Báo cáo khoa học: Effect of ecdysone receptor gene switch ligands on endogenous gene expression in 293 cells docx

Effect of ecdysone receptor gene switch ligands on
endogenous gene expression in 293 cells
Siva K. Panguluri
1
, Bing Li
2
*, Robert E. Hormann
2
and Subba R. Palli
1
1 Department of Entomology, College of Agriculture, University of Kentucky, Lexington, KY, USA
2 Intrexon Corporation, Norristown, PA, USA
Gene therapy is used to correct a defect in the expres-
sion of a gene by transferring a gene expression cas-
sette containing a promoter, a terminator, and the
coding region of a gene whose absence or defect causes
a disease. Current technology uses constitutive promot-
ers, such as the cytomegalovirus promoter, for expres-
sion of transgenes. Such an ‘always on’ arrangement is
not desirable because it can exacerbate pleiotropic
effects and also leaves no option for remediation in the
event of medical complications due to transgene
expression. To address this issue, regulated expression
of the additive or corrective gene becomes attractive
for various gene therapy applications. Despite extra
complexity, the regulated expression system can
Keywords
diacylhydrazine; ecdysone; gene therapy;
microarray; RSL-1
Correspondence
S. R. Palli, Department of Entomology,

College of Agriculture, University of
Kentucky, Lexington, KY 40546 USA
Fax: +1 859 323 1120
Tel: +1 859 257 4962
E-mail:
*Present address
MicroBiotiX, Inc., Worcester, MA, USA
(Received 24 June 2007, revised 13 August
2007, accepted 4 September 2007)
doi:10.1111/j.1742-4658.2007.06089.x
Regulated gene expression may substantially enhance gene therapy. Corre-
lated with structural differences between insect ecdysteroids and mamma-
lian steroids, the ecdysteroids appear to have a benign pharmacology
without adversely interfering with mammalian signaling systems. Conse-
quently, the ecdysone receptor-based gene switches are attractive for appli-
cation in medicine. In the present study, the effect of inducers of ecdysone
receptor switches on the expression of endogenous genes in HEK 293 cells
was determined. Four ligand chemotypes, represented by a tetrahydroquin-
oline (RG-120499), one amidoketone (RG-121150), two ecdysteroids
[20-hydroxyecdysone (20E) and ponasterone A (Pon A)], and four diacyl-
hydrazines (RG-102240, RG-102277, RG-102398 and RG-100864), were
tested in HEK 293 cells. The cells were exposed to ligands at concentra-
tions of 1 lm (RG-120499) or 10 lm (all others) for 72 h and the total
RNA was isolated and analyzed using microarrays. Microarray data
showed that the tetrahydroquinoline ligand, RG-120499 caused cell death
at concentrations ‡ 10 lm.At1lm, this ligand caused changes in the
expression of genes such as TNF, MAF, Rab and Reprimo.At10lm, the
amidoketone, RG-121150, induced changes in the expression of genes such
as v-jun, FBJ and EGR, but was otherwise noninterfering. Of the two ste-
roids tested, 20E did not affect gene expression, but Pon A caused some

changes in the expression of endogenous genes. At lower concentrations
pharmacologically relevant for gene therapy, intrinsic gene expression
effects of ecdysteroids and amidoketones may actually be insignificant.
A fortiori, even at 10 lm, the four diacylhydrazine ligands did not cause
significant changes in expression of endogenous genes in 293 cells and
therefore should have minimum pleiotropic effects when used as ligands
for the ecdysone receptor gene switch.
Abbreviations
AMK, amidoketone; qRT-PCR, quantitative real-time reverse transcription PCR; STAT 6, signal transducer and activator of transcription 6;
THQ, tetrahydroquinoline.
FEBS Journal 274 (2007) 5669–5689 ª 2007 The Authors Journal compilation ª 2007 FEBS 5669
substantially raise the level of safety and might even
be essential to control the expression levels of proteins
that have narrow therapeutic indices, such as cyto-
kinins and hormones.
The main purpose of a regulated gene expression
system is to control the timing and levels of transgene
expression in vivo. Whether the expressed protein
remains within the cell or, more commonly, is secreted
and ⁄ or distributed in extracellular compartments, it
will undergo elimination according to pharmacokinetc
principles [1]. Thus, too little protein will be subthera-
peutic, too much will be potentially toxic. Therefore, a
successful therapy would be characterized by regulated
expression of the transgene finely tuned to the chang-
ing clinical state of a patient.
Several gene switches have been developed for regu-
lating the expression of transgenes in humans [2].
More specifically, ecdysone receptor (EcR) gene
switches for medicinal purposes have been reported by

several laboratories [3–9]. Among these switches, EcR-
based gene switches display particularly low basal
activity in the absence of an inducer and strong induc-
ible activity in the presence of an inducer [2,8,10].
The relative structural dissimilarity of ecdysteroids
and mammalian steroids might suggest that binding of
the former to vertebrate steroid receptors would be too
weak for pharmacological effects, particularly adverse
ones. In support of this proposition, humans consume
significant amounts of phytoecdysteroids contained in
dietary vegetables seemingly without any apparent
detrimental effects [11]. However, Oehme et al. [12]
reported recently that ecdysteroids and ecdysone
mimics can induce and⁄ or suppress endogenous genes
in RKO and other mammalian cells and promote
apoptosis.
A more comprehensive understanding of possible
pleiotropic effects of ligand inducers and ⁄ or the switch
components is essential for successful use of the EcR
gene switch for in vivo applications such as gene ther-
apy. The diacylhydrazine [13,14] nonsteroidal ecdysone
agonists, such as Rheoswitch ligand 1 (RSL-1; Fig. 1),
are reported to be an excellent inducer for EcR gene
switches, supporting up to 9000-fold induction of
reporter activity [15]. Other steroidal ligands, such as
ponasterone A (Pon A; Fig. 1), have also been
reported as potential inducers of EcR-based gene
HO
HO
H

H OH
OH
RH
OH
Ecdysteroids (ECD)
20-hydroxyecdysone
ponasterone
N
H
N
O
O
O
Diacylhydrazines (DAH)
RG-100864 R
1
=Cl R
2
=H
R = OH
R = H
RG-102398
R
1
=2-CH
3
, 3-OCH
3
R
2

=3,5-di-CH
3
RG-102240
R
1
=2-CH
2
CH
3
, 3-OCH
3
R
2
=3,5-di-CH
3
RG-102277
R
1
=2-CH
2
CH
3
, 3-OCH
3
R
2
=3-CH
3
, 5-C(O)NH
2

N
O
HN
F
F
F
N
H
O
O
O
O
Amidoketone (AMK)
RG-121150
Tetrahydroquinoline (THQ)
RG-120499
R
1
R
2
Fig. 1. EcR ligands analyzed by microarray
and qRT-PCR analysis.
Ecdysone receptor gene switch ligands S. K. Panguluri et al.
5670 FEBS Journal 274 (2007) 5669–5689 ª 2007 The Authors Journal compilation ª 2007 FEBS
switches [8,14]. In addition, other chemotypes, such
as amidoketones (AMK) and tetrahydroquinolines
(THQ), are also being developed as inducers of EcR
gene switches [16–20] (Fig. 1).
The main goal of the present study was to determine
the intrinsic gene expression effects of EcR switch in-

ducers in mammalian cells. We studied the effect of
eight EcR ligands: four diacylhydrazines, two ecdyster-
oids, one THQ and one AMK ligand (Fig. 1) on the
expression of endogenous genes in HEK 293 cells
using microarray and quantitative real-time reverse
transcription PCR (qRT-PCR). THQ ligand caused
changes in the expression of genes such as TNF, MAF,
Rab and Reprimo. The AMK ligand induced changes
in the expression of genes such as v-jun, FBJ and
EGR. 20-Hydroxyecdysone (20E) did not affect gene
expression, but Pon A caused some changes in the
expression of endogenous genes. At lower ligand con-
centrations applicable for therapeutic use, potential
pleiotropic effects may or may not be observed. The
four diacylhydrazine ligands did not cause significant
changes in the expression of endogenous genes.
Results and Discussion
THQ ligand, RG-120499, affects 293 cells via many
pathways
Incubation of 293 cells with the THQ compound RG-
120499 at 10 lm for 3 days resulted in the death of
70% of the cells as indicated by observation of cell
morphology. To determine the possible genes and path-
ways that are affected by this ligand, we performed a
microarray experiment using RNA isolated from
293 cells treated with 1 lm RG-120499. A total of 1171
genes were up-regulated and 443 genes were down-
regulated in 293 cells treated with this compound
compared to the cells treated with dimethylsulfoxide
(Fig. 2A). Among these, 115 genes showed P £ 0.01

with a two-fold or more greater in expression levels in
ligand-treated cells compared to the levels in dimethyl-
sulfoxide-treated cells (Table 1). Among these 115
genes, 55 genes showed signal detection values of more
than 100. We selected v-maf , TNF, PNAS13, Rab, Rep-
rimo, DNAH and KIF9 genes for qRT-PCR. The qRT-
PCR data (Fig. 2B,C) showed that v-maf, TNF,
PNAS13 and DNAH mRNA levels increased, and Rab
and Reprimo mRNA levels decreased in RG-120499
treated cells, compared to the levels in dimethylsulfox-
ide-treated cells. The microarray and qRT-PCR data
showed perfect correlation for these six genes. The two-
fold down-regulation of the KIF9 gene observed in
microarray analyses was not confirmed by qRT-PCR
because this method showed a ten-fold up-regulation of
this gene in the presence of RG-102499.
MAF [v-maf musculoaponeurotic fibrosarcoma
oncogene homolog (avian)] is a basic-leucine zipper
transcription factor that plays crucial roles in gene reg-
ulation, differentiation, oncogenesis and development
in many organisms [21]. v-maf is a viral oncogene
encoding a leucine zipper motif that forms heterodi-
mers with the protein products of maf-related genes or
other proteins such as fos, jun and myc oncogenes that
have leucine zipper motifs [22]. Our microarray and
qRT-PCR data showed that, in 293 cells, RG-120499
up-regulates v-maf gene expression by 2.4- and
2.3-fold, respectively. Nishizawa et al. [22] have dem-
onstrated that the human cellular counterpart of the
v-maf (c-maf) gene is conserved across species. Addi-

tionally, Massrieh et al. [21] reported that the MAF
transcription factor transcript levels are induced by
proinflammatory cytokines in PHM1-31 myometrial
cells and that MAF transcription factor mRNA is
rapidly induced by IL-1B and TNF in primary
myometrial and PHM1-13 cells. Our data also indicate
up-regulation of the TNF gene in microarray and
qRT-PCR data (by 2.2- and 5.5-fold, respectively), an
observation consistent with TNF-induced up-regulation
of MAF transcript levels. Zheng et al. [23] reported
that the tumor necrosis factor (TNF) can mediate
mature T-cell receptor-induced apoptosis through the
p75 TNF receptor. This may be the possible reason
for the death of 293 cells when treated with 10 lm
RG-120499 ligand.
In addition to the aforementioned up-regulated
genes, two genes, namely, Rab and Reprimo, were
down-regulated by this THQ ligand in 293 cells, as
observed by both microarray and qRT-PCR tech-
niques. The Rab proteins constitute a subfamily of
Ras-related GTP-binding proteins that are localized in
distinct intracellular compartments [24]. Mutations in
the Rab gene can alter the morphology of entire organ-
elles by blocking protein transport along the exocytic
and endocytic pathways because Rab proteins plays a
key role in membrane trafficking [25]. Barbosa et al.
[26] reported that mutations in the Rab gene(s) can
cause irregularities in the protein transport machinery
leading to the formation of giant lysosomes in mouse
beige (bg) mutant and other mutant mice. It has been

suggested that Chediak–Higashi syndrome, a rare
autosomal recessive disorder in humans, is the conse-
quence of a mutation to a homolog of bg. Our micro-
array and qRT-PCR data indicate that the rab gene was
down-regulated in 293 cells by two- and 23-fold, respec-
tively, by the THQ, RG-120499. Mutation studies of the
Rab gene suggest that the Rab gene down-regulation by
S. K. Panguluri et al. Ecdysone receptor gene switch ligands
FEBS Journal 274 (2007) 5669–5689 ª 2007 The Authors Journal compilation ª 2007 FEBS 5671
this ligand may be a reason for cell death in addition to
modulation of the v-maf and TNF genes.
Tumor suppressor genes that encode transcriptional
factors can affect a variety of cellular mechanisms
underlying growth, differentiation, and apoptosis
[27,28]. Also, when cells were exposed to DNA dam-
age-inducing agents or other noxious stress, the p53
protein, which is the most commonly mutated gene in
human cancer, is induced and ⁄ or activated, resulting in
cell cycle arrest or apoptosis [29–31]. Reprimo is a
highly glycosylated protein, which will localize in the
cytoplasm and induce G2 arrest of the cell cycle when
expressed ectopically [32]. In the present study, it was
observed that RG-120499 down-regulates the Reprimo
gene by two-fold as measured by both microarray and
qRT-PCR techniques. From these observations and
previous reports, we suggest that the down-regulation
of the Reprimo gene may cause loss of DNA repair,
which, in turn, is passed on to the next generations,
thereby accumulating DNA defects. RG-120499 modu-
lates the expression of other genes as well. The

PNAS123 gene (transformation related protein 11) is
up-regulated by 2.7- and 4.3-fold as measured by
microarray and qRT-PCR, respectively. The pathways
in which these genes are involved are not known. RG-
120499 also triggers five- and 23-fold up-regulation of
the DNAH (human axonemal dynein heavy chain)
gene as measured by microarray and qRT-PCR tech-
niques, respectively. DNAH is a microtubule associ-
ated motor protein that moves cilia and flagella
[33,34]. Afzelius [35,36] showed that patients suffering
from Kartagener syndrome have cilia lacking dynein
A
BC
1E-051E-041E-03
Significance (t test p-value)
1E-021E-011E-00
/16
5000
0.025
0.02
0.015
0.01
0.005
0
2.7
2.3
5.5
4.3
-23
-2 23

10
2.4
v-Maf
DMSO
THQ
DMSO
THQ
DMSO
THQ
DMSO
THQ
DMSO
THQ
DMSO
THQ
DMSO
THQ
TNF PNAS
RAB
Reprimo
DNAH
KIF
v-Maf
DMSO
THQ
DMSO
THQ
DMSO
THQ
DMSO

THQ
DMSO
THQ
DMSO
THQ
DMSO
THQ
TNF PNAS
RAB
Reprimo DNAH
KIF
2.2
-2
-2
5.1
-2
4500
4000
3500
3000
2500
2000
Signal Values
Relative Expression
1500
1000
500
0
/8 /4 /2 /1.5
Fold Suppression/Induction

1
x1.5 x4 x8 x16
Fig. 2. The THQ ligand RG-120499 affects 293 cells via many pathways. (A) The V-plot of differentially expressed genes from microarray
data. The P-values of t-test are plotted against fold suppression or induction. The horizontal bar in the plot represents the nominal significant
level 0.001 for the t-test under the assumption that each gene has a unique variance. The vertical bars represent the genes that are mini-
mum of two-fold up- or down-regulated compared to the control dimethylsulfoxide (DMSO). (B) The signal values of v-maf, TNF, PNAS, Rab,
Reprimo, DNAH and KIF9 genes from the microarray. The signal values from the microarray analysis were plotted for each gene are
indicated as mean ± SD (n ¼ 3). The numbers above the bar represents the fold changes with this ligand against dimethylsulfoxide. (C) The
relative expressions of v-maf, TNF, PNAS, Rab, Reprimo , DNAH and KIF9 gene transcripts in qRT-PCR. The relative expression values from
the qRT-PCR analysis were plotted for each gene are indicated as mean ± SD (n ¼ 3). The numbers above the bar represents the fold
changes with this ligand against dimethylsulfoxide.
Ecdysone receptor gene switch ligands S. K. Panguluri et al.
5672 FEBS Journal 274 (2007) 5669–5689 ª 2007 The Authors Journal compilation ª 2007 FEBS
arms. This disease is characterized by chronic respira-
tory tract infections, altered position of internal
organs, and infertility arising from immotile sperm.
Milisav and Affara [37] reported that the human
dynein-related gene DNEL2 may play an important
role specifically in sperm motility and is not involved
in the movement of cilia. In summary, the use of the
THQ gene switch ligand RG-120499 may cause signifi-
cant changes in the expression of host genes.
AMK, RG-121150, affects gene expression in
293 cells
The affect of the AMK RG-121150 on gene expression
in 293 cells was analyzed using microarray and qRT-
PCR. In microarray analysis, a total of 636 genes were
up-regulated and 604 genes were down-regulated by
this ligand (Fig. 3A). Among these genes, 71 genes
showed P £ 0.01 and a two-fold or greater change in

expression (Table 1). Among 71 genes, 24 genes
showed signal detection values greater than 100.
Among these are hypothetical proteins, nuclear pro-
teins, transcriptional factors, glycogen phosphorylase,
hormone degrading enzymes, kinases and some solute
carrier proteins. To validate microarray data with
qRT-PCR, the primers for early growth response gene
(EGR), FBJ, v-jun , and a hypothetical protein gene
were designed. For three of the four genes, the micro-
array data was confirmed by qRT-PCR. The data
showed that the hypothetical protein gene was up-regu-
lated by two- and 1.5-fold in microarray and qRT-PCR
experiments, respectively. EGR and v-jun showed sup-
pression of their expression by the ligand in analyses by
both methods (Fig. 3B,C). The gene FBJ mRNA levels
were down-regulated in microarray experiments and up-
regulated in qRT-PCR experiments.
The EGR gene product is a transcription factor that
plays a role in differentiation and growth. EGR genes
are transiently and coordinately induced upon activa-
tion of peripheral blood T lymphocytes [38,39]. These
EGR genes are also expressed in a wide range of cell
types, including lymphoid cells, myeloid cells such as
thymocytes, B cells, monocytes, and nonlymphoid cells
such as fibroblasts, kidney cells and neurons [40,41].
Huang et al. [42,43] showed that the expression of the
EGR gene exogenously in various tumor cells unex-
pectedly and markedly reduces growth and tumorige-
nicity, whereas the suppression of endogenous EGR
by antisense RNA enhances growth and promotes

phenotypic transformation. From our microarray and
qRT-PCR data, the AMK RG-121150 caused down-
regulation of this EGR gene by five- and six-fold,
Table 1. Differentially expressed genes in 293 cells treated with different ligands.
Ligand P-value
Total genes Genes with signal values 100 or more
Up-regulated Down-regulated Total Up-regulated Down-regulated Total
RG-120499 (THQ) < 0.0001 1 0 1 1 0 1
< 0.001 12 5 17 8 0 8
< 0.01 71 26 97 39 7 46
RG-121150 (AMK) < 0.0001 0 1 1 0 1 1
< 0.001 4 9 13 0 6 6
< 0.01 26 31 57 6 11 17
20E (steroid) < 0.0001 0 0 0 0 0 0
< 0.001 1 5 6 0 1 1
< 0.01 15 30 45 0 4 4
Pon A (steroid) < 0.0001 0 1 1 0 0 0
< 0.001 2 4 6 0 0 0
< 0.01 20 15 35 1 2 3
RG-100864
(halofenozide, diacylhydrazine)
< 0.0001 0 0 0 0 0 0
< 0.001 1 3 4 0 0 0
< 0.01 19 15 34 2 1 3
RG-102398
(methoxyfenozide, diacylhydrazine)
< 0.0001 0 0 0 0 0 0
< 0.001 3 4 7 0 0 0
< 0.01 20 25 45 1 2 3
RG-102240

(RSL-1, diacylhydrazine)
< 0.0001 0 1 1 0 1 1
< 0.001 2 2 4 0 0 0
< 0.01 27 14 41 3 2 5
RG-102277 (diacylhydrazine) < 0.0001 0 0 0 0 0 0
< 0.001 3 3 6 1 1 2
< 0.01 21 18 39 0 3 3
S. K. Panguluri et al. Ecdysone receptor gene switch ligands
FEBS Journal 274 (2007) 5669–5689 ª 2007 The Authors Journal compilation ª 2007 FEBS 5673
respectively, indicating a possible effect RG-121150 on
this pathway. Overall, these studies showed that AMK
ligand RG-121150 could cause significant changes in
the expression of genes in host cells. However, the cor-
responding toxicological potential could be substan-
tially mitigated or even erased by the likelihood that
actual gene therapy dosage would be significantly
lower than the 10 lm studied here. Likewise, judicious
choice of the specific member of the AMK chemotype
may confer intrinsic benignity.
Ecdysteroid ligand, 20E, shows little effect on
293 cells but Pon A affects cell division
Two steroid ligands Pon A and 20E were tested in
293 cells to determine their effect on gene expression
using microarray and qRT-PCR. In 20E-treated
293 cells, a total of 542 genes were up-regulated and
627 genes were down-regulated compared to dimethyl-
sulfoxide-treated cells (Fig. 4A). Out of these, 51 genes
showed P £ 0.01 and a change in gene expression of
two-fold or greater (Table 1). Among these 51 genes,
only five genes had signal detection values greater than

100. We selected signal transducer and activator of
transcription 6 (STAT6) genes to perform qRT-PCR
analyses. Although the STAT6 gene was down-regu-
lated by 20E in microarray analysis, the qRT-PCR
analyses showed that the mRNA levels did not change
in treated cells compared to untreated cells
(Fig. 4B,C). Experiments in our laboratory (S. R. Palli,
unpublished results) showed that, in mammalian cells,
20E does not induce reporter genes placed under the
A
BC
1E-051E-041E-03
Significance (t test p-value)
1E-021E-011E-00
/16 /8 /4 /2 /1.5
Fold Suppression/Induction
1 x1.5 x4
1.5
-6.14
-9
3.3
x8 x16
18000
0.2500
0.2000
0.1500
0.1000
0.0500
0.0000
16000

14000
12000
10000
8000
6000
4000
2000
-5.4
DMSO
DMSO
Amido
DMSO
Amido
DMSO
Amido
DMSO
Amido
DMSO
Amido
DMSO
DMSO
2.05
-21
-58
Signal Values
0
Hypo EGR v-jun FBJ
Hypo EGR v-jun FBJ
Relative Expression
ketone

Amido
ketone
Amido
ketone
Amido
ketone
Fig. 3. AMK RG-121150 affects endogene expression in 293 cells. (A) The V-plot of differentially expressed genes from microarray analysis.
The P-values of t-test are plotted against fold suppression or induction. The horizontal bar in the plot represents the nominal significant level
0.001 for the t-test under the assumption that each gene has a unique variance. The vertical bars represent the genes that are minimum of two-
fold up- or down-regulated compared to the control dimethylsulfoxide (DMSO). (B) The signal values of hypothetical protein, EGR, v-jun and FBJ
genes from microarray. The signal values from the microarray analysis were plotted for each gene are indicated as mean ± SD (n ¼ 3). The
numbers above the bar represents the fold changes with this ligand against dimethylsulfoxide. (C) The relative expressions of hypothetical
protein, EGR, v-jun and FBJ gene transcript levels in qRT-PCR. The relative expression values from the qRT-PCR analysis were plotted for each
gene are indicated as mean ± SD (n ¼ 3). The numbers above the bar represents the fold changes with this ligand against dimethylsulfoxide.
Ecdysone receptor gene switch ligands S. K. Panguluri et al.
5674 FEBS Journal 274 (2007) 5669–5689 ª 2007 The Authors Journal compilation ª 2007 FEBS
control of the EcR gene switch. The microarray data
corroborate evidence for an overall benign influence of
20E on gene expression and metabolism in mammalian
cells [44]. It is conceivable that 20E is excluded by
mammalian cells, or perhaps some other factors in the
mammalian cells inhibit the activity of 20E.
A total of 639 genes were up-regulated and 638 were
down-regulated in 293 cells treated with Pon A com-
pared to the cells treated with dimethylsulfoxide
(Fig. 5A). Among these, 41 genes showed P £ 0.01 and
two-fold or greater induction (up ⁄ down-regulated;
Table 1). Only three genes showed signal detection val-
ues greater than 100. Out of these three genes, we
selected tousled-like kinase (Tlk) gene for qRT-PCR

analysis. The data from both methods showed that the
Tlk gene was induced by Pon A by two-fold
(Fig. 5B,C). In proliferating human cells, Tlks are
maximally active during the S phase but rapidly inacti-
vated in response to inhibitors of DNA replication
[45]. Sillje and Nigg [46] showed that the Asf1 (anti-
silencing function 1) proteins are phosphorylated by
Tlks both in vivo and in vitro during the S phase and
dephosphorylated with inactivation of Tlks. Constan-
tino et al. [47] used the ecdysone inducible gene expres-
sion system in hematopoietic cells and found that the
two steroids, muristerone A and Pon A, altered the
signaling pathways induced by IL-3 in the pro-B cell-
line, Ba ⁄ F3. Indeed, they also showed that these ecdy-
steroids potentiate the IL-3-dependent activation of
the PI 3-kinase ⁄ Akt pathway, an effect that could ulti-
mately interfere with the growth, and ⁄ or survival of
these cells. Our data do not reveal any affect of Pon A
on the PI 3-kinase ⁄ Akt pathway in 293 cells. A possi-
ble reason is that these genes might be expressed only
1E-05 1E-04 1E-03
Significance (t test p-value)
1E-02 1E-01 1E-00
/16
250
0.006
0.005
0.004
0.003
Relative Expression

0.002
0.001
0
-2.86
20E
DMSO
STAT6
20E
DMSO
STAT6
-0.7
B
C
200
150
100
S
ignal Values
50
0
A
/8 /4 /2 /1.5
Fold Suppression/Induction
1 x1.5 x4 x8 x16
Fig. 4. Steroidal ligand 20E does not have much effect on 293 cells. (A) The V-plot of differentially expressed genes from microarray analy-
sis. The P-values of t-test are plotted against fold suppression or induction. The horizontal bar in the plot represents the nominal significant
level 0.001 for the t-test under the assumption that each gene has a unique variance. The vertical bars represent the genes that are mini-
mum of two-fold up- or down-regulated compared to the control dimethylsulfoxide (DMSO). (B) The signal values of STAT6 gene from micro-
array. The signal values from the microarray analysis were plotted for each gene are indicated as mean ± SD (n ¼ 3). The numbers above
the bar represents the fold changes with this ligand against dimethylsulfoxide. (C) The relative expressions of STAT6 RNA transcripts as

determined by qRT-PCR. The relative expression values from the qRT-PCR analysis were plotted for each gene are indicated as mean ± SD
(n ¼ 3). The numbers above the bar represents the fold changes with this ligand against dimethylsulfoxide.
S. K. Panguluri et al. Ecdysone receptor gene switch ligands
FEBS Journal 274 (2007) 5669–5689 ª 2007 The Authors Journal compilation ª 2007 FEBS 5675
in a particular type of cells, such as hematopoietic cells,
but not 293 cells. These observations, in conjunction
with our microarray and qRT-PCR data, indicate that
Pon A may affect cell division in mammalian cells.
Diacylhydrazine ligands do not induce significant
changes in gene expression
Diacylhydrazine nonsteroidal ligands have been sub-
jected to extensive studies concerning their toxicologi-
cal effects on vertebrates for the purposes of
Environmental Protection Agency registration as com-
mercial insecticides [48,49]. We selected four represen-
tatives from the diacylhydrazine gene switch
chemotype for evaluation of their effects on expression
of endogenous genes in 293 cells: the lepidopteran
insecticide, methoxyfenozide (RG-102398); the coleopt-
eran insecticide, halofenozide (RG-100864); the Rheo-
SwitchÒ ligand RSL-1 (RG-102240, also known as
GS
TM
-E); and, finally, a more polar and water-soluble
variant of RSL-1, namely RG-102277. First, we deter-
mined the effect of dimethylsulfoxide itself on the
expression of genes in 293 cells. The expressions of a
total of 43 genes were modulated with P £ 0.01 with a
two-fold change in expression. Among the affected
genes, 38 were up-regulated and five of them were

down-regulated. Interestingly, only 13 up-regulated
genes and one down-regulated gene showed signal
detection values of more than 100 in the dimethylsulf-
oxide-treated 293 cells. In our experience, the genes
that show signal detection values less than 100 are not
reliable indicators of gene expression; therefore, we did
not consider these in our analyses.
Cells treated with the diacylhydrazine RG-102240
resulted in up-regulation of a total of 865 genes; on the
other hand, 411 genes were down-regulated in 293 cells
treated with RG-102240 (Fig. 6A). From these, we con-
sidered for further analysis only the genes with P £ 0.01
1E-051E-041E-03
Significance (t test p-value)
1E-021E-011E-00
/16
90
0.0025
0.002
0.0015
0.001
Relative Expression
0.0005
0
2
PonA
DMSO
TLK
PonA
DMSO

TLK
1.99
BC
Signal Values
0
10
20
30
40
50
60
70
80
A
/8 /4 /2 /1.5
Fold Suppression/Induction
1 x1.5 x4 x8 x16
Fig. 5. Steroidal ligand Pon A may affect cell division in 293 cells. (A) The V-plot of differentially expressed genes from microarray analysis.
The P-values of t-test are plotted against fold suppression or induction. The horizontal bar in the plot represents the nominal significant level
0.001 for the t-test under the assumption that each gene has a unique variance. The vertical bars represent the genes that are minimum of
two-fold up- or down-regulated compared to the control dimethylsulfoxide (DMSO). (B) The signal values of Tlk gene from microarray. The
signal values from the microarray analysis were plotted for each gene are indicated as mean ± SD (n ¼ 3). The numbers above the bar rep-
resents the fold changes with this ligand against dimethylsulfoxide. (C) The relative expression of Tlk transcripts as determined by qRT-PCR.
The relative expression values from the qRT-PCR analysis were plotted for each gene are indicated as mean ± SD (n ¼ 3). The numbers
above the bar represents the fold changes with this ligand against dimethylsulfoxide.
Ecdysone receptor gene switch ligands S. K. Panguluri et al.
5676 FEBS Journal 274 (2007) 5669–5689 ª 2007 The Authors Journal compilation ª 2007 FEBS
with a two-fold change in expression compared to the
expression in dimethylsulfoxide-treated cells. A total of
46 genes met these criteria. However, only six of these

genes showed signal detection values greater than 100
(Table 1). The annotations for these genes were devel-
oped by using NIH david ease software (http://david.
niaid.nih.gov/david/ease.htm). Only two out of six were
annotated; these were identified as DAB2 interacting
protein gene and kinesin family member-9 (KIF9).
The reliability of microarray results depends on sev-
eral factors such as array production, RNA extraction,
probe labeling, hybridization conditions and image
analysis [50–53]. Therefore, the genes identified as dif-
ferentially expressed by this method must be validated
with another method such as qRT-PCR, which is
quantitative, rapid, and requires 1000-fold less RNA
than conventional assays [54]. For this reason, we
designed primers for DAB2 interacting protein gene
and KIF9 from their cDNA sequences for qRT-PCR
analysis. Their expression levels were measured in
dimethylsulfoxide- and RG-102240-treated 293 cells
and compared to microarray data. The DAB2 interact-
ing protein gene showed two-fold down-regulation by
microarray and a four-fold increase by qRT-PCR
analysis in RG-102240-treated cells compared to
the expression in dimethylsulfoxide-treated cells
(Fig. 6B,C). The KIF9 gene showed 3.9-fold down-reg-
ulation by microarray and 2.8-fold down-regulation by
qRT-PCR analysis in RG-102240-treated cells com-
pared to the expression in dimethylsulfoxide-treated
cells (Fig. 6B,C). The Drosophila melanogaster Dab
(Disabled) interacting protein is the mammalian ortho-
C

1E-05
1E-04
1E-03
Significance (t test p-value)
1E-02
1E-011E-00
/16
1200
1000
800
600
400
200
0
0.008
0.007
0.006
0.005
Relative Expression
0.004
0.003
0.002
0.001
0
-3.91
-2.11
3.9
DMSO DMSOGSE GSE
DAB Kinesine
DMSO DMSOGSE GSE

DAB Kinesine
-2.8
B
Signal Values
A
/8
/4 /2
/1.5
Fold Suppression/Induction
1
x1.5
x4
x8
x16
Fig. 6. RSL-1 ligand does not cause significant changes in gene expression in 293 cells. (A) The V-plot of differentially expressed genes from
microarray analysis. The P-values of t-test are plotted against fold change in gene expression. The horizontal bar in the plot represents the sig-
nificant level of 0.001 for the t-test under the assumption that each gene has a unique variance. The vertical bars represent the genes that are
minimum of two-fold up- or down-regulated compared to the control dimethylsulfoxide (DMSO). (B) The signal values of Dab and KIF9 genes
from the microarray analysis. The signal values from the microarray analysis were plotted for each gene are indicated as mean ± SD (n ¼ 3).
The numbers above the bar represents the fold changes with this ligand against dimethylsulfoxide. (C) The relative expressions of Dab and
KIF9 genes as determined by qRT-PCR analysis. The relative expression values from the qRT-PCR analysis were plotted for each gene are
indicated as mean ± SD (n ¼ 3). The numbers above the bar represents the fold changes with this ligand against dimethylsulfoxide.
S. K. Panguluri et al. Ecdysone receptor gene switch ligands
FEBS Journal 274 (2007) 5669–5689 ª 2007 The Authors Journal compilation ª 2007 FEBS 5677
logue of D. melanogaster DabIP. The DabIP partici-
pates in a signaling complex containing various pro-
teins involved in brain development as well as other
aspects of adult brain function [55]. Although the
microarray showed two-fold down-regulation of the
Dab gene, the qRT-PCR data showed four-fold

up-regulation. The reasons for difference in the fold
regulation between the two techniques are not readily
apparent. KIF9 protein is found to interact with Ras-
like GTPase Gem and is involved in cell shape remod-
eling [56]. Both microarray data and qRT-PCR data,
showed similar results (3.9-fold and 2.8-fold down-reg-
ulation, respectively) in KIF9 gene expression levels.
Over-expression of the KIF9 mutant did not cause any
significant difference in Gem-induced cell elongation
and it does not appear to be essential for the Gem-
induced phenotypic changes [56]. From these observa-
tions, it can be concluded that, although the RSL-1
ligand down-regulates the KIF9 gene, there may not be
significant changes in cell shape remodeling.
Treatment of HEK 293 cells with RG-102398
resulted in a total of 598 down-regulated and 388
up-regulated genes (Fig. 7A). Among these, 52 genes
indicated P £ 0.01 and a change in expression of two-
fold or greater compared to dimethylsulfoxide-treated
cells (Table 1). Only three genes [ribosomal protein L13
(RPL), hypothetical protein gene FLJ38705 and leptin
receptor] out of these 52 showed signal detection values
greater than 100. The qRT-PCR and microarray anal-
ysis of RPL gene expression are weakly correlated.
The RPL gene was down-regulated by two-fold by
microarray, but was up-regulated 1.25-fold by
C
1E-05
1E-04
1E-03

Significance (t test p-value)
1E-02
1E-01
1E-00
/16
250
0.10000
0.09000
0.08000
0.07000
0.06000
0.05000
0.04000
0.03000
0.02000
0.01000
0.00000
Relative expression
-2.27
-2.05
DMSO DMSO
Zink FingerRPL
RG-102398 RG-102398
DMSO
DMSO
Zink Finger
RPL
RG-120398
RG-120398
-1.49

1.25
B
200
150
100
Signal values
50
0
A
/8 /4 /2 /1.5
Fold Suppression/Induction
1 x1.5 x4 x8 x16
Fig. 7. Effect of RG-102398 on 293 cells. (A) The V-plot of differentially expressed genes from microarray analysis. The P-values of t-test are
plotted against fold suppression or induction. The horizontal bar in the plot represents the nominal significant level 0.001 for the t -test under
the assumption that each gene has a unique variance. The vertical bars represent the genes that are minimum of two-fold up or down regu-
lated compared to the control dimethylsulfoxide (DMSO). (B) The signal values of RPL and Zink finger protein genes. The signal values from
the microarray analysis were plotted for each gene are indicated as mean ± SD (n ¼ 3). The numbers above the bar represents the fold
changes with this ligand against dimethylsulfoxide. (C) The relative expressions of RPL and Zink finger protein RNA levels as determined by
qRT-PCR. The relative expression values from the qRT-PCR analysis were plotted for each gene are indicated as mean ± SD (n ¼ 3). The
numbers above the bar represents the fold changes with this ligand against dimethylsulfoxide.
Ecdysone receptor gene switch ligands S. K. Panguluri et al.
5678 FEBS Journal 274 (2007) 5669–5689 ª 2007 The Authors Journal compilation ª 2007 FEBS
qRT-PCR analysis in RG-102398-treated cells
compared to the expression in dimetylsulfide-treated
cells (Fig. 7B,C). By contrast, a good correlation was
observed between the microarray and qRT-PCR data
with the hypothetical protein gene (down-regulated by
two-fold and 1.4-fold by microarray and qRT-PCR
analyses, respectively) (Fig. 7B,C). The hypothetical
protein FLJ38705 is zinc finger protein 41 homolog

(mouse). The pathways and interactions of this hypo-
thetical protein are not known. RLP is a ribosomal
protein which usually decorates the rRNA cores of the
subunits. Approximately two-thirds of the mass of the
ribosome consists of RNA and one-thirds comprises
protein. The proteins are named in accordance with
the subunit of the ribosome to which they belong: the
small (S1 to S31) and the large (L1 to L44). Many of
ribosomal proteins, particularly those of the large sub-
unit, feature a globular, surface-exposed domain with
long finger-like projections that extend into the rRNA
core to stabilize its structure. Most of the proteins
interact with multiple RNA elements, often from dif-
ferent domains. The crucial activities of decoding and
peptide transfer are RNA-based; proteins play an
active role in functions that may have evolved to
streamline the process of protein synthesis. In addition
to their function in the ribosome, many ribosomal
proteins have some function ‘outside’ the ribosome.
Although the RLP13 gene was affected by the RSL
ligand RG-102398 in the present study, further studies
are needed to confirm its effects, as these two tech-
niques indicate opposite regulation.
Cells treated with RG-100864 showed a total of 566
up-regulated genes and 398 down-regulated genes
(Fig. 8A). Among these, only 38 genes showed
P £ 0.01 and two-fold or greater induction (Table 1).
Only three of these genes showed signal detection
C
1E-05

1E-04
1E-03
Significance (t test p-value)
1E-02
1E-01
1E-00
/16
140
120
100
80
60
40
20
0
0.0160
0.0140
0.0120
0.0100
0.0080
0.0060
0.0040
0.0020
0.0000
Relative Expression
-2.08
RG-100864DMSO
Hypo
RG-100864DMSO
Hypo

-2.6
B
Signal Values
A
/8
/4
/2
/1.5
Fold Suppression/Induction
1
x1.5
x4
x8 x16
Fig. 8. Effect of RG-100864 on 293 cells. (A) The V-plot of differentially expressed gene from microarray analysis. The P-values of t-test are
plotted against fold suppression or induction. The horizontal bar in the plot represents the nominal significant level 0.001 for the t-test under
the assumption that each gene has a unique variance. The vertical bars represent the genes that are minimum of two-fold up- or down-
regulated compared to the control dimethylsulfoxide (DMSO). (B) The signal values of hypothetical protein gene from the microarray. The signal
values from the microarray analysis were plotted for each gene are indicated as mean ± SD (n ¼ 3). The numbers above the bar represents the
fold changes with this ligand against dimethylsulfoxide. (C) The relative expressions of hypothetical protein transcript levels as determined by
qRT-PCR. The relative expression values from the qRT-PCR analysis were plotted for each gene are indicated as mean ± SD (n ¼ 3). The
numbers above the bar represents the fold changes with this ligand against dimethylsulfoxide.
S. K. Panguluri et al. Ecdysone receptor gene switch ligands
FEBS Journal 274 (2007) 5669–5689 ª 2007 The Authors Journal compilation ª 2007 FEBS 5679
values greater than 100. A cDNA sequence is available
for only one gene out of these three: hypothetical pro-
tein gene FLJ22344 , to which we have designed primers
and performed qRT-PCR. The microarray data and
qRT-PCR data for this hypothetical protein gene are
negatively correlated. In the microarray experiment, the
hypothetical protein gene showed two-fold up-regula-

tion compared to two-fold down-regulation in qRT-
PCR analysis (Fig. 8B,C). The pathways and interac-
tions of this hypothetical protein are not known.
In 293 cells treated with RG-102277, 512 genes were
up-regulated and 456 genes were down-regulated com-
pared to dimethylsulfoxide-treated cells (Fig. 9A).
Among these genes, 45 genes showed P £ 0.01 and
two-fold or greater induction (up ⁄ down regulated)
(Table 1). Only five out of these 45 genes showed sig-
nal detection values greater than 100, of which two
genes [mannose receptor C type 2 (MRC) and
KIAA0515] had assigned cDNA sequences. MRC
expression, showed a positive correlation between
qRT-PCR and microarray data (2.8- and 3.3-fold
down-regulated, respectively; Fig. 9B,C). MRC, also
known as Endo 180, is a 180 kDa transmembrane gly-
coprotein that is a part of trimolecular cell surface
complex with urokinase-type plasmogen activator and
its receptor [57]. This trimolecular complex plays an
important role in cell guidance and chemotaxis during
normal and pathological events [58]. The expression of
MRC is restricted to stromal cells, macrophages, endo-
thelial cells, tumor endothelium and breast cancer tis-
sue in normal conditions [59–61]. The up-regulation of
this MRC is observed in tumor endothelial cells [62],
which have a potential role in the regulation of tumor
neoangiogenesis. The ligand RG-102277 showed down-
regulation of this MRC gene in 293 cells as measured
by both analytical techniques. Because only up-regula-
C

1E-051E-041E-03
Significance (t test p-value)
1E-021E-011E-00
/16
160
140
120
100
80
60
40
20
0
0.160000
0.140000
0.120000
0.100000
0.080000
0.060000
0.040000
0.020000
0.000000
Relative expression
2.01
-3.35
DMSO
MRC
KIAA
DMSORG-
102277

RG-
102277
DMSO
MRC KIAA
DMSORG-
102277
RG-
102277
-1.3
-2.8
B
Signal Values
A
/8
/4
/2
/1.5
Fold Suppression/Induction
1
x1.5
x4
x8
x16
Fig. 9. Effect of RG-102277 on 293 cells. (A) The V-plot of differentially expressed genes from microarray analysis. The P-values of t-test are
plotted against fold suppression or induction. The horizontal bar in the plot represents the nominal significant level 0.001 for the t -test under
the assumption that each gene has a unique variance. The vertical bars represent the genes that are minimum of two-fold up- or down-regu-
lated compared to the control dimethylsulfoxide (DMSO). (B) The signal values of MRC and KIAA genes from the microarray. The signal val-
ues from the microarray analysis were plotted for each gene are indicated as mean ± SD (n ¼ 3). The numbers above the bar represents
the fold changes with this ligand against dimethylsulfoxide. (C) The relative expressions of MRC and KIAA transcripts as determined by
qRT-PCR. The relative expression values from the qRT-PCR analysis were plotted for each gene are indicated as mean ± SD (n ¼ 3). The

numbers above the bar represents the fold changes with this ligand against dimethylsulfoxide.
Ecdysone receptor gene switch ligands S. K. Panguluri et al.
5680 FEBS Journal 274 (2007) 5669–5689 ª 2007 The Authors Journal compilation ª 2007 FEBS
tion will cause tumor neoangiogenesis, the down-
regulation of this gene by RG-102277 probably does
not contraindicate its use.
Overall, the effect of diacylhydrazines on the expres-
sion of genes in 293 cells was minimal as shown by
microarray as well as by qRT-PCR data. There appear
to be differences among the individual diacylhydra-
zines, but it may be premature to interpret these data
in terms of structure–activity relationships. No signifi-
cant pathway is affected by these ligands. Only one or
two genes have a cDNA sequence available for qRT-
PCR analysis and only half of the genes studied
showed positive correlation between microarray and
qRT-PCR analysis. The poor correlation between the
microarray and qRT-PCR data for these genes is due
to the fact that the small variations in the mRNA levels
can be accurately detected by qRT-PCR, but might not
be accurately reflected in microarray expression scores,
especially for genes expressed at low levels (approxi-
mately three or four copies per cell) [63]. Etienne et al.
[64] have hypothesized that, in addition to genes with
low expression levels, those with very high expression
levels or a greater percent of absent calls may show
lower levels of correlation between microarray and
semiquantitative qRT-PCR data. The level of expres-
sion differences between microarray and qRT-PCR
may also be due to the lack of specificity in the primers

designed to discriminate gene family members at the
level of primary screening by DNA arrays [54]. There-
fore, to the degree of resolution afforded by combined
qRT-PCR and microarray analysis, we concluded that
these diacylhydrazine ligands are safe for use as induc-
ers of gene switches, especially because the concentra-
tions anticipated for gene therapy would likely be lower
than the 1 lm examined in the present study.
Conclusion
The present investigation analyzed the effects of EcR
gene switch ligands on the expression of endogenous
genes. Two widely used analytical techniques, oligo
nucleotide probe microarray and qRT-PCR, were
employed to perform the analyses. Keeping in mind
that the ligand concentrations in this investigation are
high relative to the anticipated pharmacologically rele-
vant blood levels for gene therapy, the THQ RG-
120499 caused significant changes in the expression of
genes such as v-maf, TNF, PNAS13, Rab, Reprimo and
DNAH at 1 lm. An AMK, RG-121150, affected the
expression of genes such as EGR , FBJ and v-jun at
10 lm. Also at 10 lm, the steroidal ligand, Pon A
induced only the Tlk gene, known to interfere with the
growth and ⁄ or survival of cells. Otherwise, like RG-
121150, Pon A also appears to be a benign substance.
The homologue 20E did not cause significant changes
in gene expression at all. Likewise, at 10 lm, four di-
acylhydrazine nonsteroidal ligands did not cause signif-
icant changes in gene expression, except for minor
effects on a few genes of lesser significance, KIF9, RPL

and MRC. The relatively minor perturbation of gene
expression in 293 cells for the diacylhydrazines corro-
borates on a gene expression level the known favorable
mammalian toxicology.
Experimental procedures
Sources, preparation, and characterization of
ligands
General procedures
Most reagents were purchased from Aldrich (Milwaukee,
WI, USA), VWR (West Chester, PA, USA), or Fisher Scien-
tific (Pittsburg, PA, USA). Pon A and 20E were purchased
from Alexis Biochemicals (Lausen, Switzerland). Both sub-
stances were assayed without further purification. Solvents
were reagent grade unless otherwise stated. Anhydrous
solvents were used as purchased. Analytical TLC was per-
formed on Macherey–Nagel Polygram
Ò
Sil G ⁄ UV
254
0.2 mm
plates. Most plates were visualized by UV light; some plates
were developed using iodine or phosphomolybdic acid. Silica
gel chromatography was performed using Aldrich silica gel
(230–400 mesh, 60 A
˚
) in glass columns under a N
2
or argon
head pressure of approximately 30 psi. Melting points were
measured in glass capillary tubes and are uncorrected. Most

1
H NMR spectra were recorded at 400.13 MHz with a
Bruker DPX-400;
13
C NMR spectra were recorded at
100.6 MHz with a Bruker DPX-400 at NMR Services in
Rochester, NY. Some
1
H NMR was performed at 200 MHz
on a Varian instrument (Palo Alto, CA, USA) or 300 MHz
on a Bruker instrument (Billerica, MA, USA). Unless other-
wise stated, internal reference is solvent. LC-MS analysis was
performed using an Agilent 1100 LC stack coupled with an
Agilent (Foster City, CA, USA) single quad mass spectro-
meter. Solvents were (A) H
2
O ⁄ 0.1% formic acid and (B)
ACN ⁄ 0.1% formic acid in a gradient of T ¼ 0 15% B to
T ¼ 10 98% B and a stop time of 20 min on a
75 mm · 2.1 mm C18 column with a flow rate ¼ 0.2 mLÆ
min
)1
. Exact mass analysis were performed by direct infusion
into an Agilent ESI ⁄ TOF mass spectrometer. Mass spec-
trometry was performed by the Scripps Research Institute
mass spectroscopy service (La Jolla, CA, USA).
Preparation and purification of ligands
The diacylhydrazine RG-102277 (Fig. 10) was prepared in
a manner analogous to literature precedent [13] with adjust-
ments in the final phase of the synthesis to incorporate the

S. K. Panguluri et al. Ecdysone receptor gene switch ligands
FEBS Journal 274 (2007) 5669–5689 ª 2007 The Authors Journal compilation ª 2007 FEBS 5681
amide group. 2-Ethyl-3-methoxybenzoic acid 1 was con-
verted to the corresponding acid chloride and coupled with
t-butylhydrazine under controlled conditions to form a
mono-acylated hydrazine 2. The alternate ring was con-
structed from 3,5-dimethyl benzoic acid 3. Rather than
direct bromination of the acid [14], we found that esterifica-
tion to the methyl ester followed by controlled bromination
with N-bromosuccinimide and distillation provides pure
and plentiful quantities of 3-bromomethyl-5-methyl benzo-
ate. Subsequent de-esterification using HBr smoothly yields
the corresponding carboxylic acid. The bromomethyl
benzoic acid, in turn, was converted to the acid chloride 4
using thionyl chloride and then coupled with the aforemen-
tioned mono-hydrazide 2. The benzylic bromide was con-
verted to the benzyl alcohol 6 using calcium carbonate, and
the alcohol was then oxidized first to the aldehyde 7 and
then to the carboxylic acid 8 using pyridinium chlorochro-
mate and potassium permanganate, respectively [13]. In our
hands, this two-step oxidation provided purer product in
higher yields than did direct oxidation. The diacylhydrazine
carboxylic acid was then esterified to an activated ester 9
with pentafluorophenol using N¢,N¢-dicyclohexylcarbodi-
imide as a coupling agent. Finally, the pentafluorophenol
was displaced with ammonia in dioxane ⁄ water to provide
RG-102277. The diacylhydrazines, as well as the THQ
RG-120499 and AMK RG-121150 were purified by flash
chromatography and ⁄ or crystallization to 99% purity or
greater as judged by 400 MHz

1
H NMR. The ecdysteroids
were used in the form obtained from the commercial vendor.
[6-Fluoro-4-(4-fluoro-phenylamino)-2-methyl-3,4-
dihydro-2H-quinolin-1-yl]-(3-fluoro-4-methyl-phenyl)-
methanone (RG-120499)
RG-120499 was prepared according to the previously
described procedure [17,18].
1
H NMR (400 MHz, dimethyl-
sulfoxide-d
6
) d: 7.22 (t, J ¼ 8.0 Hz, 1H), 7.11 (d, J ¼
10.4 Hz, 1H), 7.04–6.96 (m, 4H), 6.88–6.79 (m, 3H), 6.64
(dd, J ¼ 4.8, 8.4 Hz, 1H), 6.02 (d, J ¼ 8.4 Hz, 1H), 4.78–
4.69 (m, 1H), 4.66–4.59 (m, 1H), 2.77 (ddd, J ¼ 3.6, 8.8,
12.8 Hz, 1H), 2.23 (s, 3H), 1.26 (ddd, J ¼ 3.2, 8.8, 12.0 Hz,
1H), 1.20 (d, J ¼ 5.6 Hz, 3H);
13
C NMR (100 MHz,
dimethylsulfoxide-d
6
) d: 167.60, 161.53, 161.11, 158.99,
158.82, 156.27, 153.86, 144.86, 140.28, 136.37, 133.40,
131.60, 128.61, 126.88, 126.62, 124.67, 116.05, 115.83,
115.68, 115.44, 114.16, 114.09, 113.70, 113.48, 111.31,
111.07, 49.25, 48.81, 21.48, 14.38; HRES (ESI) m ⁄ z calcu-
lated for C
24
H

22
F
3
N
2
O[M
+
+H]
+
411.1684, found
411.1653, calculated for C
24
H
21
F
3
N
2
NaO [M
+
+ Na]
+
433.1504, found 433.1508.
5-Ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic
acid[1-(3,5-dimethyl-benzoyl)-cyclopentyl]-amide
(RG-121510)
RG-121510 was prepared according to the previously
described procedure [15,16].
1
H NMR (400 MHz, dimethyl-

sulfoxide-d
6
) d: 7.52 (s, 2H), 7.15 (s, 1H), 6.68 (d, J ¼
8.4 Hz, 1H), 6.57 (d, J ¼ 8.4 Hz, 1H), 6.30 (s, 1H), 4.28 (s,
O
COOH
O
N
H
O
NH
O
N
H
O
N
O
Br
O
N
H
O
N
O
R
O
N
H
O
N

O
O
NH
2
COOH
Br
COCl
a, b
g
R = CH2OH
CHO
C(O)OH
C(O)OPh-F
5
h, i, j, k
l
RG-102277
c, d, e, f
12
34
5
6
7
8
9
Fig. 10. Synthesis of the diacylhydrazine RG-102277. (a) SOCl
2
, dimethylformamide; (b) t-BuNHNH
3
Cl, aqueous K

2
CO
3
,CH
2
Cl
2
; (c) CH
3
OH,
Et
3
N, CH
2
Cl
2
; (d) N-bromosuccinimide, AIBN, CCl
4
; (e) aqueous HBr, 100 °C 3 h; (f) SOCl
2
, dimethylformamide, CHCl
3
; (g) aqueous K
2
CO
3
,
CH
2
Cl

2
; (h) CaCO
3
,H
2
O, dioxane; (i) PCC, CH
2
Cl
2
; (j) KMnO
4
, t-BuOH; (k) PfOH, N¢,N¢-dicyclohexylcarbodiimide; (l) NH
4
OH, dioxane, 60 °C, 6 h.
Ecdysone receptor gene switch ligands S. K. Panguluri et al.
5682 FEBS Journal 274 (2007) 5669–5689 ª 2007 The Authors Journal compilation ª 2007 FEBS
4H), 2.68 (m, 2H), 2.42–2.35 (m, 8H), 2.15–2.10 (m, 2H),
1.94–1.87 (m, 4H), 1.00 (t, J ¼ 7.2 Hz, 3H);
13
C NMR
(100 MHz, dimethylsulfoxide-d
6
) d: 201.68, 168.63, 144.67,
141.65, 137.30, 136.85, 133.10, 132.28, 129.20, 125.99,
119.12, 114.38, 70.91, 64.22, 37.78, 24.90, 21.35, 19.62,
14.70; HRES (ESI) m ⁄ z calculated for C
25
H
30
NO

4
[M
+
+H]
+
408.2175, found 408.2177, calculated for
C
25
H
29
NNaO
4
[M
+
+ Na]
+
430.1994, found 430.1991.
Benzoic acid N-tert-butyl-N¢-(4-chloro-benzoyl)-
hydrazide (RG-100864)
RG-100864 was prepared according to the previously de-
scribed procedure [65,66].
1
H NMR (400 MHz, dimethyl-
sulfoxide-d
6
) d: 10.75 (s, 1H), 7.55–7.49 (m, 5H), 7.45 (d,
J ¼ 6.8 Hz, 2H), 7.40 (dd, J ¼ 2.4, 9.6 Hz, 2H), 1.52
(s, 9H);
13
C NMR (400 MHz, dimethylsulfoxide-d

6
) d:
171.32, 166.35, 136.98, 134.16, 132.52, 132.32, 128.92,
128.81, 127.97, 127.40, 60.91, 27.78; HRMS (ESI) m ⁄ z
calculated for C
18
H
19
ClN
2
NaO
2
[M
+
+Na] 353.1022,
found 353.1033.
3,5-Dimethyl-benzoic acid N-tert-butyl-N¢-
(3-ethoxy-2-methyl-benzoyl)-hydrazide (RG-102398)
RG-102398 was prepared according to the previously de-
scribed procedure [67].
1
H NMR (400 MHz, dimethylsulf-
oxide-d
6
) d: 10.48 (s, 1H), 7.14 (t, J ¼ 8.0 Hz, 1H), 7.09
(s, 2H), 7.07 (s, 1H), 7.00 (d, J ¼ 8.0 Hz, 1H), 6.28 (d,
J ¼ 7.6 Hz, 1H), 3.78 (s, 3H), 3.29 (s, 6H), 1.75 (s, 3H),
1.50 (s, 9H);
13
C NMR (100 MHz, dimethylsulfoxide-d

6
)
d: 172.57, 167.72, 157.60, 138.34, 137.02, 136.11, 130.86,
126.85, 124.97, 124.07, 118.63, 112.21, 60.11, 55.75, 28.19,
21.21, 11.52; HRMS (ESI) m ⁄ z calculated for
C
22
H
29
N
2
O
3
[M
+
+ H] 369.2178, found 369.2169, calcu-
lated for C
22
H
28
N
2
NaO
3
[M
+
+ Na] 391.1998, found
391.1985.
3,5-Dimethyl-benzoic acid N-tert-butyl-N¢-
(3-methoxy-2-ethyl-benzoyl)-hydrazide (RG-102240)

RG-102240 was prepared according to the previously de-
scribed procedure [67].
1
H NMR (400 MHz, dimethylsulf-
oxide-d
6
) d: 10.50 (s, 1H), 7.12 (t, J ¼ 8.0 Hz, 1H), 7.09
(s, 3H), 7.00 (d, J ¼ 8.0 Hz, 1H), 6.20 (d, J ¼ 7.6 Hz,
1H), 3.77 (s, 3H), 2.34–2.25 (m, 1H), 2.29 (s, 6H), 2.13–
2.01 (m, 1H), 1.52 (s, 9H), 0.88 (t, J ¼ 7.2 Hz, 3H);
13
C NMR (100 MHz, dimethylsulfoxide-d
6
) d: 172.61,
167.98, 157.30, 138.32, 137.09, 135.73, 130.78, 130.40,
126.81, 124.87, 118.64, 112.44, 60.52, 56.07, 28.17, 21.08,
19.98, 15.31; HRMS (ESI) m ⁄ z calculated for
C
23
H
31
N
2
O
3
[M
+
+ H] 383.2335, found 383.2320, calcu-
lated for C
23

H
30
N
2
O
3
[M
+
+ Na] 405.2154, found
405.2140.
2-Ethyl-3-methoxybenzoyl chloride
2-ethyl-3-methoxybenzoic acid [68] (60 g, 330 mmol) was
suspended in 80 mL CHCl
3
in a 500 mL round-bottom
flask equipped with condensor, addition funnel, thermo-
meter, and KOH trap. Dimethylformamide (0.5 mL) was
added, and the mixture was heated at 30 °C, thereby dis-
solving much of the acid. Thionyl chloride (30 mL) was
added through the addition funnel over a period of approx-
imately 0.5 h, maintaining temperature at approximately
30 °C. The reaction was heated at reflux for approximately
2 h. A Dean-Stark trap was attached, and excess CHCl
3
and SOCl
2
was removed by distillation. The Dean-Stark
trap was removed, and a short distillation head was
attached. Product acid chloride was distilled under vacuum
(approximately 1 mm). Two fractions of 2-ethyl-3-meth-

oxybenzoyl chloride were collected: a forerun (6.46 g,
9.8%, b.p. 93–95 °C at approximately 1 torr) possibly con-
taining a small amount of SOCl
2
, and a main fraction
(56.36 g, 86%, b.p. 93–95 °C at approximately 1 torr). The
product crystallized as it cooled to room temperature.
1
H NMR (400 MHz, CDCl
3
) d: 7.67 (d,1H), 7.29 (t, 1H),
7.08 (d, 1H), 3.87 (s, 3H), 2.88 (q, 2H), 1.15 (t, 3H).
2-Ethyl-3-methoxy-benzoic acid N¢-tert-butyl-hydrazide
(2)
A 500 mL, three-neck flask, equipped with magnetic stir
bar and thermometer was charged with 100 mL CH
2
Cl
2
and aqueous K
2
CO
3
(37.4 g, 270.6 mmol in 60 mL in
water). The biphasic mixture was cooled in an ice bath
and solid t-butylhydrazine hydrochloride (26.87 g,
215.6 mmol) was added in portions. The mixture was
cooled to )3 °C, and vigorously stirred as 2-ethyl-3-meth-
oxybenzoyl chloride (prepared from 2-ethyl-3-methoxyben-
zoic acid, 24.4 g, 135.4 mmol, after removal of thionyl

chloride and without further purification) was added drop-
wise as a solution in 50 mL CH
2
Cl
2
, while maintaining the
temperature below )2 °C. After the addition was com-
pleted, stirring was continued at )2 °Cto)5 °C for
another 2 h, and then at room temperature overnight.
Water was added to dissolve salts, the mixture was shaken
in a separatory funnel, and the aqueous phase was
removed. The remaining CH
2
Cl
2
phase was washed twice
with water and then shaken vigorously with 100 mL 10%
NaOH. This aqueous NaOH phase was chilled on ice,
acidified with 10% HCl, and extracted three times with
ethyl acetate. The ethyl acetate solution was then dried
over MgSO
4
, and solvent was evaporated to yield 2-ethyl-
3-methoxy-benzoic acid N¢-tert-butyl-hydrazide as a
slightly orange solid (10.9 g, 43.5 mmol, 32.1% yield),
quite pure as analyzed by TLC (R
f
¼ 0.28, 4 : 1 CH
2
Cl

2
:
EtOAc). The original CH
2
Cl
2
phase, now dark in color,
was dried over MgSO
4
, filtered and solvent was removed
S. K. Panguluri et al. Ecdysone receptor gene switch ligands
FEBS Journal 274 (2007) 5669–5689 ª 2007 The Authors Journal compilation ª 2007 FEBS 5683
in vacuum to yield 26 g of crude product. This material
was chromatographed on silica gel using 4 : 1 CH
2
Cl
2
:
EtOAc as eluant, to provide an additional 18.5 g
(73.9 mmol, 54.7% yield) purified product. Mp ¼ 90–
91 °C.
1
H NMR (200 MHz, CDCl
3
) d: 7.20 (t, 1H), 7.0
(br s, 1H), 6.92 (d, 2H), 4.95 (br, 1H), 3.85 (s, 3H), 2.75
(q, 2H), 1.12 (t, 3H), 1.2 (s, 9H);
13
C NMR (100 MHz,
CDCl

3
) d: 169.4, 157.8, 135.1, 131.6, 126.8, 119.1, 112.3,
56.2, 55.6, 26.9, 20.7, 14.9. A secondary fraction, 4.5 g of
less pure product was obtained.
Methyl 3,5-dimethylbenzoate
3,5-dimethylbenzoyl chloride (268 g, 1.59 mol) was dissolved
in 400 mL methylene chloride in a 1 L round bottom flask.
The mixture was cooled in an ice ⁄ water bath to 5 °C. Metha-
nol (1.91 mol, 61 g, 1.2 equivalents) was added with stirring.
A mixture of 241 g (2.38 mol) triethylamine in 100 mL meth-
ylene chloride was then carefully added via dropping funnel
over a period of approximately 45 min. The mixture was stir-
red at 5 °C for approximately 30 min, then permitted to
warm to room temperature and stirred for 2 h. The mixture
was then poured into a separatory funnel and shaken once
with 10% HCl, once with water, once with aqueous
NaHCO
3
, and finally with brine. The organic phase was
dried over Na
2
SO
4
, and solvent was removed in vacuo., to
provide a light yellow solid (225 g, 97% yield).
Methyl 3-bromomethyl-5-methylbenzoate
Methyl 3,5-dimethylbenzoate (255 g, 1.55 mol) was dis-
solved in 300 mL CCl
4
in a 2 L round bottom flask with

mechanical stirring and heated to reflux. N-bromosuccini-
mide (212 g, 1.19 mol) and 2,2¢-azobis(2-methylpropionitri-
le) (AIBN, 5 g) were added portionwise over a period of
approximately 6 h. The reaction was monitored by GC and
stopped when the quantity of dibromide approximately
equaled unreacted starting material. The heterogeneous
mixture was filtered through a bed of silica gel and solvent
was removed in vacuo. The crude product was distilled
under vacuum (b.p. 130 °C, approximately 5 mmHg) and
recrystallized from pentane to yield 65 g of white solid
(23% yield, 97–99% pure).
1
H NMR (200 MHz, CDCl
3
) d:
7.90 (s, 1H), 7.70 (s, 1H), 7.4 (s, 1H), 4.5 (s, 2H), 3.92 (s,
3H), 2.4 (s, 3H). Significantly larger quantities of slightly
less pure material were also obtained. Higher boiling frac-
tions contained methyl 3,5-bromomethylbenzoate which
was also recrystallized from pentane: (25 g, 6.5% yield,
99% pure).
3-Bromomethyl-5-methylbenzoic acid
Methyl 3-bromomethyl-5-methylbenzoate (38 g, 160 mmol),
48% HBr (300 g), and 100 mL water were mixed in a
500 mL round bottom flask and stirred at 105 °C for 3 h,
then at room temperature overnight. The mixture was
poured onto ice, and the precipitate was collected on a sin-
tered funnel, washed with water and pentane, and air-dried
to yield 31 g (85% yield) 3-bromomethyl-5-methylbenzoic
acid.

1
H NMR (200 MHz, CDCl
3
) d: 7.97 (s, 1H), 7.90
(s, 1H), 7.47 (s, 1H), 4.5 (s, 2H), 2.45 (s, 3H).
3-Bromomethyl-5-methylbenzoyl chloride (4)
3-bromomethyl-5-methylbenzoic acid (31 g 140 mmol),
oxalyl chloride (86 g, 680 mmol), and CHCl
3
(10 mL) were
mixed in a 300 mL round bottom flasked and stirred at
50 °C for 5 h, then at room temperature overnight. As the
reaction was incomplete, two drops of dimethylformamide
were added and the mixture heated at 55 °C for 2 h. Excess
oxalyl chloride and CHCl
3
were removed in vacuo, CCl
4
was added, and volatiles were removed again. The crude
product was recrystallized from hexane, then washed with
pentane to yield 10.5 g of pale yellow solid, m.p. 45–46 °C.
1
H NMR (200 MHz, CDCl
3
) d: 7.95 (s, 1H); 7.90 (s, 1H);
7.55 (s, 1H); 4.50 (s, 2H); 2.45 (s, 3H). An additional
approximately 20 g of product was recovered by repeated
recrystallizations.
N-(3-bromomethyl-5-methyl-benzoyl)-N-tert-butyl-N¢-
(2-ethyl-3-methoxy-benzoyl)-hydrazide (5)

The literature procedure was followed [19]. Into a 100 mL
round bottom flask containing 2.50 g (10 mmol) of 2-ethyl-
3-methoxy-benzoyl-N¢-tert-butyl-hydrazide was added
15 mL of methylene chloride, 2.60 g (10.5 mmol) of 3-bro-
momethyl-5-methylbenzoyl chloride in 5 mL of methylene
chloride and a solution of 2.76 g (20 mmol) of potassium
carbonate in 15 mL of water. The reaction mixture was
stirred overnight at room temperature, then diluted with
20 mL of methylene chloride and transferred to a separato-
ry funnel. The methylene chloride layer was separated and
dried, and the solvent was removed in vacuo. The crude
product was purified by column chromatography to yield
4.01 g of N-(3-bromomethyl-5-methyl-benzoyl)-N-tert-
butyl-N¢-(2-ethyl-3-methoxy-benzoyl)-hydrazide (87%
yield).
1
H NMR (300 MHz, CDCL
3
) d: 7.41 (s, 1H), 7.1
(m, 3H), 7.02 (t, 1H), 6.082 (d, 1H), 6.08 (d, 1H), 4.41 (s,
2H), 3.78 (s, 3H), 2.4 (m, 1H), 2.31 (s, 3H), 2.25 (m, 1H),
1.60 (s, 9H), 1.01 (t, 3H).
N-(3-hydroxymethyl-5-methyl-benzoyl)-N-tert-butyl-
N¢-(2-ethyl-3-methoxy-benzyl)-hydrazide (6)
The literature procedure was followed [19]. To 4.00 g
(8.68 mmol) of N-(3-bromomethyl-5-methyl-benzoyl)-N-
tert-butyl-N¢-(2-ethyl-3-methoxy-benzoyl)-hydrazide, con-
tained in a 250 mL round bottom flask, were added 40 mL
Ecdysone receptor gene switch ligands S. K. Panguluri et al.
5684 FEBS Journal 274 (2007) 5669–5689 ª 2007 The Authors Journal compilation ª 2007 FEBS

of dioxane, 40 mL of water, and 4.34 g of calcium carbon-
ate. The reaction flask was placed into an 85 °C oil bath
and the reaction was stirred and heated for 18 h. The reac-
tion mixture was cooled, transferred to a larger flask with
ethyl acetate and most of the dioxane was evaporated. The
reaction mixture was shaken with about 100 mL of ethyl
acetate and filtered. The ethyl acetate layer was separated
and the aqueous layer extracted twice with ethyl acetate.
Ethyl acetate extract was dried and evaporated to yield
2.07 g of N-(3-hydroxymethyl-5-methyl-benzoyl)-N-tert-
butyl-N¢-(2-ethyl-3-methoxy-benzyl)-hydrazide (60% yield).
1
H NMR (300 MHz, CDCl
3
) d: 7.78 (s, 1H), 7.1–7.4 (3 s,
3H), 6.96 (t, 1H), 6.8 (d, 1H), 6.08 (d, 1H), 4.53 (s, 2H),
3.77 (s, 3H), 2.35 (m, 1H), 2.32 (s, 3H), 2.2 (m, 1H), 1.60
(s, 9H), 0.96 (t, 3H).
3-Formyl-5-methyl-benzoic acid N-tert-butyl-N¢-
(2-ethyl-3-methoxy-benzoyl)-hydrazide (7)
The literature procedure was followed [19]. To 2.00 g
(5.02 mmol) of N-(3-hydroxymethyl-5-methyl-benzoyl)-N-
tert-butyl-N¢-(2-ethyl-3-methoxy-benzoyl)-hydrazide placed
in a 250 mL round bottom flask, were added 100 mL of
methylene chloride and 1.16 g of pyridinium chlorochro-
mate (PCC). The reaction mixture was refluxed for approxi-
mately 1 h, at which time TLC (1 : 1 ethyl acetate: hexane)
indicated the formation of product (R
f
¼ 0.5). The reaction

mixture was concentrated to about 20 mL and then chro-
matographed on silica gel. Elution with 30–35% ethyl
acetate in hexane yielded 1.75 g (88%) 3-formyl-5-methyl-
benzoic acid N-tert-butyl-N¢-(2-ethyl-3-methoxy-benzoyl)-
hydrazide as a white solid.
1
H NMR (300 MHz, CDCl
3
) d:
9.93 (s, 1H), 7.6–7.8 (3 s, 3H), 7.0 (t, 1H), 6.82 (d, 1H),
6.19 (d, 1H), 3.77 (s, 3H), 2.42 (s, 3H), 2.3 (m, 1H), 2.0 (m,
1H), 1.62 (s, 9H), 0.90 (t, 3H).
3-[N-tert-butyl-N¢-(2-ethyl-3-methoxy-benzoyl)-
hydrazinocarbonyl]-5-methyl-benzoic acid (8)
In a 200 mL round-bottom flask, 3-formyl-5-methyl-
benzoic acid N-tert-butyl-N¢-(2-ethyl-3-methoxy-benzoyl)-
hydrazide 3.74 g (9.43 mmol) was dissolved in 25 mL warm
t-BuOH. Phosphate buffer (25 mL, pH 7.2, Aldrich
31925.2) was added, and the mixture was stirred while
1.65 g (10.4 mmol) KMnO
4
was added. The reaction was
then stirred at 45–50 °C for 3 h. The reaction mixture was
poured into 300 mL cold water. Up to approximately
50 mL of 0.5% NaOH was used to wash glassware. The
aqueous phases were combined and filtered through What-
man #3 filter paper to remove most of the MnO
2
sludge.
The brown aqueous filtrate was extracted thrice with

150 mL CH
2
Cl
2
. The organic extracts were dried over
MgSO
4
, treated with charcoal, and evaporated to provide
2.81 g (72.2% yield) 3-[N-tert-butyl-N¢-(2-ethyl-3-methoxy-
benzoyl)-hydrazinocarbonyl]-5-methyl-benzoic acid as a
cream-colored solid. Since the product did not seem to be
very soluble in CH
2
Cl
2
, the original aqueous solution was
again extracted with 250 mL ethyl acetate. This was evapo-
rated to yield an additional 0.14 g (3.6% yield) of product.
1
H NMR (300 MHz, dimethylsulfoxide) d: 7.8 (br s, 2H),
7.5 (s, 1H), 7.15 (t, 1H), 7.0 (d, 1H), 6.22 (d, 1H), 4.2 (br,
1H), 3.74 (s, 3H), 2.42 (s, 3H), 2.2 (m, 1H), 1.87 (m, 1H),
1.51 (s, 9H), 0.8 (t, 3H).
3-[N-tert-butyl-N¢-(2-ethyl-3-methoxy-benzoyl)-
hydrazinocarbonyl]-5-methyl-benzoic acid
pentafluorophenyl ester (9)
Pentafluorophenol (0.74 g, 4 mmol as a 50% solution in
ethyl acetate) was added to a solution of 3-[N-tert-butyl-
N¢-(2-ethyl-3-methoxy-benzoyl)-hydrazinocarbonyl]-5-methyl-
benzoic acid (1.5 g, 36.4 mmol) in 30 mL ethyl acetate.

N¢,N¢-Dicyclohexylcarbodiimide (3.7 mL of a 1 m solution
in CH
2
Cl
2
, 3.7 mmol) was added and the mixture was
stirred at room temperature for 3 days. The reaction
mixture was partitioned between water and ethyl acetate.
The organic phase was separated, dried over MgSO
4
, and
filtered. Solvent was removed in vacuo to give 3.0 g of a
thick oily solid. This material was purified by silica gel
chromatography using a gradient of 0–40% ethyl acetate in
hexanes to provide 1.76 g (84% yield) 3-[ N-tert-butyl-N¢-(2-
ethyl-3-methoxy-benzoyl)-hydrazinocarbonyl]-5-methyl-ben-
zoic acid pentafluorophenyl ester.
1
H NMR (400 MHz,
CDCl
3
) d: 8.2 (s, 1H), 8.1 (s, 1H), 7.75 (s, 1H), 7.6 (br s,
1H), 7.1 (t, 1H), 6.9 (d, 1H), 6.3 (d, 1H), 3.8 (s, 3H), 2.5 (s,
3H), 2.4 (m, 1H), 2.15 (m, 1H), 1.62 (s, 9H), 0.95 (t, 3H).
3-[N-tert-butyl-N¢-(2-ethyl-3-methoxy-benzoyl)-
hydrazinocarbonyl]-5-methyl-benzamide (RG-102277)
3-[N-tert-butyl-N¢-(2-ethyl-3-methoxy-benzoyl)-hydrazino-
carbonyl]-5-methyl-benzoic acid pentafluorophenyl ester
(150 mg, 0.259 mmol) was stirred in a solution of 1 mL
concentrated NH

4
OH and 2 mL dioxane at 60 °C for 6 h.
The mixture was partitioned with water, and the organic
layer was concentrated. The residue was purified by silica
gel column chromatography using 100% ethyl acetate with
0.1–2% of triethylamine to provide 3-[N-tert-butyl-N¢-(2-
ethyl-3-methoxy-benzoyl)-hydrazinocarbonyl]-5-methyl-ben-
zamide (46 mg, 43% yield). TLC R
f
¼ 0.06, 1 : 1 ethyl ace-
tate:hexane + triethylamine).
1
H NMR (400 MHz, CDCl
3
)
d: 7.9 (s,1H), 7.75 (s 1H), 7.58 (s, 1H), 7.55 (s, 1H), 7.0 (t,
1H), 6.85 (d, 1H), 6.3 (br, 1H), 6.2 (d, 1H), 5.6 (br, 1H),
3.77 (s, 3H), 2.4 (s, 3H), 2.38 (m, 1H), 2.1 (m, 1H), 1.60 (s,
9H), 0.95 (t, 3H).
1
H NMR (400 MHz, dimethylsulfoxide-
d
6
) d: 10.58 (s, 1H), 7.96 (s, 1H), 7.78 (s, 2H), 7.44 (s, 1H),
7.37 (s, 1H), 7.12 (t, J ¼ 8.0 Hz, 1H), 7.01 (d, J ¼ 8.0 Hz,
1H), 6.23 (d, J ¼ 7.2 Hz, 1H), 3.77 (s, 3H), 2.39 (s, 3H),
S. K. Panguluri et al. Ecdysone receptor gene switch ligands
FEBS Journal 274 (2007) 5669–5689 ª 2007 The Authors Journal compilation ª 2007 FEBS 5685
2.24 (dt, J ¼ 7.2, 14.4 Hz, 1H), 1.89 (dt, J ¼ 7.2, 12.8 Hz,
1H), 1.55 (s, 9H), 0.84 (t, J ¼ 7.2 Hz, 3H);

13
C NMR
(100 MHz, dimethylsulfoxide-d
6
) d: 171.57, 167.20, 157.69,
138.34, 137.46, 135.71, 134.04, 130.49, 129.35, 127.07,
123.87, 118.71, 112.40, 61.01, 56.08, 27.81, 21.16, 20.12,
14.99; HRMS (ESI) m ⁄ z calculated for C
23
H
30
N
3
O
4
[M
+
+ H] 412.2236, found 412.2229, calculated for
C
23
H
29
N
3
NaO
4
[M
+
+ Na] 434.2056, found 434.2026.
Cell lines, reagents, and ligand treatment

The human embryonic kidney cell cultures (HEK 293) were
propagated in DMEM supplemented with 10% fetal bovine
serum. All media and serum were purchased from Life
Technologies (Rockville, MD, USA). The cells were cul-
tured to 20% confluency in 25 cm
2
flasks and ligands (1 or
10 lm) or dimethylsulfoxide control were added to each
flask. The cells were grown for three days in a CO
2
incuba-
tor at 37 °C and cell morphology was observed daily under
a light microscope.
Isolation of total RNA
The cells were harvested at 3 days after the addition of
ligand (10 lm for all ligands except RG-120499 which
was added at 1 lm). The cells were collected by scraping
with a rubber policeman and centrifuged at 2300 g for
5 min. The medium was discarded and 500 lL of Triazol
reagent (Molecular Research Center Inc., Cincinnati, OH,
USA) was added to the cell pellet. The cells were homog-
enized, 100 lL of chloroform was added, and the mixture
was gently agitated while keeping at room temperature
for 5 min, and then centrifuged at 15 800 g for 15 min at
4 °C. The supernatant was transferred into a fresh tube,
500 lL isopropanol was added, and the mixture incu-
bated at room temperature for 5 min. The mixture was
then centrifuged at 15 800 g for 15 min at 4 °C. The
supernatant was discarded slowly and the total RNA
pellet was washed with 70% ethanol, dried and dissolved

in nuclease-free water. The RNA was quantified both
visually (agarose gel electrophoresis) and UV-spectro-
photometer. cDNA synthesis by reverse transcription was
perfoemd using 2 lg of DNAse
-
1 (Ambion Inc., Austin,
TX, USA)-treated RNA and iScript cDNA synthesis kit
(Bio-Rad Laboratories, Hercules, CA, USA).
Microarray analysis
The microarray analysis was performed at the UK micro-
array core facility (University of Kentucky, Lexington, KY,
USA). The total RNA was converted into double-stranded
cDNA using a T7-oligo (dT) promoter primer. Following
RNase H-mediated second-strand cDNA synthesis, the
double-stranded cDNA was purified and served as a
template in the subsequent in vitro transcription reactions.
The in vitro transcription reaction was carried out in the
presence of T7 RNA polymerase and a biotinylated nucleo-
tide analog ⁄ ribonucleotide mix for complementary RNA
(cRNA) amplification and biotin labeling. The biotinylated
cRNA targets were then purified, fragmented, and hybrid-
ized to GeneChip expression arrays Human U133 plus 2.0
(Affymetrix Inc., Santa Clara, CA, USA). Array images
were processed to determine signals and detection calls (P,
present; A, absent; M, marginal) for each probe set using a
GeneChip Operating Software (GCOS) Computer Worksta-
tion (Affymetrix Inc.). Microarray analyses used three chips
per each treatment.
Data analysis
Data analysis was performed using SAS (SAS Institute,

Cary, NC, USA), r for Windows GUI front-end (http://
www.r-project.org), and q-value software [69]. The probe
sets with absent calls across all samples were removed to
reduce the multiple-testing problem. The expression levels
were normalized to the chip median and log transformed.
Two-way analysis of variance tests are carried out to iden-
tify differentially expressed genes. For each probe set, the
model y
ijk
¼ l + a
i
+ b
j
+ c
ij
+ e
ijk
was fit, where y
ijk
is
the log-transformed expression level of the kth chip in the
ith treatment and the jth replicate. The variable l repre-
sents the grand mean expression, a
i
is the effect due to the
treatment, b
j
is the effect due to the replicate, c
ij
is the

interaction effect between treatment and replicate, and e
ijk
is an error term, which is assumed to be normally distrib-
uted with mean 0 and variance r
2
.
Q-values computed using Q-value software indicates the
false detection rate for each probe set. Ratio comparison was
performed by dividing expression levels of expression in
ligand treated cells with the expression levels in dimethylsulf-
oxide-treated cells. The gene sets that showed two-fold induc-
tion or two-fold suppression were transferred to separate up
and down lists, respectively. The genes sets with a P £ 0.01
were identified in these lists and gene annotation was per-
formed for these probe sets at Affymetrix NetAffx analysis
center ( />Functional classification of select probe sets was performed
at the NIH DAVID server ( />upload.asp). Volcano plots were prepared using the r pro-
gram.
qRT-PCR analysis
The genes that showed signal detection values more than
100, P £ 0.01 and a change in gene expression of more than
two-fold compared to the control (dimethylsulfoxide) were
selected to study their relative expressions by qRT-PCR
using a MyiQ single color real-time PCR detection system
Ecdysone receptor gene switch ligands S. K. Panguluri et al.
5686 FEBS Journal 274 (2007) 5669–5689 ª 2007 The Authors Journal compilation ª 2007 FEBS
(Bio-Rad Laboratories). qRT-PCR was performed in a
total of 20 lL reaction volume containing 1 lL of cDNA,
1 lL of each of forward and reverse sequence specific prim-
ers (from 10 lm primer stock), 10 lL of supermix (Bio-Rad

Laboratories) and 7 lL of nuclease free water. All the
qRT-PCR reactions were performed using the following
reaction conditions: Initial incubation of 95 °C for 3 min
followed by 45 cycles of 95 °C for 10 s, 60 °C for 20 s, and
72 °C for 30 s. A fluorescence reading determined the
extent of amplification at the end of each cycle.
Standard curves were obtained using a ten-fold serial
dilution of pooled cDNA from all treatments. The expres-
sion of Homo sapiens actin gene was used as an internal
control. For all the candidate genes, the quantities of the
mRNA expression relative to actin mRNA levels were
obtained. All samples were measured in triplicate. The PCR
efficiency and correlation coefficient values were taken into
account before estimating the relative expression. Mean
and standard errors for each treatment were obtained from
all three replicates.
Acknowledgements
This work was supported in part by RheoGene Inc.
research grant to SRP. This is contribution number
07-08-102 from the Kentucky Agricultural Experimen-
tal Station.
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