A mutant selective anti-estrogen is a pure antagonist on EREs and AP-1 response elements

Article abstract of DOI:10.1016/j.bmcl.2010.06.151

Estrogen receptors (ERs) regulate gene transcription through classic estrogen response elements (EREs) as well as AP-1 responsive genes. The common SERMs Raloxifene, Tamoxifen, and ICI164384 function as ER antagonists on EREs but as ERβ agonists/partial agonists on AP-1 responsive genes. While developing a mutant selective analog of Raloxifene, that is an antagonist of ERα(E353A), we discovered an antagonist of wild-type ERα and ERβ that is also an antagonist of ERβ/AP-1 response. The analog, DRL527, represses basal AP-1 gene expression and antagonizes Raloxifene stimulated AP-1 expression. Therefore DRL527 has a unique, previously unreported, ERE/AP-1 activity profile.

Full text of DOI:10.1016/j.bmcl.2010.06.151

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5258–5261
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A mutant selective anti-estrogen is a pure antagonist on EREs and AP-1
response elements
*
Disha Jain, John T. Koh
Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Estrogen receptors (ERs) regulate gene transcription through classic estrogen response elements (EREs)
as well as AP-1 responsive genes. The common SERMs Raloxifene, Tamoxifen, and ICI164384 function
as ER antagonists on EREs but as ERb agonists/partial agonists on AP-1 responsive genes. While develop-
Received 15 April 2010
Revised 28 June 2010
Accepted 29 June 2010
Available online 24 July 2010
ing a mutant selective analog of Raloxifene, that is an antagonist of ER
a(E353A), we discovered an antag-
onist of wild-type ER and ERb that is also an antagonist of ERb/AP-1 response. The analog, DRL527,
a
represses basal AP-1 gene expression and antagonizes Raloxifene stimulated AP-1 expression. Therefore
DRL527 has a unique, previously unreported, ERE/AP-1 activity profile.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Estrogen receptor
Estrogen response element
AP-1
SERM
Estrogens play an essential role in the growth, differentiation,
and homeostasis in a broad range of target tissues, including the
male and female reproductive tracts, mammary glands, bone,
brain, liver, and cardiovascular system.1–6 The biological effects
of estrogen are mediated through two Estrogen receptor (ER) sub-
to be ERb-AP-1 agonists.7–9 Although the molecular details of the
ER/ERE pathway are well characterized, our understanding of the
molecular details of ER/AP-1 pathway, especially mechanisms of al-
tered SERM pharmacology, is limited.7,9,12–15 In the process of
exploring mutant selective ER antagonists we discovered a new
SERM DRL527 that exhibits a unique ERE/AP-1 activity profile.
Design of a new mutant selective antagonist: Previously our group
developed mutant-specific analogs of estradiol which selectively
types, ER
a and ERb, which belong to a superfamily of nuclear
receptors that act as ligand-dependent transcription factors.4–6
The classic mechanism of ER activation involves dimerization and
nuclear translocation upon ligand binding. In the nucleus ERs can
bind directly to estrogen response elements (EREs) located in the
promoter region of estrogen receptor genes (ER/ERE pathway).
ERs have also been shown to regulate gene expression in the ab-
sence of direct DNA-binding by associating with other DNA bound
transcription factors. Through interactions with transcription fac-
tors c-Jun and c-Fos, ligand bound ERs can also regulate AP-1
dependent genes (ER/AP-1 pathway) in a manner that can be dis-
tinct from ER mediated responses on classic EREs.7–9 ER ligands
that show differential responses within different tissues have been
more accurately described as Selective Estrogen Receptor Modula-
tors (SERMs).10
activate ER
a and ERb mutants, ERa(E353A) and ERb(E305A) that
are otherwise not responsive to physiological concentrations of
estradiol (<1 nM). They were designed based on the principle of ‘po-
lar-groupexchange,’ wherein a carboxylatemodified ligand comple-
mented Glu?Ala substitution at the E2/ER interface (Fig. 1). The
carboxylate-functionalized ligand (ES8) displayed a 95-fold and
400-fold preference for ERa(E353A) and ERb(E305A) over the
wild-type receptors.16 Such systems are being explored as a means
to selectively regulate ER dependent transcription pathways inde-
pendent of E2. Herein we explore the application of a similar design
strategy applied to the ER–Ral complex and explore its ability to reg-
ulate ER dependent responses on ERE and AP-1 dependent genes.
Raloxifene binds the E2 binding pocket of ER with the 6-pheno-
lic hydroxyl of the benzothiophene core mimicking the A-ring phe-
nolic hydroxyl of E2 by binding in the polar pocket between helix-3
and helix-6.17 By analogy to the mutant selective ER agonist ES8, it
was envisioned that a similar strategy could be employed to make
a mutant selective ligand receptor pair from Raloxifene. The ability
of the carboxylate-functionalized Raloxifene, DRL527 (Fig. 1), to
The ability of Tamoxifen (Tam) to function as an ER antagonist in
breast tissue, but an agonist in the uterus and bone or the ability of
Raloxifene (Ral) to act as agonist in bone while an antagonist in
breast and uterine tissues may likely result from differential activa-
tion of multiple estrogen responsive pathways.10,11 For the ER/AP-1
pathway SERMs such as Raloxifene, Tamoxifen and ICI164384 that
were originally classified as ER/ERE antagonists have been found
selectively antagonize ERa(E353A) and ERb(E305A) over the
wild-type ER’s was evaluated in cellular reporter gene assays on
* Corresponding author.
both ERE and AP-1 response elements.
E-mail address: johnkoh@udel.edu (J.T. Koh).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.151

D. Jain, J. T. Koh / Bioorg. Med. Chem. Lett. 20 (2010) 5258–5261
5259
hERα(E353A)/ES8
H
O
hERα/E2
N
H
N
O
NH
NH
E353
O
E353A
H
O
H
O
E2
ES8
O
H
O
H
H
N
O
N
H
O
H
NH
NH
H2N
N
H
H
O
O
N
OH
O
N
OH
S
S
O
HO
OH
DRL527
Mutant-Selective SERM?
RAL
Figure 1. ES8 and DRL527 design concept.
The compound DRL527 was synthesized in 4 steps from Raloxif-
ene by monobenzoylation of the 40-phenolic hydroxyl group fol-
lowed by triflate ester formation of the 6-phenolic hydroxyl
group. This approach though cumbersome, proved more effective
in our hands than a variety of strategies explored to selectively
protect or differentially deprotect the two hydroxyls.18,19 The aryl
triflate underwent Heck coupling with methyl acrylate to afford
compound DRL527 after deprotection (Fig. 2).
(IC50 = 0.3 nM), with ERa(E353A) with 3 nM ES8 (IC50 = 56 nM),
ERb(wt)+0.1 nM E2 (IC50 = 6.9 nM) and ERb(E305A)+0.3 nM ES8
(IC50 = 577 nM). Under these conditions, Raloxifene is 185-times
more selective for the ERa(wt) than the ERa(E353A) and 84-times
more selective for the ERb(wt) than the ERb(E305A) on an ERE
response element in HEK293T cells (Fig. 3a,b).
By contrast, under similar conditions, DRL527 was found to be
an almost equipotent antagonist with both wild-type and mutant
ERs of both the alpha and beta subtypes (Fig. 3b). Thus whereas
the antagonist activity of DRL527 cannot be effectively evaluated
DRL527 was evaluated in cell based reporter gene assays for its
ability to selectively antagonize wild-type ERs, ER
ERb(wt) and the ES8-selective mutants hER (E353A) and hER-
b(E305A) using an ERE-luciferase reporter in HEK293T cells. The
mutant receptors ER (E353A) and ERb(E305A) are not activated
a(wt),and
a
directly due to the inherent E2 insensitivity of ERa(E353A) and
ERb(E305A), DRL527 was effective in negating the 84- to 185-fold
selectivity bias of Raloxifene consistent with its complementary
design (Table 1).
a
by physiological concentrations of E2 (EC50 = 2.5 nM, 10.4 nM
respectively) but are activated by ES8 (EC50 = 0.24 nM, 0.07 nM
respectively) at concentrations that do not activate ER(wt).16 As
Raloxifene is a known to be an ER
ever, DRL527 while less potent that Raloxifene, was found to a
highly subtype-selective ER antagonist with (IC50b/IC50 = 300)
a
selective antagonist.20 How-
E2 is an inefficient agonist of hER
a(E353A) and hERb(E305A),
a
DRL527 was evaluated as an antagonist of these mutant ERs in
competition with ES8. In order to practically compare the intrinsic
ability of DRL527 to antagonize ER(wt)+E2 with ER(mutant)+ES8
the concentrations of E2 and ES8 were evaluated at their respective
EC90’s for each receptor. Therefore Raloxifene was evaluated as an
comparable to the some of most selective antagonists reported to
date (Fig. 3c).21 Modification of 4-hydroxyl of Raloxifene may offer
an additional means to impart subtype selectivity.
Whereas the interpretation of mutant selective antagonist
activity is complicated by the use of the corresponding mutant
selective agonist ES8, we were intrigued by the possibility that
antagonist with ER
a
(wt) by competition with 0.05 nM E2
O
O
O
N
OR1
O
N
OR1
c.
S
S
O
R2O
OR3
Ral : R1=R2=H
a.
1: R1=Bz, R2=H
3: R1=Bz, R3=CH3
DRL527: R1=H R3=H
b.
d.
2: R1=Bz, R2=Tf
Figure 2. Synthesis of DRL527. (a) NaH, BzCl, DMF, 8%; (b) TEA, Tf2O, CH2Cl2, ꢀ78 °C to rt, 38%; (c) methyl acrylate, Pd(OAc)2, DPPP, TEA, DMF, 95 °C, 22%; (d) LiOH. THF–
water, reflux, 37%.

5260
D. Jain, J. T. Koh / Bioorg. Med. Chem. Lett. 20 (2010) 5258–5261
Figure 3. Cellular reporter gene activity of (A) RAL and (B) DRL527 in competition with hERa(wt)+0.05 nM E2 ( ), ERa(E353A)+3 nM ES8 ( ), hERb(wt)+0.1 nM E2 ( ), and
ERb(E305A)+0.3 nM ES8 ( ), on an ERE response element in HEK293T cells. (C) DRL527 in competition with 0.1 nM E2 in wild-type ERa( ) and ERb( ), respectively.
Table 1
Potencies (IC50) of Raloxifene and DRL527 with wild-type and mutant ERs on ERE-reporters in cellular reporter gene assays in competition with E2 and ES8 concentrations as
indicated
ER
a+0.05 nM E2
ER
a(E353A) +3 nM ES8
Mutant Selectivity
ERb +0.1 nM E2
ERb(E305A) +0.3 nm ES8
Mutant Selectivity
RAL
DRL527
0.3 0.06 nM
82 18 nM
56 10 nM
212 32 nM
185
2.6
6.9 .8 nM
577 51 nM*
84
ꢁ1
>10
lM
>10
l
M
Error reported as SD of triplicate run. Selectivity is defined as IC50 (mutant)/IC50 (wild-type). *Denotes estimated from incomplete saturation.
Figure 4. Cellular dose–response behavior of ERb/AP-1 driven expression in transiently transfected HeLa cells. (A) Green; Raloxifene alone and brown; DRL527 alone.
(B) Green; Raloxifene alone and brown; variable concentrations of DRL527 versus 10 nM RAL. (C,D) Comparison of ligand-dependent responses on ER (4C) and ERb(4D) at an
AP-1-responsive promoter with 1 M E2, RAL and DRL527.
a
l

D. Jain, J. T. Koh / Bioorg. Med. Chem. Lett. 20 (2010) 5258–5261
5261
Table 2
Raloxifene provides a means to enhance ER subtype selectivity
and provides a new SERM DRL527 that functions as a novel ER
antagonist on AP-1 sites. To our knowledge DRL527 represents a
A new SERM profile showing DRL527 in comparison to Raloxifene and E2 at an ERE
and AP-1 site on wild-type ER alpha and beta
ER
a
ERb
unique example of an ER ligand that is an antagonist of ER
a and
ERb on EREs and an antagonist of ERb on AP-1. DRL527 or other
ER ligands that possess unique ERE/AP-1 response profiles may
serve as a unique tool to understand the role AP-1 signaling in
SERM based therapies.
ERE
AP-1
ERE
AP-1
*
*
*
E2
+
+/£
+/£
+/£
+
+
ꢀ
+
+
+
ꢀ
Tam\
Ral
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ICI\
DRL527
£
Acknowledgment
‘+’ indicates an agonist, ‘ꢀ’ an antagonist, ‘£’ indicates neither activation nor
repression, *literature results vary and show these compounds to be either agonists
or non-agonists/non-antagonists,8,12,15,22 \based on reported activites.8,12,15,22
This work was supported by the National Institutes of Health
(NIDDK R01DK54257).
Supplementary data
DRL527 may potentially serve as a mutant selective agonist of ERs
on AP-1 responsive genes because existing SERMS Raloxifene,
Tamoxifen and ICI164384 have all been shown to be ERb(wt) ago-
nists on AP-1 promoters.8,12 DRL527 was tested for its regulatory
effects on transcription from ERb(wt) and ERb(E305A) on an AP-1
based reporter in transiently transfected HeLa cells. The addition
of DRL527 afforded no significant change in AP-1 activity with ER-
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.06.151.
References and notes
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b(E305A) up to 10 lM (data not shown). However, in contrast to
Raloxifene which behaved as an agonist, DRL527 was found to be
an hERb(wt)/AP-1 antagonist, repressing basal AP-1 transcription
(EC50 = 150 43 nM) (Fig. 4a).
To further support the notion that DRL527 is indeed acting as an
ERb-AP-1 antagonist, competition experiments were performed in
the presence of 10 nM Raloxifene in transiently transfected HeLa
cells. In these experiments, DRL527 exhibited competitive inhibi-
tion of Raloxifene induced expression to below basal levels;
IC50 = 104 55 nM (Fig. 4b). The repression of ERb/AP-1 dependent
expression is ERb specific as no change in basal AP-1 driven expres-
sion is observed with ER
previously reported no ER
AP-1 activities appear to be highly context dependent and others
a
. However, whereas we observe and have
a
/AP-1 response with Tam,22 the ER
a/
have concluded that Tam, Ral and ICI can act as ERa/AP-1 ago-
nists.8,12,15 DRL527 is unique as the only reported ERb/AP-1 antag-
onist (Fig. 4c and d).
In summary, DRL527 was designed as a mutant selective antag-
onist of ERa(E353A) and ERb(E305A) based on structural analogy
to the mutant selective agonist, ES8. DRL527 functions in a mutant
selective manner towards repressing ES8/ER dependent transcrip-
tional activation at EREs. Furthermore, DRL527 was found to be a
highly subtype-selective inhibitor of ER
potency than Raloxifene. DRL527 did not alter ER
a
(wt) although with lower
(E353A) and ER-
a
b(E305A) dependent AP-1 response but was rather found to be a
antagonist of ERb(wt) dependent AP-1 activation capable of sup-
pressing basal AP-1 activity and suppressing Raloxifene induced
AP-1 expression whereas, classic ER antagonists, Tamoxifen,
Raloxifene and ICI are agonists of ERb on AP-1 sites (Table 2). Mod-
ification of the 6-phenolic hydroxyl of benzothiophene core of
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Katzenellenbogen, B. S. Endocrinology 2002, 143, 941.
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