A transcription factor hijacking to regulate RARα by using a chimeric molecule of retinoic acid and a DNA alkylator

Article abstract of DOI:10.1016/j.bmcl.2011.05.062

As a model compound for the transcription factor hijacking mechanism of action of DNA damaging agent that simultaneously bind to the nuclear receptor, we designed and synthesized a chimeric molecule, RA-mustard, which can bind with both retinoic acid rece

Full text of DOI:10.1016/j.bmcl.2011.05.062

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4248–4251
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A transcription factor hijacking to regulate RAR
molecule of retinoic acid and a DNA alkylator
a by using a chimeric
Kyung Hoon Min a, Byoung-Gu Yun b, Yoonsung Lee b, Jiho Song a, Won-Hoon Ham b,
YoungJoo Lee c, Hyun-Ju Park b,
⇑
a College of Pharmacy, Chung-Ang University, Seoul 156-756, Republic of Korea
b School of Pharmacy, Sungkyunkwan University, Suwon 449-746, Republic of Korea
c Department of Bioscience & Biotechnology, College of Life Science, Sejong University, Seoul 143-747, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 January 2011
Revised 9 May 2011
Accepted 19 May 2011
Available online 25 May 2011
As a model compound for the transcription factor hijacking mechanism of action of DNA damaging agent
that simultaneously bind to the nuclear receptor, we designed and synthesized a chimeric molecule, RA-
mustard, which can bind with both retinoic acid receptor
RA-mustard with RAR was confirmed by binding assay using RAR
mustard-modified DNA diminished the RAR -dependent luciferase expression in the RAR
Ó 2011 Elsevier Ltd. All rights reserved.
a
(RARa
) and DNA. The interaction between
-overexpressing cell extract. RA-
-abundant cells.
a
a
a
a
Keywords:
Transcription factor hijacking
Chimeric molecule
Chlorambucil
All-trans retinoic acid
Retinoic acid receptor
a
The development of small molecule regulators has attracted
considerable attention in the context of expanding our under-
standing of complicated biological processes. Chemical genetic ap-
proaches have proven to be extremely useful in studies of protein
function without any genetic manipulation. The design and devel-
opment of small molecules capable of inducing the selective loss of
a targeted protein is one of the major goals of chemical genetics.
Meanwhile, the design and development of chimeric small
molecules with two functional moieties capable of interacting
simultaneously with two molecular targets is also receiving great
attention.1 Several chimeric small molecules that regulate biologi-
cal events have been reported. STF1 (a hairpin polyamide-
wrenchnolol)2 and FkCsA (FK506-cyclosporin A)3 were designed
as transcription activators, whereas PROTACs4 were designed to
degrade target proteins.
on this notion, bifunctional ligands that alkylate DNA and bind to
specific nuclear transcription factors have been designed. Exam-
ples of such ligands include estradiol-linked genotoxicants6,7 and
11b-dichloro,8,9 which disrupt gene expression by hijacking estro-
gen receptor (ER) and androgen receptor (AR), respectively. These
activities eventually manifest as selective toxicity against cancer
cells over-expressing corresponding nuclear receptors, ER-positive
MCF6,7 and AR-positive LNCaP8,9 cells.
Our research interests include the design and development of
chimeric small molecules that regulate proteins of interest, and
verifying the transcription factor hijacking model. We chose to
target the retinoic acid receptor (RARa), a nuclear receptor
Cisplatin reacts with DNA to form 1,2-intrastrand cross-links,
and it is generally accepted to be responsible for its anticancer
activity. Interestingly, the ribosomal RNA transcription factor hUBF
binds preferentially to cisplatin-DNA adducts, thus reducing the
hUBF binding to its native DNA regulatory site and abolishing
hUBF-dependent transcription. It is evident that this hijacking of
a transcription factor could lead to functional inhibition.5 Based
chlorambucil
all-
trans
retinoic acid
O
N
H
N
Cl
Cl
⇑
Corresponding author.
E-mail address: hyunju85@skku.edu (H.-J. Park).
Figure 1. Structure of RA-mustard, a simple chimeric molecule that consists of an
all trans-retinoic acid moiety for RAR and chlorambucil for DNA alkylation.
a
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.062

K. H. Min et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4248–4251
4249
Figure 2. (a) A docking model of RA-mustard in the ligand-binding site of RAR
a. Bound antagonist BMS614 (orange) in the X-ray structure (PDB id: 1DKF) is shown for
comparison. The retinoic acid moiety of RA-mustard occupies the binding site of BMS614, whereas its nitrogen mustard moiety is exposed outside the binding pocket. (b)
Lipophilic potential surface map of the ligand-binding pocket of RAR
a
is displayed in the docking model. Lipophilicity increases from blue (hydrophilic) to brown (lipophilic).
1. ClCO2Et, Et3N, acetone,
NH2
COOH
then NaN3, 80 o
C
of ATRA to RAR
data).
a in a dose-dependent manner (see Supplementary
Cl
N
Cl
N
2. 8N HCl
Next, we explored whether the DNA adduct of RA-mustard
could be formed. Accordingly, RA-mustard was incubated with
Cl
Cl
O
[c-
32P]-50-end labeled duplex oligonucleotide DNA. As shown in
all trans-retinoic acid
N
H
Figure 3a, RA-mustard formed cross-linked and monoalkylated
DNA adducts, indicating that RA-mustard maintains the ability to
alkylate DNA. The major alkylation site of DNA by chlorambucil
is the N7 positions of guanine base.13 The DNA alkylation sites were
confirmed by strand breakage assays, in which piperidine treat-
ment generates strand breaks at the drug-modified sites.13 As com-
pared with chlorambucil, the sequence selectivity of RA-mustard
alkylation was also unchanged (Fig. 3b).
N
EDCI, HOBT, Et3N
CH2Cl2, rt
Cl
Cl
Scheme 1. Synthesis of RA-mustard.
transcription factor, which is an attractive target because it plays
essential roles in cell proliferation and differentiation.10,11
The present study, in which a hybrid molecule of an endoge-
nous ligand (retinoic acid) and a DNA alkylating agent (chlorambu-
cil) is used as a probe molecule, aims to demonstrate that a
To verify the interaction between RARa and RA-mustard modi-
fied DNA, electrophoretic mobility shift assay (EMSA)14 was con-
ducted (Fig. 4). The reaction product obtained from the binding
reaction of RAR
a-overexpressed cell extract with RA-mustard/
transcription factor hijacking mechanism works in the RAR
a-
DNA adduct migrated more slowly than the corresponding RAR
a-
abundant cells.
free RA-mustard/DNA adduct, whereas no shifted band was ob-
As an initial step, we designed a simple chimeric molecule (RA-
mustard) to hijack RAR (Fig. 1). All trans-retinoic acid (ATRA) is
an endogenous ligand for RAR , and nitrogen mustard chlorambu-
cil can form a covalent bond with DNA. ATRA (a RAR binding
served with the chlorambucil/DNA adduct, indicating that the
a
RA-mustard/DNA adduct can hijack RAR
a.
a
Taken together, these findings demonstrate that RA-mustard is
a
a bifunctional agent that interacts with both RAR
bases in duplex DNA.
a and guanine
moiety) and nitrogen mustard (a DNA binding moiety) are linked
to form a hetero-bifunctional molecule. We expected that RA-
The ability of RA-mustard to abolish the transcriptional activity
of RAR by hijacking was evaluated in COS cells using a reporter
mustard would inhibit the function of RAR
a by driving RARa to
a
ligand-modified DNA sites rather than to specific promoter
sequences.
construct in which transcription of the reporter gene was con-
trolled by retinoic acid response element (RARE) (Fig. 5a).15 ATRA
responsive luciferase reporter gene (RARE-luc) was found to be
RA-mustard was subjected to docking analysis to determine
whether it can access the binding site of RAR
a. RA-mustard was
activated by ATRA in the presence of RAR
mustard modified plasmid DNA into COS cells with RARE-luc and
plasmid expressing RAR was found to suppress the transcription
of the reporter gene in a dose-dependent manner (Fig. 5b).
In summary, we describe a transcription factor hijack system
based on the use of a chimeric small molecule that interacts with
a. Cotransfection of RA-
docked into the X-ray structure of RAR
a
(PDB ID: 1DKF)12 using
the FlexX program. The docking model showed that the retinoic
acid moiety can access the binding pocket for antagonist (BMS-
614), whereas the nitrogen mustard moiety protrudes outside
the binding site and can interact with DNA (Fig. 2). Therefore, we
synthesized the chimeric molecule, RA-mustard (Scheme 1).
Chlorambucil was readily converted to an amine using the Curtis
rearrangement. EDCI-mediated coupling of the amine with all
trans-retinoic acid gave RA-mustard (see Supplementary data for
detailed information about syntheses and spectroscopic character-
ization of the compounds).
a
both DNA and RAR
a (a nuclear receptor transcription factor). The
design of this compound by molecular modeling resulted in the
development of a bifunctional agent retaining both good affinity
for RAR
that the hybrid molecule forms cross-linked DNA adducts and hi-
jacks RAR , and that this potentially leads to the specific inhibition
of RAR -dependent gene expression. We believe that the nitrogen
a and the capacity to covalently modify DNA. We found
a
We carried out the competitive binding assay in which RA-
a
mustard competes for [3H]-labeled ATRA in the reaction with RAR
a
mustard moiety could be replaced with other useful functional
over-expressing COS cell extract. RA-mustard inhibited the binding
molecules, such as, sequence specific DNA binder polyamides or

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K. H. Min et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4248–4251
Figure 4. Electrophoretic mobility shift assay.14 A 50-end-labeled DNA fragment
and RA-mustard complex was incubated with RAR -overexpressing cell extract,
a
and the then reaction mixture was subjected to electrophoresis using a 5% low salt
nondenaturing gel.
Figure 3. DNA Alkylation with RA-mustard. (a)Autoradiogram of a 20% denaturing
polyacrylamide gel showing the drug-modified and non-modified species. After the
reaction of 50-end-labeled 16-base-pair duplex oligomers ([ 32P]-50-AATTATGGC-
c-
CATATTA-30/50-TAATATGGCCATAATT-30) with RA-mustard and chlorambucil,
respectively. CO is without drug treatment and CO0 is from the reaction with
DMSO. The numbered bands were excised and the DNA was extracted by 10%
annealing buffer. (b)Autoradiogram of 12% denaturing polyacrylamide gels showing
the DNA strand-breakage patterns of the oligonucleotides (1–4 and 10–30) excised
from the gel in (a). AG and TC represent the purine and pyrimidine specific chemical
cleavage reaction. Asterisks indicate the drug-modified bases.
Figure 5. (a) A schematic of the ATRA responsive reporter gene assay. RA-mustard
modified plasmid DNA was co-transfected with an ATRA-responsive luciferase
reporter gene (RARE-luc reporter plasmid) into COS cells. (b) Mixtures of various
ratios of RA-mustard modified to non-modified plasmid DNA were then co-
transfected with RAR vector and RARE-Luc reporter plasmid into COS cell. All cells
were pre-treated with ATRA except for CO (control). After incubation for 24 h,
luciferase activities were measured.15 ⁄p <0.05 versus the sample with 0:4.
other alkylating agents. Furthermore, we believe that our results
provide potentially useful information for the design of program-
mable transcription inactivator.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.05.062.
Acknowledgments
This study was supported by a Grant of the Korea Healthcare
technology R&D Project, Ministry for Health & Welfare, Republic
of Korea (Grant A092006), and in part by Basic Science Research
Program through the National Research Foundation of Korea
(NRF) funded by the Ministry of Education, Science and Technology
(Grants 2010-0029358 and 314-2008-1-E00305).
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for 20 min. Reaction mixtures were loaded onto 5% low salt gels were dried for
40 min and exposed to a molecular imaging plate. Images were recorded using
a BAS-250 imager.
15. Transient transfection and luciferase assays. COS cells were seeded in 24-well
plates at a density of 7 ꢀ 104 cells/well. After 24 h, plasmids were transiently
transfected into cells using a calcium phosphate-DNA coprecipitation method.
A total of 0.5
25
lg of DNA in 25 ll of CaCl2ꢁH2O (250 mM CaCl2) was mixed with
l
l of 2ꢀ HBS (280 mM NaCl, 10 mM KCl, 1.5 mM Na2HPO4ꢁ2H2O, 12 mM
dextrose, 50 mM HEPES) with constant bubbling and within 5–10 min this
solution was added to each well. The next day, transfected cells were washed
with PBS and add enough 1X lysis buffer to cover the cells. Cell lysate was
stored at ꢂ70 °C. Luciferase activities were determined using the luciferase
assay system using 20 ll of cell lysate with 100 ll of Luciferase Assay Reagent
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14. Electrophoretic mobility shift assay (EMSA). Nuclear extracts were isolated from
(Promega, Madison, WI) and an AutoLumat LB9507 luminometer. Results are
expressed in relative light units. The means and standard deviations of
triplicate samples are shown. All transfection experiments were repeated three
or more times and similar results were obtained. Statistical analyses were
performed by Student’s t-test for luciferase assays using the SPSS 12.0
statistical software program (SPSS, Chicago, IL). The results were considered
to be statistically significant at p <0.05.
COS cells overexpressing RARa, according to the manufacturer’s instructions
(Activemotif, USA). Protein concentrations were determined using the Bradford
method. Gel retardation assays were performed as reported. Probe DNA (2000–
5000 cpm/ll) from [c-
32P]-50-end-labeled plasmid (pUC18) fragmented with
restriction enzyme EarI and HindIII, 10 mM RA-mustard or 1 mM chlorambucil
were mixed in 10% annealing buffer (1 mM Tris–HCl, 10 mM NaCl) and were