification conditions with the degenerate primers were
20s at 94?C, 30 s at 50?C and 50 s at 72?C for 40 cycles for a first
round of amplification and the same amplification conditions were
used in a second round of amplification with the degenerate
primers and with the obtained PCR product from the first round as
Gene specific primers (GSPs) and nested gene specific primers
(NGSPs) were designed in the partial sequence obtained with the
degenerate primers, and the 50- and 30- end sequences of AgEcR
were obtained by rapid amplification of cDNA ends (RACE) using
SMART? RACE cDNA Amplification Kit (Clontech). The GSP 50-
CGAACCCTGGTAACCTCTTGGCGAACTC-30and NGSP 50-CTCATGCCC
ACCGTCAAAC-30were used for 50RACE, and GSP 50-GCAGTGCGCC
ATCAAGAGGAAAGAGAAG-30and NGSP 50-TCAGTTGATCGTGGAGTT
CGC-30were used for 30RACE. The primary PCR was performed
with GSPs under the following amplification conditions: 30 s at
94?C and 2 min at 72?C for 5 cycles, and then 30 s at 94?C, 30 s
at 70?C and 90 s at 72?C for 5 cycles, followed by 30 s at 94?C, 30 s
at 68?C and 90 s at 72?C for 30 cycles. The secondary PCR was held
with NGSPs under the condition of 30 s at 94?C, 30 s at 60?C and
90 s at 72?C for 25 cycles. Finally, the sequence of the PCR products
was obtained as mentioned above.
2.5. 3D-modeling of the ligand-binding pocket of AgEcR and ligand
Multiple amino acid sequence alignments were carried out with
CLUSTAL-X (Thompson et al., 1997) using the Risler’s structural
matrix for homologous amino acid residues (Risler et al., 1988).
Molecular modeling of the EcR-LBD from the coleopterans
L. decemlineata (Chrysomelidae, GenBank accession no. BAD99296)
LdEcR-LBD (Ogura et al., 2005), T. molitor (Tenebrionidae, GenBank
accession no. CAA72296) TmEcR-LBD (Mouillet et al., 1997) and
A. grandis (Curculionidae, GenBank accession no. ACK57879)
AgEcR-LBD (because our sequence is not full length, it is supple-
mented with the last 15 amino acids from TcEcR-LBD), was carried
out on a Silicon Graphics O2 R10000 workstation, using the
programs InsightII, Homology and Discover3 (Accelrys). The atomic
coordinates of T. castaneum TcEcR-LBD in complex with the
ecdysteroid PonA (RCSB Protein Data Bank code 2NXX) (Iwema
et al., 2007) were used to build the 3D-model of the receptors. The
high percentages of both identity (w50%) and similarity (w81%)
that LdEcR-LBD, TmEcR-LBD and AgEcR-LBD share with the
template TcEcR-LBD allowed us to build rather accurate 3D-models.
Steric conflicts were corrected during the model building proce-
dure using the rotamer library (Ponder and Richards,1987) and the
search algorithm implemented in the Homology program (Mas
et al., 1992) to maintain proper side-chain orientation. An energy
minimization of the final model was carried out by 300 cycles of
steepest descent using the cvff forcefield of Discover. PROCHECK
(Laskowski et al.,1993) was used to assess the geometric quality of
the 3D-model. In this respect, about 80% of the residues of the
modeled LdEcR-LBD were correctly assigned on the best allowed
regions of the Ramachandran plot, the remaining residues being
located in the generously allowed regions of the plot except for
three residues (Glu6, Ser39 and Arg188) which occur in the non-
allowed region (result not shown). Molecular cartoons were drawn
with PyMol (W.L. DeLano, http://pymol.sourceforge.net) and the
UCSF Chimera package (Pettersen et al., 2004). The fold recognition
html) (Bennett-Lovsey et al., 2008) that also used 2NXX and
structurally-related proteins as templates yielded readily super-
posable 3D-models. However, some discrepancies that essentially
deal with the shape of the loops connecting the a-helical stretches
of EcR-LBD were observed with our lab-made modeled structures.
Nevertheless, these discrepancies occur outside the groove
responsible for the binding of PonA.
The ConSurf server (http://consurf.tau.ac.il) (Landau et al., 2005)
was used to identifyconserved residues on the molecular surface of
the EcRmolecules. Conservation of residueswas represented on the
molecular surface according to a color scale code (from blue: not
conserved to red: strictly conserved) using PyMol. The 51 amino
acid sequences available for the EcR-LBD from insects include
Coleoptera (4 accessions), Orthoptera (1 accession), Dictyoptera
(1 accession), Hymenoptera (5 accessions), Hemiptera (4 acces-
sions), Diptera (20 accessions), Lepidoptera (13 accessions),
Neoptera (2 accessions) and Mecoptera (1 accession).
TcEcR-LBD in complex with PonA was taken as a template for
docking both PonA and tebufenozide to LdEcR-LBD, TmEcR-LBD
and AgEcR-LBD. Docking was performed with InsightII using
Discover3 as a forcefield. Clipping planes of TcEcR-LBD, LdEcR-LBD,
TmEcR-LBD and AgEcR-LBD complexed with PonA were rendered
3.1. Testing 20E and ecdysone agonists on transfected or
baculovirus-mediated transduced coleopteran cell lines
The efficiency of the transfection with Lipofectin? of the Colo-
rado potato beetle cell line BCIRL-Lepd-SL1 and the cotton boll
weevil cell line BRL-AG-3A with the ecdysone responsive reporter
plasmid was too low (data not shown). On the other hand, the
T. Soin et al. / Insect Biochemistry and Molecular Biology 39 (2009) 523–534 525
efficiency of the transfection of the BRL-AG-3C cell line was suffi-
ciently high to obtain good results. A dose–response curvewith 20E
and PonA as ligand (Fig. 1) and respective corresponding EC50-
values of 244 nM (95% confidence interval (CI): 213–278 nM) and
4.40 nM (95% CI: 3.47–5.59 nM) were calculated with GraphPad
Prism 4.00 (sigmoid dose–response with variable slope and
constraint bottom ¼ 0). We tested the following DBH analogues on
the transfected cells: RH-5849, tebufenozide, halofenozide and
methoxyfenozide (Fig. 2A). Their activity was calculated as the
percentage activity compared to the activity of 1 mM 20E. None of
these DBHs reached an activity of more than 50% and at 1 mM none
of them showed an activity >5%. The order inpotency (EC50-values)
to inducethe reporterconstruct
tebufenozide > halofenozide > RH-5849 (Table 1). The order of
efficacy (maximal induction capacity) was methoxyfenozide ?
tebufenozide > halofenozide ? RH-5849 (Table 1).
It was possible to transduce the BRL-AG-3A (data not shown)
and BRL-AG-3C (Fig. 2B) (but not the BCIRL-Lepd-SL1) cell lines
using the modified baculovirus BmNPV/A.GFP/ERE-b.act.luc and to
test the ecdysone responsiveness of (potential) ecdysone agonists
on these cells. In comparison with the transfection assay there is
a higher basal level of luciferase activity (treatment with ethanol)
present after infection of these Anthonomus cell lines; around 2–3%
of the activity of 1 mM 20E for transfected BRL-AG-3C cells
compared to around 10% for infected BRL-AG-3C cells. Although
methoxyfenozide and tebufenozide had approximately the same
potency in this assay as with the reporter plasmid based assay, they
could reach activities higher than 50% compared to 1 mM 20E.
Methoxyfenozide reached an activity around 90% at 100 mM.
A dose–response curve of methoxyfenozide with BRL-AG-3A cells
(data not shown) was similar as with BRL-AG-3C cells. Although
we were not able to transfect or infect the L. decemlineata cell
line, this cell line is sensitive to ecdysone (agonist). 20E and
methoxyfenozide have a cell proliferation inhibition activity on
these cells (data not shown).
To test if the results of the reporter assays were influenced by
cytotoxic effects of the non-steroidal ecdysone agonists, the cyto-
toxicity was determined by using an MTS assay. RH-5849,
halofenozide, tebufenozide and methoxyfenozide showed no
cytotoxic effects on the BRL-AG-3C cells after an incubation of 24 h
at the tested concentrations with this MTS assay. The cells werealso
visually examined under the microscope and also no cytotoxic
effects were observed at 100 and 10 mM. At 1 mM these ecdysone
agonists crystallize out in the medium (data not shown).
3.2. Cloning and molecular analysis of the AgEcR
and 30RACE were performed. However, we did not obtain the full
length AgEcR because the oligodT adapter for 30RACE hybridized to
probably miss around 39 bp at the end of the coding sequence.
The cloned AgEcR corresponds to the B1-isoform type of EcRs.
-11 -10-9-8-7-6-5 -4-3
Log (concentration (M))
% of maximum response
Fig. 1. A sigmoid dose–response curve of BRL-AG-3C cells transfected with a plasmid
with an ecdysone responsive reporter construct treated with 20E and PonA. Error bars
present the SEM (n ¼ 3).
Concentration (µ µM)
125 10 2050
Concentration (µ µM)
% activity compared to 1 µ µM 20-E
% activity compared to 1 µ µM 20-E
Fig. 2. Dose–response curves of BRL-AG-3C cells transfected with a plasmid with an
ecdysone responsive reporter construct (A) and infected with a modified baculovirus
containing an ecdysone responsive reporter construct (B) treated with non-steroidal
ecdysone agonists. Error bars present the SEM (n ¼ 3).
Potency (expressed as EC50) and efficacy (expressed as top of the curve, in
comparison to 1 mM 20E) of DBHs on BRL-AG-3C cells transfected with a plasmid
with an ecdysone responsive construct.
DBHEC50(mM) (95% CI)a
Top (%) (95% CI)a
aCalculated with GraphPad Prism 4.00 (sigmoid dose–response with variable
slope and constraint bottom ¼ 0).
T. Soin et al. / Insect Biochemistry and Molecular Biology 39 (2009) 523–534526
An alignment of this partial AgEcR (GenBank accession no.
ACK57879) with the other available coleopteran and two dipteran
and lepidopteran EcR-B1 sequences is presented in Fig. 3. The
partial AgEcR sequence showed high homology to other coleop-
teran EcR-B1 sequences: 83%, 84% and 80% amino acid identity to
the TcEcR (GenBank accession no. NP_001135390), LdEcR (GenBank
accession no. BAD99297) and TmEcR (GenBank accession no.
CAA72296), respectively. If we consider the amino acid identity
between the LBDs, the AgEcR shows 91%, 91% and 88% identity to
TcEcR, LdEcR and TmEcR, respectively. The amino acid identity
between the AgEcR-LBD and the LBD from dipterans and lepidop-
terans is much lower: for example 58% and 61% with the LBD from
Bombyx mori (GenBank accession no. AAA87340) and Heliothis
virescens (GenBank accession no. CAA70212) respectively, and 64%
and 66% with the LBD from Drosophila melanogaster (GenBank
accession no. AAA28498) and Aedes aegypti (GenBank accession no.
In addition, a phylogenetic tree of EcR-LBDs was generated by
neighbor-joining method using CLUSTAL-X multiple alignment
program with a bootstrap value of 1000 trials for each branch
position and exclusion of gap position. In the tree the AgEcR-LBD
sequence is grouped together with the other coleopteran receptors
3.3. Model of ligand-binding domain of coleopteran EcR
Like TcEcR-LBD from T. castaneum (Iwema et al., 2007), the EcR-
LBD from L. decemlineata (LdEcR-LBD), T. molitor (TmEcR-LBD) and
A. grandis (AgEcR-LBD) also consist of twelve a-helices tightly
packed around a ligand-binding groove that specifically anchor
PonA and other ecdysteroids (Fig. 5A–D).
Nine amino acid residues of TcEcR-LBD participate in the
binding of PonA through a network of 8 hydrogen bonds (Glu330,
Thr362, Thr365, Arg402, Val417 and Tyr427) and stacking interac-
tions (Phe416 and Trp543) (Fig. 5E). Another residue Asn 521
interacts with PonAvia a water-mediated bound. Due to the strictly
conserved character of these residues (Fig. 6), docking experiments
performed with PonAyielded a very similar binding scheme for the
modeled LdEcR-LBD, TmEcR-LBD and AgEcR-LBD (Fig. 5F–H).
Besides the EcR-LBD from Coleoptera, this high degree of conser-
vation extends to residues contributing both to the three-dimen-
sional fold and to the ecdysone-binding groove of insect EcR-LBD.
As shown from a survey of the conservation of amino acid residues
on the surface of EcR-LBD from different taxa of insects, most of the
residues are rather well conserved (Fig. 6A–C). Variable residues
essentially occur at the surface of the molecules in regions corre-
sponding to extended loops exposed to the solvent (Fig. 6A and B).
Similarly, 6 out of the 9 residues building the ecdysone-binding
groove are strictly conserved (conservation score of 9), the 3 others
if not conserved being replaced by structurally homologous resi-
dues (conservation score of 8) (Fig. 6C).
For the LBD of HvEcR there are two different crystal structures
described, one with PonA and one with the DBH analogue
BYI06830 as ligand. The LBPs of these two crystal structures are
only partially overlapping (Billas et al., 2003). There is only one
crystal structure of the EcR-LBD of T. castaneum described and this
structure has PonA as ligand. Thus the TcEcR-LBP with PonA as
ligand is used to dock tebufenozide in the LBP of TcEcR and AgEcR.
Different solutions resulted from the docking of tebufenozide to
the ecdysone-binding site of TcEcR-LBD. A solution which seems
particularly relevant consists of the superimposition of the A-ring
of tebufenozide tothealkylchain of ecdysone in suchawaythat the
ethyl-phenyl ring (B ring of the DBH) of the non-steroidal ecdysone
agonist occupies a cavity extending the binding site of TcEcR-LBD
opposite to the cavity harboring the alkyl chain of the ecdysteroid
(Fig. 7A–C). The anchoring of tebufenozide to the binding cavity
results from predominantly hydrophobic (Met, Leu and Ile resi-
dues) and stacking interactions (Phe, Tyr and Trp residues) in
association with a few hydrogen bonds linked to the backbone
carbonyl group of tebufenozide (Fig. 7D). A very similar docking
pattern was obtained with AgEcR-LBD (Fig. 7E–H).
While the model of AgEcR-LBD docked with tebufenozide was
generated using as template the structure of TcEcR-LBD bound with
PonA, it appears that the AgEcR-LBPs with tebufenozide and PonA
as ligands (Fig. 7F) overlap slightly more than the HvEcR-LBPs with
the non-steroidal ecdysone agonist BYI06830 and PonA as ligands
(Billas et al., 2003). On the other hand, it is clear that the AgEcR-LBD
model with docked tebufenozide (Fig. 7H) is more similar to the
HvEcR-LBD crystal structure with BYI06830 than PonA as ligand
(Billas et al., 2003). However, it is difficult to assess whether the
differences between the model of AgEcR-LBD bound with tebufe-
nozide and the structure of HvEcR-LBD bound with BYI06830 are
a consequence of the initial template that was used to create the
AgEcR-LBD model (i.e. TcEcR complexed with PonA) or reflect true
differences between the LBD structures. In particular, it is not
known whether the structure of AgEcR-LBD is as flexible as the
HvEcR-LBD structure such that an alternative LBP might be created
upon binding of a DBH analogue.
We demonstrated that the activity of the ecdysone agonists
RH-5849, tebufenozide, halofenozide and methoxyfenozide in
A. grandis cells transfected with an ecdysone responsive reporter
first isolated from the plant Podocarpus nakaii (Nakanishi et al.,
similar to the difference in relative binding affinity of 20E and PonA
to ecdysteroid receptor protein extracts from A. grandis cells (Dha-
dialla and Tzertzinis, 1997). The order of potency of the DBH
analogues was methoxyfenozide ? tebufenozide > halofenozide
> RH-5849. These data correspond with the results of the ligand-
binding assay with invitrotranslated L. decemlineataEcR-A and RXR
of Ogura et al. (2005). They demonstrated the following order
of binding affinities: 20E > methoxyfenozide > halofenozide
> tebufenozide > RH-5849. This means that the amino acid
sequence of the receptor is probably the main cause if a DBH
analogue can induce EcR-USP/RXR mediated transcription. Metab-
olism and transport through the cell membrane of the Ag3C cells
apparently play a minor role. The order of potency methoxy-
most in vivo or in vitro lepidopteran bioassays. But in Lepidoptera,
methoxyfenozide and tebufenozide are active in vitro at very low
2004). This is expected since their high activity led to their devel-
opment as lepidopteran specific insecticides. The low activity of
halofenozide on the transfected A. grandis cells is remarkable
because it is used to control coleopteran (scarabid larvae) and lepi-
dopteran insects in turf and ornamentals (Dhadialla et al., 1998).
However, halofenozide is not in all coleopteran insects more active
than the other DBH insecticides. For example, halofenozide and
methoxyfenozide are equally toxic to Harmonia axyridis (Carton
et al., 2003). Actually, there are only a few articles published that
report toxicity tests of insects with halofenozide. We also do not
know the in vivo activity of halofenozide on A. grandis. It cannot be
excluded that there exist compounds that are more active in vivo
analogues found with a higher potency than 20E against Coleoptera
to design DBH analogues or other non-steroidal ecdysone agonists
T. Soin et al. / Insect Biochemistry and Molecular Biology 39 (2009) 523–534 527
T. Soin et al. / Insect Biochemistry and Molecular Biology 39 (2009) 523–534528
with higher activity than 20E on a coleopteran or dipteran EcR and
that can be used as highly effective coleopteran and dipteran
In transfection experiments, it is notable that none of these
ecdysone agonists reached 50% activity compared to 1 mM 20E at
any concentration tested. The maximal response of 20E and PonA
was the same on the transfected A. grandis cells. Toya et al. (2002)
have reported that the maximal induction of chromafenozide in Sf9
cells transfected with a similar reporter plasmid was about 4 times
lower than the maximal induction of 20E or PonA. However at
a concentration of 1 nM, chromafenozide and PonA have almost the
same activity. The authors suggested that chromafenozide binds
a lepidopteran EcR with comparable affinity to PonA, but it may
activate the EcR in a different manner. Mosallanejad et al. (2008)
have transfected B. mori Bm5 cells with the same reporter plasmid
as used for the experiments in this research article and they
observed that methoxyfenozide and chromafenozide could not
reach 60% of induction activity although these compounds are still
active at about 0.1 nM.
Thereare also other factors that influence the bindingof a ligand
to a receptor. Browning et al. (2007) demonstrated an additional
hydrogen bond in the 20E-bound HvEcR compared to the PonA-
bound HvEcR. Paradoxically, PonA has a significantly higher affinity
for EcR than 20E. Theoretical studies based on docking and free
energy methods indicate that the favorable contribution from the
extra H-bond made by 25-OH of 20E is counterbalanced by its
larger desolvation cost compared with that of PonA.
Infecting the same cell line with a modified baculovirus that has
results with respect to the relative activities of DBH analogues. The
virus could also be used to infect the BRL-AG-3A (data not shown)
and the Spodoptera exigua BCIRL/AMCY-SeE-CLG4 cell line (Swevers
et al., 2008) that are hard totransfect bylipofection. Nevertheless, it
seems that the method does not work for every cell line: the
L. decemlineata BCIRL-Lepd-SL1 cell line was found to be refractory
approximately the same potency in this assay compared to the
50% compared to 1 mM 20E. Methoxyfenozide at 100 mM reached an
this higher efficacy, we can only hypothesize. The BmNPV/A.GFP/
ERE-b.act-luc baculovirus possesses an ecdysteroid UDP-glucosyl
transferase (egt) gene that catalyzes the transfer of glucose from
thus preventing larval molting and pupation. It is well established
that Bombyx mori baculovirus (BmNPV) has a narrow host range
(Maeda et al., 1990), and will normally not propagate in a coleop-
teran cell line. However, it cannot be excluded that some early virus
genes are transcribed in non-host cell lines and the Epiphyas post-
vittana nucleopolyhedrovirus contains an egt with upstream early
Fig. 3. (continued).
Fig. 3. Multiple alignment of the EcR of the coleopterans A. grandis (AgEcR, GenBank accession no. ACK57879), T. castaneum (TcEcR, GenBank accession no. NP_001135390), T.
molitor (TmEcR, GenBank accession no. CAA72296) and L. decemlineata (LdEcR, BAD99297), the lepidopterans B. mori (BmEcR, GenBank accession no. BAA07890) and H. virescens
(HvEcR, GenBank accession no. CAA70212), and the dipterans A. aegypti (AeEcR, GenBank accession no. AAA87394) and D. melanogaster (DmEcR, GenBank accession no. AAA28498).
The F-domain of the dipteran and lepidopteran EcRs is not completely presented. The strictly conserved amino acid residues forming the ecdysone-binding site are indicated by ;
(residues involved in hydrogen bonds), C (residue involved in a water-mediated hydrogen bond) and + (residues involved in stacking interactions).
Fig. 4. A phylogenetic tree was generated based on the amino acid sequences of the
EcR-LBD except for C-terminal 15 amino acids from the coleopterans A. grandis (AgEcR,
GenBank accession no. ACK57879) T. castaneum (TcEcR, GenBank accession no.
CAL25730), T. molitor (TmEcR, GenBank accession no. CAA72296) and L. decemlineata
(LdEcR, GenBank accession no. BAD99297), the lepidopterans B. mori (BmEcR, GenBank
accession no. AAA87340) and H. virescens (HvEcR, GenBank accession no. CAA70212),
and the dipterans A. aegypti (AeEcR, GenBank accession no. AAA87394) and D. mela-
nogaster (DmEcR, GenBank accession no. AAA28498). This tree was made by neighbor-
joining method using CLUSTAL-X multiple alignment program with a bootstrap value
of 1000 trials for each branch position and exclusion of gap position. The indicated
numbers are bootstrap values as percentage of a 1000 replicates and the scale bar
indicates 0.05 change per residue.
T. Soin et al. / Insect Biochemistry and Molecular Biology 39 (2009) 523–534 529
promoter motifs present (Caradoc-Davies et al., 2001). This means
that it is possible that egt is active in the BRL-AG-3C cell line and
causes inactivation of 20E resulting in a higher relative efficacy of
methoxyfenozide and tebufenozide compared to 20E. However, the
relative increase of the efficacy of methoxyfenozide is higher than
Taken together, our studies of transfection and transduction of
coleopteran cell lines have established that the BRL-AG-3C cell line
of A. grandis can be engineered to incorporate an EcR reporter
construct for the development of a high-throughput screening
system for ecdysone agonists specific for coleopteran insects, in
a manner similar to that described for lepidopteran and dipteran
cell lines (Swevers et al., 2004; Wheelock et al., 2006; own
unpublished results). To our knowledge, this is the first time that
such a system specific for coleopteran insects is described. The use
of a coleopteran cell line has the advantage that it expresses
endogenous EcR and Ultraspiracle (USP), coactivators and core-
pressors, as well as the complete transcription machinery specific
to coleopteran receptors and thus corresponds to the ‘natural
environment’ for function of coleopteran EcRs. This is considered
an advantage compared to the development of screening systems
based on overexpression of EcR in mammalian or insect cell lines
(Henrich et al., 2003; Maki et al., 2004) or introduction of the
coleopteran EcR in dipteran or lepidopteran insect cell lines that are
deficient for their endogenous receptor.
We have cloned the A. grandis EcR and the N-terminal amino
acid sequence corresponds well to the B1-isoform of other cole-
opteran EcRs. The A-isoform was not identified with the SMART?
RACE technique in the BRL-AG-3C cell line.
As already reported in Iwema et al. (2007), the high level of
residue conservation observed in arthropod EcR-LBD accounts for
a conserved canonical ligand-binding activity with high affinity
towards 20E and other ecdysteroids such as PonA. Carmichael et al.
(2005) reported the crystal structure of the EcR-LBD heterodimer of
the hemipteran Bemisia tabaci in complex with PonA and they
observed that the overall mode of PonA binding is very similar in
comparison with the lepidopteran HvEcR-LBD heterodimer crystal
structure in complex with PonA. If we look at the conservation of
the AA residues of the lepidopteran HvEcR that interact with the
DBH ecdysone agonist BYI06830 (Billas et al., 2003), we find only
two different residues in coleopteran EcRs. Residue V402 (HvEcR),
conserved in lepidopteran insects but replaced by methionine in
other insects, is considered to be essential for the specificity of
BYI06380 to Lepidoptera. All possible methionine rotamers would
sterically interfere with the BYI06380 methyl groups on the A-ring
(Billas et al., 2003). This methionine also hydrophobically interacts
with tebufenozide in our model of the AgEcR-LBD docked with
tebufenozide (Fig. 7). Halofenozide does not have methyl groups on
the A-ring and would not experience such steric interference but
apparently has a low affinity for EcRs of both Coleoptera and
Lepidoptera (Swevers et al., 2004; Ogura et al., 2005). Second, the
apolar residue V434 (HvEcR), to our knowledge also conserved in
all lepidopteran EcRs, is substituted by the polar threonine (T383
AgEcR) in all coleopteran EcRs. Thus, the substitution of this residue
in coleopteran receptors could possibly interfere with the binding
of DBH analogues in coleopteran EcRs.
From all the AA residues in the AgEcR-LBD model that interact
with tebufenozide, there are only two residues not conserved in the
HvEcR sequence: AgEcR M351, which is already discussed above,
and AgEcR I362 which corresponds to HvEcR V413. But the latter
one does not interact with BYI06830 in the LBP of HvEcR. Also the
H-bridges are quite different between HvEcR-LBP bound BYI06830
and AgEcR-LBP bound tebufenozide. But these two different DBH
analogues may be positioned slightly different in the LBP.
For the LBD of HvEcR there are two different crystal structures
described, one with PonA and one with the DBH analogue
BYI06830 as ligand. The LBPs of these two crystal structures are
only partially overlapping and seem to be induced by the binding of
different types of ligand, illustrating the flexibility of the EcR-LBD in
lepidopteran receptors (Billas et al., 2003). There is only one crystal
structure of the EcR-LBD of T. castaneum described and this struc-
ture has PonA as ligand. Thus the TcEcR-LBP with PonA as ligand
Fig. 5. Ribbon diagram of TcEcR-LBD (A), LdEcR-LBD (B), TmEcR-LBD (C) and AgEcR-LBD (D). The twelve a-helices building the three-dimensional fold of the receptors are differently
colored and numbered H1-H12; the two short strands of b-sheet are colored purple and numbered b1 and b2. N and C correspond to the N- and C-terminus of the polypeptide chain,
respectively. PonA complexed to the EcR-LBD is represented in pink stick. E–H: Clips showing the binding of PonA to the ligand-binding groove of TcEcR-LBD (E), LdEcR-LBD
(F), TmEcR-LBD (G) and AgEcR-LBD (H). Residues interacting with PonA by direct hydrogen bonds (light pink colored sticks), water-mediated hydrogen bonds (light green colored
sticks) and stacking interactions (light orange colored sticks) are labeled. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version
of this article.)
Fig. 6. Sphere (A) and ribbon diagram (B) representation of conserved residues on the
molecular surface of EcR-LBD from insects. The color scale used is as follows from non-
conserved (blue) to strictly conserved (red) residues: blue (conservation ¼ 1), medium
blue (conservation ¼ 2), cyan (conservation ¼ 3), light blue (conservation ¼ 4), pale
yellow (conservation ¼ 5), light pink (conservation ¼ 6), pink (conservation ¼ 7), hot
pink (conservation ¼ 8), red (conservation ¼ 9). C: Histogram showing the distribution
of conserved residues on the molecular surface of EcR-LBD from insects. The 9 amino
acid residues (indicated by ‘‘3’’ and ‘‘6’’) forming the ecdysone-binding site (they
correspond to 3 the Glu330, Phe416 and Trp543 residues, and 6 the Thr362, Thr365,
Arg402, Val417, Tyr427 and Asn523 residues of TcEcR-LBD, respectively) occur among
the best conserved residues of EcR-LBD. (For interpretation of the references to colour
in this figure legend, the reader is referred to the web version of this article.)
T. Soin et al. / Insect Biochemistry and Molecular Biology 39 (2009) 523–534 531
Fig. 7. Clips (dotted red lines) made across the ligand-binding groove envelopes to show the respective orientation of tebufenozide (pink stick) and PonA (blue stick) in both cavities
located at the bottom of the ligand-binding groove of TcEcR-LBD (A–C) and AgEcR-LBD (E–G). The ligand-binding groove is outlined by dashed red line. Docking of tebufenozide to
the binding pocket of TcEcR-LBD and AgEcR-LBD is shown at (D) and (H). Residues that hydrophobically interact with tebufenozide are in yellow sticks. Aromatic residues involved
in stacking interactions are colored orange. Hydrogen bonds are represented by dashed blue lines. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
was used to dock tebufenozide in the LBP of TcEcR and AgEcR. This
is maybe not the ideal approach but there is no coleopteran EcR-
LBD crystal structure available with a DBH analogue as ligand.
Future studies are requiredtoassess the flexibilityof the EcR-LBD of
coleopteran receptors and if binding of DBH analogue can also
induce an alternative LBP in coleopteran EcRs.
Based on our docking studies of tebufenozide in the LBP of
coleopteran EcRs, there are small differences in the binding mode
of tebufenozide in the LBP compared to that of BYI06830 in the LBP
of HvEcR. Our model for the coleopteran receptor is incapable of
explaining why DBH analogues have much higher activation
properties in lepidopteran receptors than in coleopteran receptors.
Thus, rather subtle differences between coleopteran and lepidop-
teran EcR-USP/RXR-ligand complexes may be translated into rather
large differences in activation potential by the different receptors.
Also in other nuclear receptor complexes it was observed that
binding of ligands with different types of activity (agonist, antag-
onist) results in only minor differences in overall structure of
nuclear receptor complexes, including the binding of coactivator
peptides (Chandra et al., 2008). The LBD can also have an important
function in modulating DNA binding depending on the manner in
which the complex is organized. The EcR is by itself incapable of
high-affinity DNA binding or transcriptional activation. Rather,
these activities are dependent on heterodimer formation with USP.
Moreover, 20E promotes the heterodimerization between EcR and
USP (Yao et al., 1993). Again, small differences in the overall
structure of the nuclear receptor complex can maybe influence the
heterodimerization necessary for high-affinity DNA binding.
Additional structural studies, employing complexes of whole
nuclear receptors bound to their response elements and coac-
tivators, therefore may be required to reveal how small changes in
binding of DBH analogues to EcRs of different insect orders are
translated into large differences in activity with respect to target
In conclusion, wereport in this researcharticle the development
and the evaluation of two coleopteran-specific reporter-based
screening systems to discover and evaluate ecdysone agonists: for
the first one we transfected an A. grandis cell line with a plasmid
with an ecdysone responsive reporter construct, for the second one
we infected the same cell line with a modified baculovirus with the
same reporter construct. We cloned the almost full length coding
sequence of the EcR of this cell line and made an initial 3D-model,
based on the crystal model of T. castaneum, of the LBD specific for
coleopteran receptors, and herein we docked PonA and tebufeno-
zide. Our studies therefore provide the basis for the development of
a cell-based high-throughput screening system for ligands specific
for coleopteran EcRs. In addition, refinements of the model of the
LBD of TcEcR, AgEcR, LdEcR and TmEcR may allow docking studies
of ligands in silico to allow the rational design of non-steroidal
This research was supported by a PhD Grant (no. 43628) from
the Institute for the Promotion of Innovation by Science and
Technology in Flanders (IWT, Belgium) to T. Soin, and support by
the Fund for Scientific Research (FWO-Vlaanderen, Belgium) to
G. Smagghe. Research on ecdysone agonists at the Insect Molecular
Genetics and Biotechnology group at the Institute of Biology,
National Center for Scientific Research ‘‘Demokritos’’ was sup-
ported by two bilateral scientific and technological cooperation
grants (Greece-Japan & Greece-Spain) from the General Secretariat
for Research and Technology, Ministryof Development, in Greece. P.
Rouge ´ gratefully acknowledges the financial support of Universite ´
Paul Sabatier and CNRS. We thank C.L. Goodman (USDA-ARS,
Columbia, MO) for providing the beetle cell lines.
Bennett-Lovsey, R.M., Herbert, A.D., Stemberg, M.J.E., Kelley, L.A., 2008. Exploring
the extremes of sequence/structure space with ensemble fold recognition in the
program Phyre. Proteins 70, 611–625.
Billas, I.M.L., Iwema, T., Garnier, J.M., Mitschler, A., Rochel, N., Moras, D., 2003.
Structural adaptability in the ligand-binding pocket of the ecdysone hormone
receptor. Nature 426, 91–96.
Browning, C., Martin, E., Loch, C., Wurtz, J.M., Moras, D., Stote, R.H., Dejaegere, A.P.,
Billas, I.M.L., 2007. Critical role of desolvation in the binding of 20-hydrox-
yecdysone to the ecdysone receptor. J. Biol. Chem. 282, 32924–32934.
Caradoc-Davies, K.M.B., Graves, S., O’Reilly, D.R., Evans, O.P., Ward, V.K., 2001.
Identification and in vivo characterization of
Nucleopolyhedrovirus ecdysteroid UDP-glucosyltransferase. Virus. Genes 22,
Carmichael, J.A., Lawrence, M.C., Graham, L.D., Pilling, P.A., Epa, V.C., Noyce, L.,
Lovrecz, G., Winkler, D.A., Pawlak-Skrzecz, A., Eaton, R.E., Hannan, G.N., Hill, R.J.,
2005. The X-ray structure of a hemipteran ecdysone receptor ligand-binding
domain. J. Biol. Chem. 280, 22258–22269.
Carton, B., Smagghe, G., Tirry, L., 2003. Toxicity of two ecdysone agonists, hal-
ofenozide and methoxyfenozide, against the multicoloured Asian lady beetle
Harmonia axyridis (Col., Coccinellidae). J. Appl. Entomol. 127, 240–242.
Chandra, V., Huang, P., Hamuro, Y., Raghuram, S., Wang, Y., Burris, T.P., Rastinejad, F.,
2008. Structure of the intact PPAR-g-RXR-a nuclear receptor complex on DNA.
Nature 456, 350–357.
Cory, A.H., Owen, T.C., Barltrop, J.A., Cory, J.G., 1991. Use of an aqueous soluble
tetrazolium formazan assay for cell-growth assays in culture. Cancer Commun.
Dhadialla, T.S., Carlson, G.R., Le, D.P., 1998. New insecticides with ecdysteroidal and
juvenile hormone activity. Annu. Rev. Entomol. 43, 545–569.
Dhadialla, T.S., Tzertzinis, G., 1997. Characterization and partial cloning of ecdys-
teroid receptor from a cotton boll weevil embryonic cell line. Arch. Insect
Biochem. Physiol. 35, 45–57.
Douris, V., Giokas, S., Lecanidou, R., Mylonas, M., Rodakis, G.C., 1998. Phylogenetic
analysis of mitochondrial DNA and morphological characters suggest a need for
taxonomic re-evaluation within the Alopiinae (Gastropoda: Clausiliidae).
J. Molluscan Stud. 64, 81–92.
Henrich, V.C., Burns, E., Yelverton, D.P., Christensen, E., Weinberger, C., 2003.
Juvenile hormone potentiates ecdysone receptor-dependent transcription in
a mammalian cell culture system. Insect Biochem. Mol. Biol. 33, 1239–1247.
Iwema, T., Billas, I.M.L., Beck, Y., Bonneton, F., Nierengarten, H., Chaumot, A.,
Richards, G., Laudet, V., Moras, D., 2007. Structural and functional character-
ization of a novel type of ligand-independent RXR-USP receptor. EMBO J. 26,
Landau, M., Mayrose, I., Rosenberg, Y., Glaser, F., Martz, E., Pupko, T., Ben-Tal, N.,
2005. ConSurf 2005: the projection of evolutionary conservation scores of
residues on protein structures. Nucleic Acids Res. 33, W299–W302.
Laskowski, R.A., MacArthur, M.W., Moss, D.S., Thornton, J.M., 1993. PROCHECK:
a program to check the stereochemical quality of protein structures. J. Appl.
Crystallogr. 26, 283–291.
Long, S.H., McIntosh, A.H., Grasela, J.J., Goodman, C.L., 2002. The establishment of
a Colorado potato beetle (Coleoptera: Chrysomelidae) pupal cell line. Appl.
Entomol. Zool. 37, 447–450.
Maeda, S., Mukohara, Y., Kondo, A., 1990. Characteristically distinct isolates of the
nuclear polyhedrosis virus from Spodoptera litura. J. Gen. Virol. 71, 2631–2639.
Maki, A., Sawatsubashi, S., Ito, S., Shirode, Y., Suzuki, E., Zhao, Y., Yamagata, K.,
Kouzmenko, A., Takeyema, K., Kato, S., 2004. Juvenile hormones antagonize
ecdysone actions through co-repressor recruitment to EcR/USP heterodimers.
Biochem. Biophys. Res. Commun. 320, 262–267.
Mas, M.T., Smith, K.C., Yarmush, D.L., Aisaka, K., Fine, R.M., 1992. Modeling the anti-
CEA antibody combining site by homology and conformational search. Proteins
Struct. Func. Genet. 14, 483–498.
Mosallanejad, H., Soin, T., Iatrou, K., Nakagawa, Y., Smagghe, G., 2008. Non-steroidal
ecdysteroid agonist chromafenozide: gene induction activity, cell proliferation
inhibition and larvicidal activity. Pest. Biochem. Physiol. 92, 70–76.
Mouillet, J.F., Delbecque, J.P., Quennedey, B., Delachambre, J., 1997. Cloning of two
putative ecdysteroid receptor isoforms from Tenebrio molitor and their devel-
opmental expression in the epidermis during metamorphosis. Eur. J. Biochem.
Nakagawa, Y., 2005. Nonsteroidal ecdysone agonists. Vitam. Horm. 73, 131–173.
Nakanishi, K., Koreeda, M., Sasaki, S., Chang, M.L., Hsu, H.Y., 1966. Insect hormones.
The structure of ponasterone A, insect-moulting hormone from the leaves of
Podocarpus nakaii Hay. Chem. Commun. 24, 915–917.
Ogura, T., Minakuchi, C., Nakagawa, Y., Smagghe, G., Miyagawa, H., 2005. Molecular
cloning, expression analysis and functional confirmation of ecdysone receptor
and ultraspiracle from the Colorado potato beetle Leptinotarsa decemlineata.
FEBS J. 272, 4114–4128.
O’Reilly, D.R., Miller, L.K., 1989. A baculovirus blocks insect molting by producing
ecdysteroid UDP-glucosyl transferase. Science 245, 1110–1112.
T. Soin et al. / Insect Biochemistry and Molecular Biology 39 (2009) 523–534 533
Pettersen, E.F., Goddard, T.D., Huang, C.C., Couch, G.S., Greenblatt, D.M., Meng, E.C., Download full-text
Ferrin, T.E., 2004. UCSF chimera – a visualization system for exploratory
research and analysis. J. Comput. Chem. 25, 1605–1612.
Ponder, J.W., Richards, F.M., 1987. Tertiary templates for proteins. Use of packing
criteria in the enumeration of allowed sequences for different structural classes.
J. Mol. Biol. 193, 775–791.
Risler, J.L., Delorme, M.O., Delacroix, H., Henaut, A., 1988. Amino-acid substitutions
in structurally related proteins. A pattern recognition approach. Determination
of a new and efficient scoring matrix. J. Mol. Biol. 204, 1019–1029.
Smith, J.W., Swink, W.D., 2003. Boll weevil eradication: a model for sea lamprey
control? J. Gt. Lakes Res. 29, 445–455.
2008. Juvenile hormone analogs do not affect directly the activity of the ecdys-
teroid receptor complex in insect culture cell lines. J. Insect Physiol. 54, 429–438.
Stiles, B., Heilmann, J., Sparks, R.B., Santoso, A., Leopold, R.A., 1992a. Transfection of
cultured cells of the cotton boll weevil, Anthonomus grandis, with a heat--
shock-promoter-chloramphenicol-acetyltransferase construct. Insect Mol. Biol.
Stiles, B., McDonald, I.C., Gerst, J.W., Adams, T.S., Newman, S.M., 1992b. Initiation
and characterization of 5 embryonic-cell lines from the cotton boll weevil
Anthonomus grandis in a commercial serum-free medium. In Vitro Cell. Dev.
Biol. Anim 28, 355–363.
Swevers, L., Kravariti, L., Ciolfi, S., Xenou-Kokoletsi, M., Ragoussis, N., Smagghe, G.,
Nakagawa, Y., Mazomenos, B., Iatrou, K., 2004. A cell-based high-throughput
screening system for detecting ecdysteroid agonists and antagonists in plant
extracts and libraries of synthetic compounds. FASEB J. 18, 134–136.
Swevers, L., Soin, T., Mosallanejad, H., Iatrou, K., Smagghe, G., 2008. Ecdysteroid
signaling in ecdysteroid-resistant cell lines from the polyphagous noctuid pest
Spodoptera exigua. Insect Biochem. Mol. Biol. 38, 825–833.
Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The
CLUSTAL_X windows interface: flexible strategies for multiple sequence align-
ment aided by quality analysis tool. Nucleic Acids Res. 25, 4876–4882.
Toya, T., Fukasawa, H., Masui, A., Endo, Y., 2002. Potent and selective partial ecdy-
sone agonist activity of chromafenozide in Sf9 cells. Biochem. Biophys. Res.
Commun. 292, 1087–1091.
Triglia, T., Peterson, M.G., Kemp, D.J., 1988. A procedure for in vitro amplification of
DNA segments that lie outside the boundaries of known sequences. Nucleic
Acids Res. 16, 8186.
Wheelock, C.E., Nakagawa, Y., Harada, T., Oikawa, N., Akamatsu, M., Smagghe, G.,
Stefanou, D., Iatrou, K., Swevers, L., 2006. High-throughput screening of ecdy-
sone agonists using a reporter gene assay followed by 3-D QSAR analysis of the
molting hormonal activity. Bioorg. Med. Chem. 14, 1143–1159.
Yanagi, M., Tsukamoto, Y., Watanabe, T., Kawagishi, A., 2006. Development of
a novel lepidopteran insect control agent, chromafenozide. J. Pest. Sci. 31,
Yao, T., Forman, B., Jiang, Z., Cherbas, L., Chen, J., McKeown, M., Cherbas, P., Evans, R.,
1993. Functional ecdysone receptor is the product of EcR and Ultraspiracle
genes. Nature 366, 476–479.
T. Soin et al. / Insect Biochemistry and Molecular Biology 39 (2009) 523–534534