Discovery of a small molecular compound simultaneously targeting RXR and HADC: Design, synthesis, molecular docking and bioassay

Article abstract of DOI:10.1016/j.bmcl.2013.04.067

Retinoid X receptor (RXR) and Histone deacetylase (HDAC) are considered important targets for anti-cancer therapy due to their crucial roles in genetic or epigenetic regulations of cancer development and progression. Here, we have designed and synthesized a novel compound which targets both RXR and HADC. This dual-targeting agent is derived from bexarotene and suberoylanilide hydroxamic acid (SAHA), prototypical RXR agonist and HDAC inhibitor, respectively. Molecular docking studies demonstrate that this agent has a relatively strong affinity to RXR and HADC. Importantly, it presents the potentials of activation of RXR and inhibition of HDAC in both cell-free and whole-cell assays, and displays anti-proliferative effect on representative cancer cell lines and drug-resistant cancer cell lines.

Full text of DOI:10.1016/j.bmcl.2013.04.067

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3891–3895
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of a small molecular compound simultaneously targeting
RXR and HADC: Design, synthesis, molecular docking and bioassay
Guo-Liang Chen a, , Li-Hui Wang b,c, , Jian Wang a, Kang Chen b,c, Man Zhao a, Zhao-Zhu Sun a,
Shuang Wang b,c, Hong-Li Zheng b,c, Jing-Yu Yang b,c, , Chun-Fu Wu b,c,
⇑
⇑
a Key Laboratory of Structure-Based Drugs Design & Discovery of Ministry of Education, Shenyang Pharmaceutical University, 103 Wenhua Road, 110016 Shenyang, PR China
b Department of Pharmacology, Shenyang Pharmaceutical University, 103 Wenhua Road, 110016 Shenyang, PR China
c Benxi Institute of Pharmaceutical Research, Shenyang Pharmaceutical University, 103 Wenhua Road, 110016 Shenyang, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Retinoid X receptor (RXR) and Histone deacetylase (HDAC) are considered important targets for anti-can-
cer therapy due to their crucial roles in genetic or epigenetic regulations of cancer development and pro-
gression. Here, we have designed and synthesized a novel compound which targets both RXR and HADC.
This dual-targeting agent is derived from bexarotene and suberoylanilide hydroxamic acid (SAHA), pro-
totypical RXR agonist and HDAC inhibitor, respectively. Molecular docking studies demonstrate that this
agent has a relatively strong affinity to RXR and HADC. Importantly, it presents the potentials of activa-
tion of RXR and inhibition of HDAC in both cell-free and whole-cell assays, and displays anti-proliferative
effect on representative cancer cell lines and drug-resistant cancer cell lines.
Received 9 December 2012
Revised 9 April 2013
Accepted 25 April 2013
Available online 7 May 2013
Keywords:
Retinoid X receptors
Histone deacetylase
Dual-target
Ó 2013 Elsevier Ltd. All rights reserved.
Anti-cancer
Mechanism-based targeted therapies as a treatment for human
cancers has been heralded as one of the major breakthroughs in
human cancer research in the past three decades. However, the
clinical effectiveness of such treatment is generally unsustainable
in the long run and relapses are almost inevitable after the treat-
ment.1 One explanation is that the core hallmark capabilities of
cancer are regulated by partially redundant signaling molecules.
As such, a targeted therapeutic agent inhibiting only one key mol-
ecule in a tumor may not completely shut off the core hallmark
capability, allowing some cancer cells to survive with residual
function.1 To address this problem, the next-generation of anti-
cancer medicine will have to incorporate, within a single molecule,
elements that simultaneously tackle multiple targets for cancer
therapy.2
Retinoid X receptor (RXR) is a promising target for anti-cancer
therapy. RXR belongs to a member of the nuclear receptor super-
family that regulates many physiological functions, including cell
growth, differentiation and apoptosis.3 RXR act as ligand-depen-
dent transcription factor that functions as heterodimer with reti-
noid acid receprtor (RAR) to modulate the transcriptional activity
of retinoid receptor target genes associated with cell growth and
differentiation. Recently, RXR agonist has been identified as an
important agent for cancer treatment.3 A synthetic RXR agonist
bexarotene has been approved by FDA for the treatment of cutane-
ous T-cell lymphoma (CTCL).4 In addition, several clinical trials are
ongoing to assess the potential of bexarotene for treatment of
other tumors. However, a large number of the known RXR agonists
have elicited only limited in vivo antitumor activities and have not
progressed beyond clinical trials.5,6 The RXR agonists that modu-
late the functions of additional intracellular targets, other than
the RXR, may be able to ameliorate the shortcomings of current
RXR agonists.
Histone deacetylase (HDAC), which deacetylate lysines on core
histones and other cellular proteins,7 plays the crucial roles in the
epigenetic regulation of gene transcription and controlling other
cellular functions, particularly on cancer.8 HDAC inhibitors have
been demonstrated to induce cell-cycle arrest, terminal differenti-
ation and apoptosis in a broad spectrum of human tumor cell lines
in vitro8, and have antiangiogenic and antitumor activity in human
xenograft models.8,9 Two HDAC inhibitors, SAHA (vorinostat) and
FK228 (romidepsin), have been approved by FDA for the treatment
of CTCL. Preclinical data with numerous cancer cell lines has shown
synergistic and additive effects when combining HDAC inhibitors
with various antitumor therapies, suggesting HDAC might be an
ideal target for combination.10
In theory, hyperacetylation of histone proteins induced by
HDAC inhibitors could increase the accessibility of DNA within
chromatin, loosen gene promoter and consequently potentiate
the anticancer activities of RXR agonist. Moreover, recent research
⇑
Corresponding authors. Tel./fax: +86 24 23986339 (C.-F.W.).
E-mail addresses: yangjingyu2006@gmail.com (J.-Y. Yang), wucf@syphu.edu.cn
(C.-F. Wu).
These two authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.04.067

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G.-L. Chen et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3891–3895
a
b
CONHOH
COOH
COCl
DW22
Bexarotene
Scheme 1. Reagents and condition: (a) SOCl2, CH2Cl2, 89%; (b) NH2OHÁHCLÁKOH, MeOH, 92%.
has shown that the combination of RXR agonist and HDAC inhibi-
tor synergistically induced apoptosis and inhibited the invasion of
colon cancer cells.11 More importantly, clinical trials demonstrated
that HDAC inhibitor could augment the antitumor effect of RXR
agonist in CTCL patients.12 This evidence suggests that simulta-
neous RXR activation and HDAC inhibition could be a promising
approach in cancer therapy.
potent anti-proliferative activity across a broader range of human
cancer cell lines.
Hydroxamic acid derivatives are key HADC inhibitors due to
their powerful zinc-binding ability. Taking bexarotene as a lead
compound, we designed bexarotene hydroxamic acid (DW22) by
incorporating the hydroxamic acid moiety into its structure in or-
der to obtain a molecular with dual-target on RXR and HDAC. The
preparation of bexarotene hydroxamic acid (DW22) is shown in
Scheme 1.13
Here, we disclosed a small molecular with dual acting RXR acti-
vation and HDAC inhibition. In addition, the compound exhibited
Table 1
The structures and binding free energies of bexarotene and DW22
Compd
Structure
Binding free energy (kcal/mol)
HDAC-2(3MAX)
Binding free energy (kcal/mol)
RXR- (3H0A)
a
Bexarotene
DW22
À8.74
À12.93
À13.07
COOH
À10.47
CONHOH
Figure 1. Binding modes of compound bexarotene (A), displayed with ball and stick with green color) and bexarotene_hydroxamic (B), displayed with ball and stick with pink
color) with RXR and their bioactive-conformational alignments (C) within the receptor site.
Figure 2. Binding modes of compound bexarotene (A), displayed with ball and stick with green color) and bexarotene_hydroxamic (B), displayed with ball and stick with pink
color) with HDAC-2 and their bioactive-conformational alignments (C) within the receptor site.

G.-L. Chen et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3891–3895
3893
Molecular docking studies were performed to investigate the
binding affinities and interaction modes for the dual-targeting
compound using AutoDock 3.05.14 The ligand structures were built
with the Sybyl software package15 and minimized using the Tripos
force field and assigned charges using the Gasteiger–Hückel meth-
od. The crystal structures of RXR (PDB code: 3H0A16) and HDAC-2
(PDB code: 3MAX17) were selected as the receptor. Polar hydrogens
were added and partial charges were loaded for protein using the
Kollman United Atom charges. Atomic solvation parameters and
fragmental volumes for the proteins were assigned using the
addsol utility in the program package. A grid map of dimensions
22.5 Â 22.5 Â 22.5 Å, with a grid spacing of 0.375 Å was generated
for falcipain-2. Affinity grid fields were generated using the
auxiliary program AutoGrid3.0.
The Lamarckian genetic algorithm (LGA) was used to find the
appropriate binding positions, orientations, and conformations of
the ligands. The resulting data was taken from docking experi-
ments in which the lowest total docking energy was obtained.
Ten LGA runs, each with 300 individuals in the population, were
performed. Results differing by less than 2 Å in a positional root
mean square deviation (rmsd) were grouped together. In each
group, the lowest binding energy configuration with the highest%
frequency was selected as the group representative.
The predicted binding free energies for the compounds bexaro-
tene and DW22 were À8.74 and À10.47 kcal/mol for HDAC-2, and
À12.93 and À13.07 kcal/mol for RXR, respectively (Table 1). Within
the RXR binding site, both bexarotene and DW22 formed hydrogen
bonds with Arg 316 and Ala 327, sharing a similar binding mode (
Fig. 1). Within the HDAC-2 binding site, bexarotene formed hydro-
gen bonds with Tyr308, while compound Asp181, Asp269 and
Gly306. They also bound with similar poses (Fig. 2).
To further understand the activities of DW22 against RXR-in-
volved transcriptions, we carried out cell-based transactivation as-
says by cotransfection of RXRa with its specific response element
reporter.18 As shown in Figure 3, DW22 activated RXR reporter
with EC50 values 19.5 3.6 nmol/L, which is lower than that of
bexarotene (EC50 = 149.0 2.9 nmol/L). These results demonstrate
that DW22 has improved biological activity on RXR activation as
compared to bexarotene.
Figure 3. The effects of DW22 and bexarotene on RXR activation.
Figure 4. The effects of DW22 and SAHA on HDAC inhibition in cell free assay (A), HL60 cell assay (B), acetylated H3 and p21WAF1/CIP1 (C). Histone 3 and b-actin were used as
loading controls, respectively, and protein expression was quantified by densitometry relative to control. Quantification of the effect of exposure to increasing concentrations
of DW22 on acetylated H3 and p21WAF1/CIP1 expression is shown column diagram.

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G.-L. Chen et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3891–3895
We subsequently tested the HDAC inhibition activity of DW22
and SAHA with IC50 of 80.3 5.2 lmol/L for A549/SAHA
against crude HeLa cell nuclear extract HDACs using a cell free
cell(Table 3), the bifunctional compound has significant activity
against bexarotene-resistant and SAHA-resistant cancer cell
growth.
assay.19 DW22 showed a significant inhibition against HeLa cell
nuclear extract HDACs (IC50 = 6.7 0.1
lmol/L), which are compa-
rable to that of the standard SAHA (IC50 = 2.8 0.2
l
mol/L)
In conclusion, we designed and synthesized a bifunctional com-
pound DW22 which displayed an enhanced binding ability to both
RXRa and HDAC-2 in molecular docking analysis. In addition,
(Fig. 4A). More importantly, DW22 also exhibited HDAC inhibitory
activity in a concentration-dependent manner in HL60 cells.20 In
the same concentration (5
ability to inhibit HDAC as compared with SAHA in HL60 cells
(Fig. 4B).
In order to verify the HDAC inhibition activity of DW22, the ef-
fect of DW22 on the level of acetylated histone protein in the HL60
cells was examined by Western blot analysis using the specific
antibodies against acetylated H3.21 DW22 treatment significantly
increased the levels of acetylated H3 in the HL60 cells (Fig. 4C) in
l
mol/L), DW22 displayed the similar
DW22 exhibited an improved biological activity on RXR activation
than bexarotene, and as similar biological activity on HDAC inhibi-
tion as SAHA. On the other hand, DW22 showed a strong anti-tu-
mor effect in several solid cancer cell lines and drug-resistant
cancer cell lines. Thus, DW22 can be considered as an appropriate
lead bifunctional compound to developing more potent anti-tumor
agent for cancer therapy.
a
concentration-dependent manner. Recent studies have
Acknowledgements
demonstrated that the HDAC inhibitor-mediated induction of
p21WAF1/CIP1 is the result of increased H3 acetylation associated
with the p21WAF1/CIP1 gene promoter22. Therefore, we further mea-
sured the levels of p21WAF1/CIP1 expression to confirm the effect of
DW22 on the HDAC. The result showed that DW22 could increase
the expression of p21WAF1/CIP1 in a concentration-dependent man-
ner (Fig. 4C). These results suggest that DW22 might be an inhibi-
tor of HDAC.
Further evaluation of their anti-proliferative activities was car-
ried out in a broader range of human cancer cell lines representing
gastric (HGC-27), gallbladder (GBC-SD), prostate (DU-145) and liver
(Hep-3B) cancer by MTT assay with bexarotene and SAHA as posi-
tive control (Table 2).23 The positive control compounds bexarotene
and SAHA inhibit the proliferation of these tumor cell lines, respec-
tively. Bifunctional compound DW22 decreased the viability of
HGC-27 the most, while they showed the least cytotoxicity against
GBC-SD. Although a little bit lower than that seen with SAHA,
DW22 displays enhanced cytotoxicity as compared with bexaroten-
e. The similar anti-proliferative activities between DW22 (Mean
The authors gratefully acknowledge financial support from the
National High Technology Research and Development Program of
China (863 Program) (No. 2012AA020305), National Key Scientific
Project for New Drug Discovery and Development (No.
2010ZX09401-304), China Postdoctoral Science Foundation funded
project (20100481213, 2012T50474), Liaoning Science and Tech-
nology Program (No. 2011404012-1) and Education Department
of Liaoning Province (No. L2011174).
References and notes
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3. Altucci, L.; Leibowitz, M. D.; Ogilvie, K. M.; de Lera, A. R.; Gronemeyer, H. Nat.
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4. Mann, B. S.; Johnson, J. R.; Cohen, M. H.; Justice, R.; Pazdur, R. Oncologist 2007,
12, 1247.
5. Blumenschein, G. R.; Khuri, F. R.; von Pawel, J.; Gatzemeier, U.; Miller, W. H.;
Jotte, R. M.; Le Treut, J.; Sun, S. L.; Zhang, J. K.; Dziewanowska, Z. E.; Negro-Vilar,
A. J. Clin. Oncol. 1879, 2008, 26.
IC50 = 16.1 lmol/L) and SAHA (Mean IC50 = 13.2 lmol/L) suggest
6. Esteva, F. J.; Glaspy, J.; Baidas, S.; Laufman, L.; Hutchins, L.; Dickler, M.;
Tripathy, D.; Cohen, R.; DeMichele, A.; Yocum, R. C.; Osborne, C. K.; Hayes, D. F.;
Hortobagyi, G. N.; Winer, E.; Demetri, G. D. J. Clin. Oncol. 2003, 21, 999.
7. Thiagalingam, S.; Cheng, K. H.; Lee, H. J.; Mineva, N.; Thiagalingam, A.; Ponte, J.
F. Ann. N.Y. Acad. Sci. 2003, 983, 84.
that HDAC inhibition is the dominating mode of antiproliferative
activities of this compound. In addition, to demonstrate the advan-
tage of the bifunctional compound, we also detected the effects of
DW22 on bexarotene-resistant human oral squamous carcinoma
KB cell line (KB/ bexarotene) and SAHA-resistant human lung can-
cer A549 cell line (A549/SAHA).23 Our data indicated that DW22
inhibited KB/bexarotene and A549/SAHA cell growth with IC50 of
8. Kim, H. J.; Bae, S. C. Am. J. Transl. Res. 2011, 3, 166.
9. Mottet, D.; Castronovo, V. Curr. Cancer Drug Targets 2010, 10, 898.
10. Ai, T.; Cui, H.; Chen, L. Curr. Med. Chem. 2012, 19, 475.
11. Papi, A.; Ferreri, A. M.; Guerra, F.; Orlandi, M. Anticancer Res. 2012, 32, 2855.
12. Dummer, R.; Beyer, M.; Hymes, K.; Epping, M. T.; Bernards, R.; Steinhoff, M.;
Sterry, W.; Kerl, H.; Heath, K.; Ahern, J. D.; Hardwick, J. S.; Garcia-Vargas, J.;
Baumann, K.; Rizvi, S.; Frankel, S. R.; Whittaker, S. J.; Assaf, C. Leuk. Lymphoma
2012, 53, 1501.
16.8 2.4 and 3.8 1.3 lmol/L, respectively. Compared with
bexarotene with IC50 of 46.3 0.6 lmol/L for KB/bexarotene cell
13. The synthesis of DW22 was carried out in a straightforward two-step synthesis
from
4-(1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-
yl)vinyl)benzoic acid (bexarotene). Sequential treatment of the prepared
bexarotene with refluxing thionyl chloride and dry dichloromethane (CH2Cl2)
to afford the intermediate chloride in 89% yield, which was converted to DW22
by a freshly prepared hydroxylamine in 92% yield, DW22: white solid, total
yield 82%. 1H NMR (DMSO-d6, 300 MHz): d(ppm) 11.17 (1H, s, CONHOH), 8.99
(1H, s, CONHOH), 7.71 (2H, d, J = 8.4 Hz, –CH@CH–), 7.29 (2H, d, J = 8.4 Hz, –
CH@CH–), 7.14 (1H, s, Ar-H), 7.06 (1H, s, Ar-H), 5.88 (1H, s, @CH2), 5.22 (1H, s,
@CH2), 1.91 (3H, s, Ar-CH3), 1.66 (4H, s, CH2), 1.25 (12H, d, CH3); 13C NMR
(DMSO-d6, 75 MHz): d 163.79, 148.03, 143.58, 142.62, 141.70, 137.80, 131.95,
131.72, 127.72, 127.14, 127.03, 125.92, 116.43, 34.53, 33.53, 33.39, 31.55,
31.52, 19.37. MS (ESI) m/z 364 [M+H]+, 362 [MÀH]À.
Table 2
Anti-proliferative activities of DW22 on human cancer cell lines
Compd
IC50(lM)
HGC-27
GBC-SD
DU-145
Hep-3B
Mean
DW22
SAHA
Bexarotene
8.6 1.2
5.4 1.1
32.7 0.5
24.1 0.5
14.8 1.2
73.3 1.6
14.1 1.3
27.2 0.3
61.6 2.6
17.4 0.9
5.4 0.2
191.8 3.5
16.1
13.2
89.9
14. Connors, R. V.; Wang, Z.; Harrison, M.; Zhang, A.; Wanska, M.; Hiscock, S.; Fox,
B.; Dore, M.; Labelle, M.; Sudom, A.; Johnstone, S.; Liu, J.; Walker, N. P.; Chai, A.;
Siegler, K.; Li, Y.; Coward, P. Bioorg. Med. Chem. Lett. 2009, 19, 3550.
15. SYBYL 6.91 Manual. Tripos Inc., St. Louis, MO, USA.,2004.
Table 3
Anti-proliferative activities of DW22 on bexarotene-resistant and SAHA-resistant cell
lines.
16. Bressi, J. C.; Jennings, A. J.; Skene, R.; Wu, Y.; Melkus, R.; De Jong, R.; O’Connell,
S.; Grimshaw, C. E.; Navre, M.; Gangloff, A. R. Bioorg. Med. Chem. Lett. 2010, 20,
3142.
17. Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew, R. K.;
Olson, A. J. J. Comput. Chem. 1998, 19, 1639.
Compd
IC50
(
lM)
KB
KB/bexarotene
A549
A549/SAHA
18. The 293 cells were cultured in Dulbecco’s modified Eagle’s medium containing
5% fetal bovine serum. The cells were cotransfected with CRBPII-tk-luc (a gift
from makishima.makoto, Nihon University School of Medicine, JAPAN), pCMX–
hRXR and pRL Renilla Luciferase Reporter Vector (Promega Corporation). The
DW22
SAHA
Bexarotene
6.1 0.2
—
8.8 1.1
16.8 2.4
—
46.3 0.6
9.4 1.5
17.9 0.2
—
3.8 1.3
80.3 5.2
—

G.-L. Chen et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3891–3895
3895
ratio of these three plasmids is 10:3:2. 5 h after transfection, the cells were
treated with different concentrations of DW22 and bexarotene. After 48 h
transfection, the RXR activity was detected with Dual-Luciferase Reporter
Assay System (Promega Corporation). RXR activity is presented as the
means SD of three determinants.
measured using a microplate reader (Molecular Devices). HDAC activity is
presented as the means SD of three determinants.
21. About 1 Â 107 HL60 cells were gathered after pre-treatment DW22 and SAHA
for 48 h. Nuclear protein was prepared by a commercial kit (Thermo Scientific).
In brief, an equal amount of total protein extracts from cultured cells were
fractionated by 10–15% SDS–PAGE, then electrically transferred onto
polyvinylidene difluoride (PVDF) membranes and probed with antibodies
against acetylated H3 (Cell Signaling, MA, USA), histone H3 (Cell Signaling),
p21WAF1/CIP1 (Cell Signaling) or b-actin antibody (Sigma). The bound secondary
antibodies on the PVDF membrane were reacted with ECL detection reagents
(Thermo Scientific) and exposed to X-ray films.
19. The HeLa nuclear protein concentration was measured by BCA protein assay
(Beyontime, CHN). The in vitro HDAC assay was performed with an HDAC
fluorescent activity assay kit (Biovison, USA). Briefly, proteins were incubated
with different concentrations of DW22 and SAHA (Sigma, USA) at 37 °C for
30 min in the presence of an HDAC fluorimetric substrate. The HDAC assay
developer (which produces a fluorophore in reaction mixture) was added, and
the fluorescence was measured using a microplate reader (Molecular Devices).
HDAC activity is presented as the means SD of three determinants.
20. The HL60 cell was treated with different concentrations DW22 and SAHA for
24 h before assays. Proteins were isolated by using cell lysis buffer (Beyontime,
CHN). The protein concentration was measured by BCA protein assay
22. Glozak, M. A.; Seto, E. Oncogene 2007, 26, 5420.
23. The anti-proliferative activity of the compounds was tested on a panel of
human cancer cell lines. The bexarotene and SAHA-resistant cell sublines were
generated by step-wise exposures of the KB cell line and the A549 cell line to
increasing concentrations of either bexarotene or SAHA, starting with 2
lM for
(Beyontime, CHN). The HDAC activity was measured with
a
HDAC
bexarotene or 5 M for SAHA. Cells were seeded in 96-well plates at a density
l
fluorescent activity assay kit (Biovison, USA). In brief, cell lysates were
incubated with the HDAC substrate Boc-Lys(Ac)-pNA (10 mM) in HDAC assay
of 3000 cell/well and incubated with diluted DW22, SAHA and bexarotene for
48(for drug-resistant cells) or 72 h. The final DMSO concentration in the assay
was 0.1%. The final number of cells per well was assessed using the MTT dye
(Sigma, M5655) following the manufacturer instructions.
buffer. After 90 min at 37 °C, reactions were stopped by adding 10
lL of Lysine
Developer and further incubated for 30 min at 37 °C. Absorbance was