Discovering Small-Molecule Estrogen Receptor α/Coactivator Binding Inhibitors: High-Throughput Screening, Ligand Development, and Models for Enhanced Potency

Article abstract of DOI:10.1002/cmdc.201000507

Small molecules, namely coactivator binding inhibitors (CBIs), that block estrogen signaling by directly inhibiting the interaction of the estrogen receptor (ER) with coactivator proteins act in a fundamentally different way to traditional antagonists, which displace the endogenous ligand estradiol. To complement our prior efforts at CBI discovery by denovo design, we used high-throughput screening (HTS) to identify CBIs of novel structure and subsequently investigated two HTS hits by analogue synthesis, finding many compounds with low micromolar potencies in cell-based reporter gene assays. We examined structure-activity trends in both series, using induced-fit computational docking to propose binding poses for these molecules in the coactivator binding groove. Analysis of the structure of the ER-steroid receptor coactivator (SRC) complex suggests that all four hydrophobic residues within the SRC nuclear receptor box sequence are important binding elements. Thus, insufficient water displacement upon binding of the smaller CBIs in the expansive complexation site may be limiting the potency of the compounds in these series, which suggests that higher potency CBIs might be found by screening compound libraries enriched with larger molecules.

Full text of DOI:10.1002/cmdc.201000507

MED
DOI: 10.1002/cmdc.201000507
Discovering Small-Molecule Estrogen Receptor
a/Coactivator Binding Inhibitors: High-Throughput
Screening, Ligand Development, and Models for Enhanced
Potency
Aiming Sun,*[a] Terry W. Moore,[b] Jillian R. Gunther,[b] Mi-Sun Kim,[a] Eric Rhoden,[c]
Yuhong Du,[c] Haian Fu,[c] James P. Snyder,[a] and John A. Katzenellenbogen*[b]
Small molecules, namely coactivator binding inhibitors (CBIs),
that block estrogen signaling by directly inhibiting the interac-
tion of the estrogen receptor (ER) with coactivator proteins act
in a fundamentally different way to traditional antagonists,
which displace the endogenous ligand estradiol. To comple-
ment our prior efforts at CBI discovery by de novo design, we
used high-throughput screening (HTS) to identify CBIs of novel
structure and subsequently investigated two HTS hits by ana-
logue synthesis, finding many compounds with low micromo-
lar potencies in cell-based reporter gene assays. We examined
structure–activity trends in both series, using induced-fit com-
putational docking to propose binding poses for these mole-
cules in the coactivator binding groove. Analysis of the struc-
ture of the ER–steroid receptor coactivator (SRC) complex sug-
gests that all four hydrophobic residues within the SRC nuclear
receptor box sequence are important binding elements. Thus,
insufficient water displacement upon binding of the smaller
CBIs in the expansive complexation site may be limiting the
potency of the compounds in these series, which suggests
that higher potency CBIs might be found by screening com-
pound libraries enriched with larger molecules.
Introduction
The estrogen receptor (ER), a member of the superfamily of
ligand-regulated nuclear transcription factors, mediates the
action of estrogens, including the primary endogenous ligand
17b-estradiol (E2).[1] ERs exist as two subtypes, ERa and ERb,
and because they are found in both reproductive (uterus,
ovary, and breast) and non-reproductive (bone, brain, and the
cardiovascular system) tissues, they have emerged as attractive
therapeutic targets for the treatment of breast cancer, the pre-
vention of osteoporosis, and the mitigation of menopausal
symptoms via hormone replacement.[2] The binding of an es-
trogen to the ligand binding domain (LBD) of the ER forms a
complex that interacts with specific DNA binding sites and re-
cruits coregulators of the p160 class of steroid receptor coacti-
vators (SRCs).[3] Because the SRC proteins mediate important al-
terations in chromatin structure and facilitate assembly of the
transcriptional machinery, the recruitment of these proteins to
the ER is essential for expression of ER-regulated genes.
Traditionally, the inhibition of ER activity has been achieved
using antagonist molecules that bind to the ligand binding
pocket in place of estradiol and trigger a conformational
change that indirectly blocks coactivator binding by an intra-re-
ceptor or allosteric process.[4] An alternative and yet underex-
ploited approach to blocking estrogen action involves small
molecules that disrupt the interaction between agonist-activat-
ed ER and the SRC by an extra-receptor or direct competitive
process. Such molecules are termed coactivator binding inhibi-
tors (CBIs).[5]
SRCs, which exist as three subtypes (SRC-1, -2, and -3), pos-
sess multiple copies of a conserved, signature sequence motif,
LXXLL (L is leucine and X is any amino acid), known as a nu-
clear–receptor interaction box (NR box). X-ray crystal structures
of several nuclear hormone receptor–agonist complexes
bound to protein fragments of p160 coactivators or to pep-
tides having one or more NR boxes have been solved. The co-
activators bind to the nuclear receptor LBD through a two-turn
amphipathic a-helical motif encompassing the NR box LXXLL
signature sequence, with the ER–coactivator complex being
further stabilized by interactions between the intrinsic dipole
moment of the helical coactivator peptide backbone and
charged residues from the ER at either end of the binding
groove. The X-ray structure of the ERa complex with the
[a] Dr. A. Sun, M.-S. Kim, Dr. J. P. Snyder
Department of Chemistry, Emory University
1515 Dickey Drive, Atlanta, GA 30322 (USA)
Fax: (+1)404-727-6689
E-mail: asun2@emory.edu
[b] Dr. T. W. Moore, Dr. J. R. Gunther, Dr. J. A. Katzenellenbogen
Department of Chemistry, University of Illinois
600 South Mathews Avenue, Urbana, Illinois 61801 (USA)
Fax: (+1)217-333-7325
E-mail: jkatzene@uiuc.edu
[c] E. Rhoden, Dr. Y. Du, Dr. H. Fu
Department of Pharmacology, Emory University
1510 Clifton Road, Atlanta GA 30322 (USA)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201000507.
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Estrogen Receptor a/Coactivator Binding Inhibitors
second NR box of SRC-2 shows this interaction in detail (Fig-
ure 1a).[6] From this image, it is evident that the first and third
leucine residues of the SRC-2 NR-2 box ILHRLL peptide proj-
Given the recent availability of chemical libraries and screening
facilities to academic researchers,[7] we were hopeful that we
might use high-throughput screening (HTS) to discover CBIs of
novel structures with higher affinities that might be more bio-
logically useful.
To this end, we developed and optimized a time-resolved
fluorescence resonance energy transfer (TR-FRET) assay to
screen large compound libraries for nonpeptidic compounds
that would show ERa CBI activity.[8] In this assay, the interaction
between a europium-labeled ERa LBD and a Cy5-labeled frag-
ment of SRC-3, induced upon estradiol binding to the ER, was
monitored by TR-FRET, and an 86000-member library of small
molecules was screened for the ability to disrupt this interac-
tion, monitored by a decrease in TR-FRET signal. This activity,
followed by confirmatory assays we have previously de-
scribed,[8] identified four distinct ERa CBI scaffolds (1–4) with
IC50 values of 5–30 mm that were selected for follow-up chemis-
try and structure–activity relationship (SAR) development (Fig-
ure 1b). All four compounds were resynthesized and re-evalu-
ated in the primary TR-FRET assay.
Curiously, samples of compounds 2, 3 and 4 resynthesized
in our laboratories showed no activity in the TR-FRET assay.
The activity of resynthesized 1 diminished somewhat com-
pared with the original library sample, but it nevertheless
showed distinct activities in both the TR-FRET assay and in a re-
porter gene assay (see below). Gratifyingly, analogues prepared
in parallel with the resynthesis of 4 showed activity, even
when the resynthesized version of the original hit compound
was inactive within the concentration limits of our assay.
Herein, we describe the optimization of two new series of
CBIs, namely those based on the scaffolds of 1 and 4. In prob-
ing the SARs in these series, we used a cell-based ERa-mediat-
ed luciferase reporter gene assay to demonstrate that the com-
pounds are both cell permeable and active in a more biologi-
cally relevant assay. In addition, we used two different concen-
trations of estradiol in the reporter gene assay to indirectly
confirm that the inhibitors do not bind at the ligand binding
pocket, thereby supporting our proposed mechanism for the
action of these compounds.
We found that the structural changes we made to the 1 and
4 scaffolds in developing these two series have significant ef-
fects on their potencies as CBIs. We then performed in silico
analysis of the interaction of these molecules with the hydro-
phobic coactivator binding groove shelf region of the ER co-
activator interaction site, using induced-fit-based modeling
methods, and we obtained binding interaction models that
provided a satisfying rationale for our observed SAR data. In
further analysis of the molecular size and the spatial extent of
the interaction of these CBIs with ERa, compared to that of
the ILHRLL sequence of the SRC NR box peptide, we noted
that the interaction surface area of our CBIs was much less
than that of the peptide ligand. This suggests that the poten-
cies of the CBIs explored thus far are limited by the entropic
component of free energy, due to the less complete displace-
ment of bound surface waters by these smaller molecules. It
further suggests that higher potency CBIs might be found by
screening compound libraries enriched with larger molecules.
Figure 1. a) Crystal structure of GRIP1 peptide (red) on the surface of the
ERa (brown=hydrophobic, green/blue=neutral to hydrophilic); b) HTS hits
of ERa coactivator binding inhibitors identified by a TR-FRET assay.
ect downward into a short, but deep “hydrophobic groove”
made up of several residues from helices 3, 4, 5, and 12 of the
LBD. Furthermore, the second leucine and the preceding iso-
leucine residue (ILHRLL) rest on a largely “hydrophobic shelf”
adjacent to the groove. All of these interactions are likely con-
tributors to the high-affinity binding of the SRC to the ER.
In spite of this detailed molecular portrayal of the site of
receptor–coactivator interaction, only a few small molecules
have been found that bind to this hydrophobic surface groove
shelf region of the ER and block the interaction with coactiva-
tor (i.e., act as a CBI).[5a,b,e,6b,c] With one exception,[5b] the ER
CBIs reported thus far have been discovered using de novo
design, and they only possess micromolar affinities for ER.
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A. Sun, J. A. Katzenellenbogen et al.
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Results and Discussion
Analogue design
In deciding which building blocks to use to prepare analogues
of 1 and 4, we chose hydrophobic alkyl and aryl substituents
we believed would be well suited for binding in the coactiva-
tor binding groove shelf region, based on both the natural leu-
cine and isoleucine residues of the NR box motif and other
work that has shown the coactivator binding groove capable
of binding other aliphatic and aromatic substituents.[5e,6b,9] Be-
cause we were not certain of the binding orientation of 1 or 4
within the coactivator binding groove, we chose various hy-
drophobic substituents to append to the cores. We hoped that
using an array of substituents would give differential biological
results that would, in turn, reveal the binding orientation of
the molecules within the groove through induced-fit computa-
tional modeling of ligand binding poses.
Scheme 1. General synthesis of thioxo-quinazolinone 1 and derivatives.
Reagents and conditions: a) EtOH, reflux, >48 h, 40–72%; b) KOH, EtOH,
reflux, 6 h, quantitative; c) 1n HCl, RT, 5 min, quantitative; d) EDCI, HOBt,
CH2Cl2/DMF (6:1), RT, 20 h, 53%.
The piperazine rings of 1k, 1t and 1u were constructed by
reaction of a substituted aniline (3-nitrile or 3-nitro aniline)
with bis-(2-chloroethyl)amine hydrochloride. Piperazine 8 was
further coupled with quinazolinone acid 7 to afford derivatives
1k, 1t and 1u with 3-isopropyl-, 3-cyano- and 3-nitro-substi-
tuted groups, respectively (Scheme 2).
Chemical Synthesis
Quinazolinone series 1
Modifications of CBI quinazolinone hit 1 were explored in four
sectors: the chlorophenyl moiety, the piperazine ring, the
linker, and the quinazolinone (see Figure 2). Synthesis of the
Scheme 2. Synthesis of quinazolinone derivatives 1k, 1t and 1u. Reagents
and conditions: a) K2CO3, chlorobenzene, reflux, 48–72 h, 45%; b) EDC, HOBt,
7, CH2Cl2, DMF, RT, 24 h, 55–90%.
Figure 2. Strategy for the elucidation of structure–activity relationships for 1.
Structural modifications were made to four regions: chlorophenyl (A), piper-
azine (B), linker (C), and the benzo ring of quinazolinone (D).
Modification of phenyl piperazine (A and B): A common syn-
thetic procedure was used to prepare a small library of com-
pounds assembled from a diverse series of aromatic piper-
azines (9a–f), as shown in Figure 3. In addition, a second series
of substituted piperidines replacing the piperazines has been
synthesized by reductive amination, followed by coupling with
acid 7. Reductive amination of tert-butyl 4-oxopiperidine-1-car-
boxylate with aniline gave phenyl amine 11, which was alkylat-
ed with benzyl bromide to generate compound 12. Boc-depro-
tection of 12 delivered piperidine 13, which was coupled with
acid 7a by the procedure described above to obtain piperi-
dines 10a–c (Scheme 3).
Modification of phenyl quinazolinone (D): In addition to exam-
ining the effect of differentially substituted piperazine and
piperidine rings on sector B, we synthesized a small group of
compounds with substituted phenyl quinazolinone rings by
decorating the sector D phenyl ring with -OMe, -COOMe and
-COOH groups (16a–e and 17, Figure 4, and scheme S1 in the
Supporting Information).
quinazolinone 1 was initiated by combining 2-methoxycarbon-
yl phenyl isothiocyanate and g-amino butyric acid 5a and ana-
logues to obtain thiourea derivative 6, followed by base-pro-
moted cyclization to give quinazolinone 7. Acid 7 was further
coupled with piperazine 8 to provide quinazolinone 1 and its
derivatives (Scheme 1).
Modification of the chlorophenyl (A) and linker (C): In the first
stage of hit optimization, we introduced a variety of substitut-
ed phenyl rings as 3-chlorophenyl replacements (sector A).
Compounds 1a–n were readily prepared by the same proce-
dure as shown in Scheme 1 by coupling various phenyl pipera-
zines 8 with acid 7a. At the same time, a series of linkers (sec-
tor C) was introduced to bridge the phenyl piperazine and qui-
nazolinone moieties. Compounds 1o–x incorporate longer
five-carbon chains; derivatives 1y, 1z, and 1aa employ a
seven-carbon linker, and 1bb has a one-carbon spacer be-
tween the two rings (Table 1).
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Estrogen Receptor a/Coactivator Binding Inhibitors
Benzothiazole series 4
Table 1. Optimization of sector A: Luciferase activity (Luc) of 1, 1a–z, 1aa and 1bb in reporter gene assay.
Two obvious points for introduc-
ing diversity into the benzothia-
zole scaffold can be seen in the
library hit 4: introduction of vari-
ous sulfides and amides at each
of the two a-thioamides in the
molecule. Analogues of benzo-
thiazole 4 were synthesized be-
ginning with the nitration of
commercially available 2-mer-
captobenzothiazole, followed by
reduction to give 6-amino-2-
Compd
R2
n
Luc
IC50 [mm]
Compd
R2
n
Luc
IC50 [mm]
1
3-Cl
4-Cl
3,4-dichloro
4-OMe
4-OH
2-OH
3-OH
4-CF3
3-CF3
2-CF3
3-CF3, 4-Cl
4-iPr
3-NO2
4-CN
2-CN
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
14.8[a]
7.1
5.5
7.4
Inactive[b]
4.8
10.4
2.3
35[a]
1o
1p
1q
1r
1s
1t
1u
1v
1w
1x
1y
1z
3,4-dichloro
4-OH
4-CF3
4-CN
2-CN
3-CN
3-NO2
3-Cl
4-Cl
3-CF3
4
4
4
4
4
4
4
4
4
4
6
6
6
0
4.2
10.8[a]
4.4
8.0
3.5
9.9
6.6
5.2[a]
9.3[a]
25[a]
1a
1b
1c
1d
1e
1 f
1g
1h
1i
1j
1k
1l
1m
1n
mercaptobenzothiazole
18
(Scheme 4).[10] Alkylation of the
thiol with N-alkyl a-chloroamides
(prepared from the simple addi-
tion of amine and chloroacetyl
chloride) gave 19,[11] which was
followed by an acylation reac-
tion with chloroacetyl chloride
to give a-chloroamide 20. Nucle-
11.9
Inactive[b]
26[a]
3-Cl
2-CN
3,4-dichloro
4-CF3
4.2
2.7[d]
Inactive[b]
Inactive[b]
8.2[c]
1aa
1bb
Inactive[b]
7.5[c]
[a] Compound showed partial inhibition in the reporter gene assay. [b] No activity at 20 mm. [c] Some toxicity in
the b-galactosidase assay (never more than 50% at 20 mm). [d] Significant toxicity in the b-galactosidase assay.
Scheme 3. Synthesis of thioxo-quinazolinone derivatives 10a–d. Reagents
and conditions: a) aniline, NaB(OAc)3H, CH2Cl2, RT, 2 h, 92%; b) BnBr, iPr2EtN,
THF, RT to reflux, 8 h, 86%; c) TFA/CH2Cl2 (1:4), RT, 10 h, 70%; d) EDC, HOBt,
CH2Cl2, DMF, RT, 24 h, 65–86%.
Figure 3. Replacing substituted phenyl ring with other aromatic rings.
Figure 4. Modification of phenyl quinazolinone.
ophilic displacement of the chloride with thiols gave
the desired benzothiazoles 4 (see Table 2), usually in
limited yields due to their often sparing solubilities in
many organic solvents.
Scheme 4. General synthesis of benzothiazole 4 and derivatives. Reagents and conditions:
a) HNO3, H2SO4, 08C, 2 h; b) Na2S, NaSH, elemental sulfur, H2O, reflux, 24 h, 73% (two
steps); c) ClCH2CONHR1, NEt3, 508C, 18 h; d) chloroacetyl chloride, Et3N, RT, 8 h, 50–60%
(two steps); e) R2SH, Et3N, 508C, 16 h, 15–76%.
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To verify that activity in this
assay was not due to a conven-
tional antagonist mechanism, a
CBI titration was performed
using two concentrations of the
agonist ligand, estradiol, 1 nm
and 100 nm. This 100-fold in-
crease in estradiol concentration
should have no effect on the IC50
value of a compound acting by
a coactivator binding inhibition
mechanism, because it is directly
competing with the SRC, not the
ligand. On the other hand, the
100-fold increase in estradiol
Table 2. Activities and structures of benzothiazole 4 analogues in a cell-based luciferase assay.
Compd
R1
R2
Luc
Compd
R1
R2
Luc
IC50 [mm]
IC50 [mm]
4
c-Pr
c-Pr
c-Pr
c-Pr
iBu
c-Pr
iBu
iBu
iBu
iBu
iBu
iBu
iBu
tBu
iPr
iPr
iPr
iPr
c-Pr
iPr
iBu
c-Pr
iBu
c-Pr
iPr
N-MeIm
Im
20–50
>50
6.3
20–50
9.1
>50
>50
>50
4y
4z
iBu
c-Pr
iPr
iBu
iPr
iBu
c-Pr
iPr
iBu
c-Pr
iPr
iBu
c-Pr
iPr
iBu
iPr
iBu
iBu
iPr
p-tBuPh
p-OMePh
p-OMePh
p-OMePh
p-ClPh
>50
20–50
>50
>50
20–50
>50
6.0
15
17
20–50
20–50
>50
8.7
4a
4b
4c
4d
4e
4 f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
o-CF3Ph
m-CF3Ph
2,5-diClPh
o-MePh
o-MePh
o-iPrPh
o-tBuPh
o-ClPh
m-ClPh
o-CF3Ph
m-CF3Ph
o-EtPh
4aa
4bb
4cc
4dd
4ee
4 ff
4gg
4hh
4ii
4jj
4kk
4ll
4mm
4nn
4oo
4pp
4qq
4rr
p-ClPh
p-CF3Ph
p-CF3Ph
p-CF3Ph
p-BrPh
p-BrPh
p-BrPh
2-Naph
2-Naph
2-Naph
3,4-diClPh
3,4-diClPh
2,4-diMePh
2,4-diMePh
p-EtPh
5.8
20–50
20–50
20–50
>50
20–50
20–50
6.3
20–50
>50
20–50
>50
>50
20–50
>50
20–50
>50
10
12
concentration would cause
a
o-ClPh
marked right shift in the inhibi-
tion curve of a compound that
was operating as a conventional
antagonist.
m-ClPh
o-CF3Ph
m-CF3Ph
p-MePh
p-MePh
p-MePh
p-EtPh
p-iPrPh
p-tBuPh
p-tBuPh
>50
20–50
20–50
20–50
20–50
>50
20–50
>50
6.1
4s
4t
4u
4v
4w
4x
iPr
Using these assays, we found
no significant change in inhibito-
ry potency of the best two com-
pounds in both series; IC50
values were 2.4 and 2.0 mm for
1g, and 3.6 and 6.0 mm for 4o,
at 100 and 1 nm estradiol, re-
spectively (see Figure 5 for com-
4ss
4tt
4uu
4vv
–
iBu
c-Pr
iPr
iPr
–
p-EtPh
p-iPrPh
p-iPrPh
2,5-diClPh
–
–
Biological Activities
petition curves and Tables 1 and 2 for IC50 values). By contrast,
as we have previously shown, conventional antagonists such
as tamoxifen are subject to competition by estradiol and show
the expected 100-fold right shift in IC50 value when evaluated
in this format.[13] These results indicate that the inhibition of
reporter gene transcription in cells that we are observing with
our new compounds is occurring by a coactivator binding
inhibition mechanism, not by conventional antagonism.
Verification of locus of CBI interaction with the ER LBD
The inhibition of coactivator binding by a small molecule, as
measured by our time-resolved FRET assay, could involve either
direct competition with the coactivator peptide for the ER sur-
face (i.e., CBI mechanism) or binding within the ligand binding
pocket and restructuring the ER in a way that prevents coacti-
vator recruitment, that is, via an allosteric-type mechanism
through which conventional antagonists function.[5e,8] To rule
out the latter mechanism, we first performed careful control
experiments using the reporter gene assay. A similar investiga-
tion of the alternative mode of activity in our CBI assays was
also made by molecular modeling.
By reporter gene assays: A reporter gene assay was devel-
oped to evaluate the inhibitory potential of these compounds
in a cellular context and confirm that they are, in fact, cell per-
meable.[12] To accomplish this, a human endometrial cancer
(HEC-1) cell line expressing endogenous nuclear receptor coac-
tivators but not ER was transfected with a plasmid set compris-
ing a full-length ERa expression vector, a luciferase reporter
gene plasmid fused to an estrogen response element (2ERE
Luc), and pCMV b-galactosidase (b-gal; internal control). The
transfected cells were incubated with estradiol (1 nm) together
with increasing concentrations of CBI; luciferase activity was
then measured. Active compounds were identified as those
that inhibited ER-mediated transcription of the reporter gene,
measured at 1 nm estradiol, and effected a concentration-
dependent decrease in the luciferase output.
Figure 5. Luciferase reporter gene assay. Representative compounds 1g (*,
[E2]=10À7 m; ~, [E2]=10À9 m) and 4o (!, [E2]=10À7 m; ^, [E2]=10À9 m)
show dose-dependent inhibition of ERa luciferase activity that for each com-
pound is similar at 10À9 m E2 (c) and 10À7 m E2 (a). For IC50 values, see
text.
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Estrogen Receptor a/Coactivator Binding Inhibitors
By molecular modeling: As a prelude to our induced-fit dock-
ing studies by molecular modeling, described below, we per-
formed an additional probe of the site of action of our active
molecules by modeling. To test the possibility that these com-
pounds might be competing with estradiol and functioning as
conventional antagonists, we examined their fit into the ligand
binding pocket of the X-ray structure of ERa complexed with
the classical antiestrogen, 4-hydroxytamoxifen (HT). HT occu-
pies the ER ligand binding site and stabilizes an antagonist
conformation of the LBD in which helix 12 is repositioned into
the coactivator site, blocking coactivator binding (PDB: 3ERT).[3]
Since HT and the high-potency test compound 1g have very
similar molecular volumes, in principle, both could separately
occupy the expansive ligand binding pocket in ER. Glide dock-
ing of 1g into the emptied HT site suggests that the phenyl
piperazine-quinazolinone scaffold is able to penetrate the site,
but the best binding pose is quite different from that of HT.
The predicted molecular orientation occupies the pocket only
partially, while much of the structure resides in solvent avoid-
ing contact with the receptor (Figure 6).
Structure–activity relationships from cell-based reporter
gene assays
Quinazolinone Series 1: The key structural features of hit 1 are
the phenyl quinazolinone and the substituted phenyl piper-
azine moieties connected by a carbon chain linker (Figure 1b
and Figure 7). Hit optimization focused on these two hetero-
cyclic units. A number of hydrophobic and hydrophilic sub-
stituents at various positions of the aromatic ring (sector A)
were introduced, and compounds with variable carbon chain
linkers (sector C) were also examined (Table 1).
Figure 6. Alignment of 1g (blue) and 4-hydroxytamoxifen (HT) (maroon) in
PDB: 3ERT (hERa–HT complex) as determined by Glide docking. Much of 1g
is predicted to bind well outside the ligand binding site.
Figure 7. a) Induced-fit docked pose for 1b at the coactivator binding site of
the ERa receptor (X-ray structure PDB: 3ERD). Displacements of Glu542 and
Ile358 side chains resulting from docking and hydrogen bond distances (ꢁ)
are indicated in parentheses; brown = hydrophobic; green = neutral; blue
= polar; b) Alignment of 1b (stick) and the coactivator peptide (purple
ribbon; PDB: 3ERD). Hydrophobic residues Leu690 and Leu694 are matched
by ligand hydrophobes.
In addition, MM-GBSA calculations[14] were carried out to
compare estimated binding free energies (DGbinding
=
E
complex(minimized)ÀEligand(minimized)ÀEreceptor) for HT and 1g.
The latter complex, as pictured in Figure 6, is posited to be
much less stable than that for HT (DDGbinding ~17 kcalmolÀ1).
These calculations are in qualitative agreement with conclu-
sions from the mechanistic studies in the reporter gene assays,
described above. This consistency between biology and mod-
eling offers a measure of confidence that the CBI docking re-
sults, reported later, might be useful for guiding future design
strategies.
Most of the compounds in this series showed activities in
the low-micromolar range (~5–10 mm). Although hit 1 with 3-
chlorophenyl substitution and the analogue with a five-carbon
linker (1v) both showed only weak activity, the analogue with
a seven-carbon linker (1y) had good activity with an IC50 value
of 4.2 mm. Repositioning of the chloro group from the meta-
(1) to the para-position (1a) increased activity, with compound
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1a showing full inhibition with an IC50 value of 7.1 mm. Howev-
er, the corresponding longer chain compound 1w had de-
creased activity. Further substitution of the phenyl ring with
two chloro groups (1b) increased activity slightly, but a longer
chain length led to complete loss of activity (1aa). Introduction
of a hydrophilic hydroxy group at the ortho-position (1e) in-
creased activity twofold compared to the meta-hydroxy com-
pound (1 f), but all activity was lost with a para-hydroxy group
(1d). Accordingly, the longer linker analogue with a para-
hydroxy substitution (1p) had poorer activity.
Since the CBI binding site is highly hydrophobic, we antici-
pated that compounds bearing a CF3 group would fit into the
hydrophobic coactivator groove more effectively than those
bearing hydrophilic groups. Indeed, an analogue incorporating
a para-CF3 group (1g) had good activity, but potency de-
creased progressively with the ortho (1i) and meta (1h) ana-
logues. The six-carbon chain meta-CF3 compound (1x) showed
very weak partial inhibition, but compound 1q with a linker
only two carbons longer than 1g had only slightly poorer ac-
tivity. On the other hand, reducing the linker flexibility by two
carbons (1bb) led to complete loss in activity. These results
imply that CBI binding to the hydrophobic groove involves a
subtle interplay of effects sensitive to small alterations in sub-
stituent placement and composition. In the modeling section
below, we suggest that reorganization of the side chains of
the NR-box helix and displacement of water from the coactiva-
tor binding groove are two important factors contributing to
CBI binding.
Table 3. Activities of analogues with substituted piperazines (9a–f), pi-
peridines (10a–c), quinazolinones (16a–e) and compound 17 in a cell-
based reporter gene assay.
Compd
Reporter gene
Compd
Reporter gene
IC50 [mm]
IC50 [mm]
9a
9c
9d
9e
9 f
8.6
10.9
6.6
10c
16a
16b
16c
16d
16e
17
Inactive[a]
7.3
21[b]
Inactive[a]
7.0[b]
Inactive[a]
22[b]
Inactive[a]
Inactive[a]
10a
10b
Inactive[a]
23.5
[a] No activity at 20 mm. [b] Compound showed partial inhibition in the
reporter gene assay.
bined different aromatic rings and heterocycles (Figure 3), for
instance, naphthalene (9a, 9b), pyridine (9d), pyrimidine (9e)
and pyrazine (9 f). Three compounds in this series (9a, 9c and
9d) delivered fairly good activities (Table 3), though not superi-
or to previous analogues. To explore the SAR around the
phenyl piperazine moiety, several derivatives were prepared
with a piperidine ring instead of piperazine, either increasing
the hydrophilicity (10a) or introducing an extra potential
hydrogen-bonding center (e.g., N-phenylpiperidine-4-hydroxy
derivative, 10c) as shown in Scheme 3, but neither substance
showed appreciable activity (Table 3).
A small set of compounds were prepared with substituents
on the quinazolinone benzene ring, and their activities are
best understood when compared to the unsubstituted ana-
logues 1 and 1g (Table 1). Compound 16b with dimethoxy
substitution (Table 3) experienced a severe loss of activity com-
pared to 1g (Table 1), as did compounds 16c, d and e, having
dimethoxy substitution. These results suggest there is limited
tolerance for steric bulk at this site. A methyl ester at C7 elicits
an improvement in activity (16a; Table 3); however, a carboxyl-
ic acid at the same position ablates activity (17; Table 3), illus-
trating the hydrophobic requirement of the CBI binding site.
The three-dimensional binding models described below sug-
gest that the quinazolinone benzene ring is sandwiched by a
small hydrophobic protein patch and solvent. Thus, introduc-
ing methoxy groups in the ring appears to add hydrophobicity
in a solvent bath, leading to reduced potency.
To our surprise, incorporation of both meta-CF3 and para-Cl
substituents on the same phenyl ring (1j) led to complete loss
of activity. This is consistent with activity domination by CF3
and a binding penalty resulting from directing the hydropho-
bic meta-CF3 moiety into solvent (cf.: binding model shown in
Figure 7). Substituting the phenyl ring with a cyano-group in
the para- (1r), ortho- (1s), and meta- (1t) positions (n=4) led
to analogues with good cellular assay activities, as indicated by
IC50 values ranging from 3 to 10 mm. Interestingly, compound
1s, with ortho-substitution, is the most active isomer (IC50 =
3.5 mm). This series complies with the binding model in which
an edge of this substituted phenyl ring bearing the ortho-
cyano group is directed into the aqueous shell around the re-
ceptor (see below).
The linker-shortened hydroxy series 1d–f (n=2) shows simi-
lar behavior and can be rationalized equally well (cf.: the
model shown in Figure 7). Unfortunately, the similar ortho-
cyano analogues with a shorter carbon-chain linker (1n) and
the longer carbon-chain linker (1z) both cause nonspecific cel-
lular toxicity, shown by a decrease in the internal control b-gal-
actosidase reporter gene. A strong electron-withdrawing influ-
ence as represented by the 3-NO2 analogue 1u delivered good
cellular activity (IC50 =6.6 mm), but the corresponding shorter
chain analogue (1l) showed some toxicity. Installation of the
hydrophobic isopropyl substituent (1k) represents a limited
mimic of the LXXLL helix backbone, but this compound
showed only partial inhibition in the cell-based assay (Table 1).
To examine tolerance of aromatic diversity within the bind-
ing site, a small library of compounds was prepared that com-
Benzothiazole series 4: Upon resynthesis of the original ben-
zothiazole hit 4, we discovered that the resynthesized com-
pound was inactive (IC50 >50 mm) in the TR-FRET assay but
showed modest activity (IC50 =20–50 mm) in the reporter gene
assay. HPLC analysis of the original library hit showed it to be
only about 85% pure, and thus it is believed that the impuri-
ties contributed to the false positive activity of the library com-
pound in the TR-FRET assay. The NMR spectrum of a commer-
cial sample of this compound, which we assume to be from
the same source that provided the sample in the original
library, was consistent with the a-chloroamide precursor of
compound 4 that would be too cysteine-reactive to make a
useful probe. Analogues prepared in parallel with our efforts
to confirm that activity of compound 4 did, however, show ac-
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Estrogen Receptor a/Coactivator Binding Inhibitors
tivity in both TR-FRET and cell-based assays; thus, they were
examined further.
The benzothiazole scaffold 4 contains two a-thioamides,
each of which can be diversely functionalized with different
amines and thiols. R1 was functionalized with different alkyl
amines, and there is no clear preference for any of them. R2
was functionalized with a number of arenethiols. Some com-
pounds containing substituents at the ortho- and meta-posi-
tions on the benzene ring showed increased activity over the
resynthesized hit (4b; Table 2); however, those compounds
containing a para-oriented substituent, particularly hydropho-
bic ones, were among the most consistently potent com-
pounds assayed in this series. For instance, the CF3-substituted
compounds showed consistently lower IC50 values when this
substituent was in the para-position, as opposed to the ortho-
or meta-position (4ee–4gg; Table 2). Surprisingly, the CF3-con-
taining compounds were much more potent than the CH3-con-
taining compounds. In fact, all compounds with ortho- or para-
methyl groups experienced complete loss of activity.
Introduction of halogens (Cl and Br) at the para-position
gave compounds showing moderate activity, but placing a
chloro substituent in the meta-position of the ring also led to
good-to-moderate binders (4n, IC50 =20–50 mm; 4o, IC50 =
6.3 mm; Table 2). Other potent compounds incorporated a 2-
naphthyl ring, rather than a substituted phenyl, at R2, and all
of these derivatives showed IC50 values of 9–12 mm (4kk–
4mm; Table 2). Replacing the naphthyl group with the bioiso-
steric 3,4-dichlorophenyl compound gave a marked reduction
in activity (4nn–oo; Table 2). Since there are neither p–p nor
p–cation interactions in the binding model for benzothiazoles
(see model in Figure 8), the origin of the difference may arise
from selective desolvation (see below).
Figure 8. a) Docked pose of 4o showing three hydrogen-bond anchors and
side chain movement upon docking (ꢁ) in parentheses; b) Alignment of 4o
(stick) and coactivator peptide (purple ribbon).
The binding site seems to be tolerant of various alkyl groups
introduced at the R1 position, as changing the alkyl substituent
in the best compounds did little to change activities. In gener-
al, however, the c-Pr substituent at R1 tends to increase the po-
tency of the compounds, sometimes by twofold, relative to iBu
and iPr (4b vs 4k and 4p; 4ee vs 4 ff and 4gg; Table 2). While
R1 is most likely deep in the hydrophobic CBI binding cleft, R2
may be outside of it. This has unexpected consequences for
the observed SAR that is noted below in the context of bind-
ing site water displacement.
GBSA,[14] a method that provides ligand binding energies with
reasonable accuracy;[16] the same method was used to evaluate
the interaction of compound 1g with the ERa ligand binding
pocket (Figure 6). For additional details, see the Molecular
Modeling subsection in the Experimental Section.
Phenyl piperazine scaffold
All active compounds in Table 1, with the exception of 1e and
1 f (both of which bear a hydroxy hydrogen donor), exhibit
similar docked poses at the coactivator binding groove. These
show good alignment with the X-ray structure of the bound
ERa coactivator peptide and simultaneously form productive
hydrophobic contacts and hydrogen bonds with Gln375,
Lys362 or Val368 (Figure 7). In addition, as a result of the in-
duced-fit docking, each is accompanied by side-chain move-
ments of Glu380, Glu542, Met543, Ile358 and/or Leu372, in
order to better match dipoles of the functional groups on the
phenyl ring associated with the piperazine ring or hydrophobic
contacts with the benzene center of the quinazolinone ring.
Notably, the center of mass of 1b does not fall along the axis
of the peptide helix, but below it and deeper into the groove
(Figure 7b). The docking has provided an intuitively reasonable
Induced-fit molecular modeling to identify binding poses
for CBIs
To identify the putative binding poses of the compounds in
the phenyl piperazine and benzothiazole series, we employed
the crystal structure of the human estrogen receptor a com-
plexed by both diethylstilbestrol (DES; ligand binding pocket)
and a peptide from the NR-box II region of the coactivator
GRIP1 (CBI site; PDB: 3ERD). The peptide was deleted, and
docking of the ligands to the coactivator site was performed
with the Prime induced-fit docking method.[15] This procedure
allows for flexibility of both ligand molecule and protein side
chains within the target coactivator binding groove. The ener-
getically favored pose is subsequently derived with MM-
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spatial overlap by superposing the dichlorobenzene ring onto
Leu690 and the piperazine ring onto Leu694, hydrophobic
onto hydrophobic.
group, tolerant of both polar and nonpolar environments, ap-
pears to be a superior substituent.[17] Replacement of CF3 with
CH3 or OMe depletes activity in accordance with water layer-
ing. One puzzling observation is the complete loss of activity
when the methyl group of 4 is removed (compound 4a). In
the context of the model shown in Figure 7, this might have
its origin in the observed loss of efficacy for carboxylic acid 17
relative to its methyl ester 16a. Both the quinazolinone COOH
and NH are exposed to solvent (Figure 7b). Just as we specu-
lated that 17 may be extracted into solvent and out of the
binding cleft, such a phenomenon may operate for 4a as well.
To test the possibility that docking might insert 1b arbitrari-
ly deep within the groove, the coactivator peptide from the
X-ray crystal structure (PDB: 3ERD) was rigidly docked into the
binding site. In none of the resulting poses do the leucine and
isoleucine side chains penetrate more deeply into the pocket
than observed in the X-ray structure, indicating that the hydro-
phobic basin of the protein is most likely the arbiter of hydro-
phobe location.
Pronounced side chain relocation can also occur for Lys362,
a cationic charge clamp residue. For example, H-bonding to
Lys362 by the piperazine-associated carbonyl group in 1b (3-
carbon linker, n=2) can contribute to modifying the locus of
the side chain. Figure 7 depicts the docked pose of 1b in
which the A-sector 3,4-dichlorophenyl piperazine moiety slips
deeply into the hydrophobic groove, while the D-sector
phenyl quinazolinone resides outside of it. During ligand fit-
ting, Glu542 and Ile358 were relocated by 1.7 and 2.3 ꢁ, re-
spectively, to accommodate the two chlorines and the pipera-
zine ring. In addition to the Lys362···O=C hydrogen bond
(1.9 ꢁ), the quinazolinone NH and the Val368 C=O interact sim-
ilarly at 2.1 ꢁ.
What the current CBIs offer and what they lack: Entropic
contribution to binding free energy originating from water
displacement
In our models, compounds 1b, 1e and 4o populate the coacti-
vator peptide binding groove with differential but productive
hydrogen bonds and favorable hydrophobic contacts. Accord-
ing to induced-fit docking, these structural similarities are ach-
ieved by a reasonable spatial match of the organic structures
and the part of the coactivator peptide that binds most deeply
in the groove. In spite of these common and favorable fea-
tures, all three compounds derived by different strategies fail
to deliver potencies, with IC50 values of 1 mm or higher in the
reporter gene assay. This could be the result of poor penetra-
tion of the cellular membrane, although an estimate of Caco-2
membrane permeabilities suggests that the values for 1b, 1e
and 4o do not differ significantly from those for the potent ER
ligands DES and HT (see the Molecular Modeling subsection in
the Experimental Section). More likely, the CBIs are simply too
small to compete effectively with the a-helical coactivator pep-
tide.
For certain active CBI analogues in this series, such as 1q
(IC50 =4.4 mm), induced-fit docking leads to displacements of
the surrounding amino acid side chains by 2–4 ꢁ. By and large,
however, compounds in this family appear to exhibit quite
similar binding poses by adopting compensating local adjust-
ments resulting from linker length variation. An exception is
compound 1e (IC50 =4.8 mm), which assumes a U-shaped pose
overlapping Leu690 and Leu694 side chains and a section of
the coactivator peptide backbone. Details of the modeled
binding poses and side chain relocations for 1q and 1e are
described in figures S1 and S2 in the Supporting Information.
The docked poses of the synthetic inhibitors reveal an im-
portant characteristic which speaks to this point. Assuming
that compounds 1b (Figure 7b), 1e (figure S2B in the Support-
ing Information) and 4o (Figure 8b) are representative of each
class, they are predicted to fill important parts of the groove,
particularly those occupied by the Leu690 and Leu694 side
chains of the coactivator peptide. However, none of the pres-
ent analogues provide structural elements that contact the
protein shelf on which the Ile689 and Leu693 hydrophobes
reside. Given the importance of entropic contributions to the
free energy of binding, the principal focus on groove-only
inhibitors may be misplaced.
Benzothiazole scaffold
Induced-fit modeling of compound 4o, one of the most active
members of this series, provides a satisfying CBI binding pose,
as illustrated by Figure 7. The structure not only fits well in the
hydrophobic groove, but also forms good hydrogen-bonding
interactions with the charge clamp residues Lys362, Glu380
and Gln375 (2.0 ꢁ). Like inhibitors 1b and 1e (Figure 7 and fig-
ure S2 in the Supporting Information, respectively), this struc-
ture offers hydrophobic features that appear to serve as
Leu690 and Leu694 side-chain surrogates (i.e., CH2S, one edge
of the thiazole ring, and the centrally placed benzene ring).
The model accommodates a number of the SAR trends dis-
cussed above. For example, activities appear to be relatively
unperturbed by replacement of the terminal iPr group in 4o
with other hydrophobes. Figure 8 illustrates the deep apolar
pocket of the CBI site in which the NHR1 is situated. At the
other end of the molecule, the SR2 moiety extends beyond this
pocket so as to become exposed to both a rather hydrophobic
protein surface and aqueous solvent, suggesting that amphi-
philic substitution might best serve binding. Indeed, the CF3
Many studies have shown that ligand binding to receptors is
frequently entropy driven,[18] and that the source of the large
ÀTDS contribution to DG arises by the release of water mole-
cules from the binding pocket during the ligand complexation
event.[19] For very tightly bound waters, the contribution has
been estimated to be as much as 2 kcalmolÀ1 per water at
300 K,[20] with lesser values for bound waters having a greater
degree of freedom. With the goal of developing a relative sem-
iquantitative estimate of the importance of this phenomenon
in the present case, the coactivator-bound X-ray structure
(PDB: 3ERD) was relieved of the coactivator peptide, surround-
ed by a box containing 8742 SPC waters 10 ꢁ from the ERa
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Estrogen Receptor a/Coactivator Binding Inhibitors
Figure 9. Number of overlapped waters within 2 ꢁ of a) coactivator peptide and the three ligands b) 1b, c) 1e and d) 4o. Reporter gene assay IC50 values in
parentheses.
protein, and then subjected to 1 ns molecular dynamics at
3008C with the Desmond protocol (see the Experimental Sec-
tion).[21] The X-ray structure of the coactivator peptide and the
docked structures of 1b, 1e and 4o were separately super-
posed on the solvated binding site and, for each structure, all
waters were deleted except those that overlapped the ligand
severely or displayed oxygen atom–ligand atom distances
below the sum of the van der Waals radii.[22] The superposi-
tions are depicted in Figure 9.
those that interact with the hydrophobic shelf, not the groove)
appear able to displace almost as many water molecules (13
total) as each of the three micromolar active inhibitors.
Libraries available for HTS often comprise molecules that
conform to specific size and lipophilicity criteria associated
with typical drug-like molecules,[23] and while these libraries
have obviously given rise to many successful screening cam-
paigns, there have been recent calls for a re-examination of
this process for targets not conventionally sighted by HTS
(e.g., inhibition of protein–protein interactions, as we are at-
tempting to do with our CBIs).[24] Based on this seeming limita-
tion of our screening library and on modeling data, we suggest
that insufficient water displacement by the low molecular
weight CBIs at the coactivator binding groove is likely to be
responsible for the limited potency of our compounds.
Clearly, the peptide is suggested to displace fivefold more
water molecules than the nonpeptidic CBI systems: The 13
water molecules that line the shelf adjacent to the binding
groove occupied by the bound coactivator side chains of
Ile689 and Leu693 are displaced by the peptide, but they are
modeled to be unaffected by the binding of our CBIs (see fig-
ures S3 and S4 in the Supporting Information). If a conservative
but average 0.2 kcalmolÀ1 were assigned to each displaced
water molecule at ambient temperatures, the peptide then
contributes about 14 kcalmolÀ1 more ERa binding free energy
from ÀTDS than do compounds 1b, 1e or 4o. It is equally sig-
nificant that Ile689 and Leu693 residues of the peptide (i.e.,
Conclusions
The development of compounds that can block the interaction
between the estrogen-activated ER and important coactivator
proteins could provide unique pharmacological tools for inter-
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A. Sun, J. A. Katzenellenbogen et al.
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rupting the signal transduction cascade by which this tran-
scription factor regulates gene activity and might provide a
lead for novel therapeutic agents. Nevertheless, inhibition of
protein–protein interactions with small molecules remains a
significant challenge.[25] In this report, we described the synthe-
sis and follow-up cell-based assays of a selection of hits that
came from a HTS effort to search for small-molecule coactiva-
tor binding inhibitors (CBIs) in a large compound library. The
screening protocol employed a TR-FRET assay that we have de-
scribed previously for inhibition of the interaction of ERa with
the important coactivator SRC3.[8]
Experimental Section
Chemistry
Mass spectrometric analysis was provided by the Emory University
Mass Spectrometry Center (Atlanta, GA, USA). Routine H and 13C
1
NMR spectra measured during synthesis were obtained on a Varian
Inova-400 (400 MHz). Deuterated solvents used for NMR were
CDCl3 (residual shifts: d=7.26 for 1H; d=77.7 for 13C) and
[D6]DMSO (residual shift: d=2.5 for 1H). The residual shifts were
taken as internal references. Chemical shifts (d) and reported in
parts per million (ppm). Thin-layer chromatography (TLC) and prep-
arative TLC (PTLC) were performed on precoated, glass-backed
plates (silica gel 60 F254; 0.25 mm thickness; EM Science) and were
visualized under UV light. Column chromatography was performed
with silica gel (230–400 mesh ASTM) using the “flash” method. Ele-
mental (CHN) analyses were performed by Atlantic Microlab Inc.
(Norcross, GA, USA). All solvents and other reagents were pur-
chased from Aldrich Chemical Co. (Milwaukee, WI, USA). Reagents
were used as received. All reactions were performed under an an-
hydrous N2 atmosphere in oven-dried glassware. All compounds
for which CHN analysis data is not reported were judged at least
95% pure by HPLC (Waters 4.6 mmꢂ150 mm C18 5 mm column
(WAT045905) with UV detection at 254 nm; flow rate=
1.00 mLminÀ1 of various mixtures of CH3CN/H2O).
Compounds 1 and 4 were identified as the most promising
hits, and optimization through analogue synthesis and further
biological evaluation in a cell-based reporter gene assay yield-
ed several compounds that were active in the low micromolar
range (e.g., 1b, 1g, and 4o). The mechanism of action of the
potential CBIs was further examined, both experimentally and
by modeling, to verify that the inhibitory activity of these com-
pounds results from direct competition with coactivator for
binding rather than by competition with estradiol at the ligand
binding site.
Compounds 1b, 1q, 1e and 4o were subjected to extensive
induced-fit docking experiments. The resulting binding models
are characterized by protein side-chain movements tailored to
each ligand (see Figures 7 and 8, and figures S1 and S2 in the
Supporting Information), a situation parallel to that suggested
for side-chain rearrangements involving different ligands that
perturb G-protein coupled receptors.[26] The models not only
provide insights into a variety of aspects of the evolving CBI
General procedures for synthesis of compound 1 and ana-
logues: Compound 7 (0.2 mmol, 1.0 equiv), EDCI (0.22 mmol,
1.1 equiv), HOBt (0.22 mmol, 1.1 equiv) in a mixed solvent of
CH2Cl2 (5 mL) and DMF (0.5 mL) were stirred at RT for at least 10 h.
The product was purified by either filtration followed by washing
with solvent or by chromatography to obtain the final product 1
and analogues as white solids.
1: 1H NMR (400 MHz, [D6]DMSO): d=12.97 (s, 1H), 7.94 (d, J=
8.0 Hz, 1H), 7.73 (t, J=7.2 Hz, 1H), 7.38 (d, J=8.0 Hz, 1H), 7.32 (t,
J=8.0 Hz, 1H), 7.22 (t, J=8.0 Hz, 1H), 6.96 (d, J=1.6 Hz, 1H), 6.90
(dd, J=8.4, 1.6 Hz, 1H), 6.80 (d, J=8.0 Hz, 1H), 4.45 (t, J=6.8 Hz,
2H), 3.54 (br s, 4H), 3.21 (m, 2H), 3.13 (m, 2H), 2.45 (t, J=7.2 Hz,
2H), 1.96 ppm (t, J=7.2 Hz, 2H); Anal. calcd for C22H23
ClN4O2S·0.5H2O: C, 58.46; H, 5.35; N, 12.40; found: C, 58.42; H,
5.10; N, 12.32.
structure–activity relationships, but illuminate that
a key
aspect of SRC blockade is mimicry of the two most deeply
buried hydrophobic leucine side chains (Leu690 and Leu694).
To the extent that water release is a contributor to the free
energy of binding, it is clear from Figure 9 that the coactivator
peptide is far more effective than the CBIs we have explored,
and that the remaining two shelf-oriented apolar side chains
(Ile689 and Leu693) of the peptide that displace 13 water mol-
ecules have no counterparts in our new compounds. These re-
sults suggest that the next generation of small molecule CBIs
should span more of the peptide space, particularly on the
shelf adjacent to the deep binding groove bordered by helices
3, 4, 5 and 12. This might well come at the cost of inhibitor
molecular weights beyond the Lipinski ideal of 500,[23] but if
other molecular properties are satisfactory, the potency gain
could certainly compensate.[24]
General procedure for the synthesis of compound 4 and ana-
logues: A round-bottom tube (50 mL) was charged with a-chloroa-
cetamidobenzothiazole 20 (1.0 equiv) and dissolved, typically with
the aid of heat, in anhyd DMF. Et3N (2.0 equiv) was added to the
solution, and then the appropriate arenethiol (1.2 equiv). The solu-
tion was heated for 16 h at 508C and then cooled to RT. The solu-
tion was added to a 70% saturated NaCl solution, and extracted
with EtOAc. The organic layer was dried (MgSO4), filtered and
evaporated. The resulting solid was recrystallized from boiling
CH3NO2.
The only other two CBIs that have been discovered through
an HTS approach are an ERa CBI[5b] and a thyroid hormone re-
ceptor (TR) CBI.[27] Both compounds have IC50 values compara-
ble to those reported here, and follow-up medicinal chemistry
has produced only modestly more potent compounds.[13,28]
Based on these results and on those of our modeling studies,
we believe that a different approach to discovering potent
CBIs by HTS is warranted—an approach that utilizes targeted
libraries enriched in higher molecular weight compounds.
4: mp: 168–1698C; 1H NMR (500 MHz, [D6]DMSO): d=10.61 (s,
1H), 8.39 (d, J=3.9 Hz, 1H), 8.33 (d, J=2.2 Hz, 1H), 7.74 (d, J=
8.8 Hz, 1H), 7.46 (dd, J=8.8, 2.2 Hz, 1H), 7.24 (d, J=1.0 Hz, 1H),
6.96 (d, J=1.2 Hz, 1H), 4.04 (s, 2H), 3.88 (s, 2H), 3.59 (s, 3H), 2.63
(app octet, J=3.7 Hz, 1H), 0.62 (td, J=7.0, 4.9 Hz, 2H), 0.41 ppm
(m, 2H); 13C NMR (125 MHz, [D6]DMSO): d=168.0, 167.4, 149.5,
140.8, 140.2, 136.4, 136.1, 129.3, 124.3, 121.8, 119.2, 112.1, 39.1,
37.3, 33.7, 23.4, 6.4 ppm; HRMS (ESI): m/z [M+H]+ calcd for
C18H20N5O2S3, 434.0779; found, 434.0760.
664
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ChemMedChem 2011, 6, 654 – 666

Estrogen Receptor a/Coactivator Binding Inhibitors
X-ray structure)[30] over alternative poses by computing an estimate
of the free energy of binding. The procedure is superior to Glide-
Score, the scoring function employed by Prime to rank poses
during their generation. Consequently, it is prudently used to re-
score the list of ligand–receptor docking geometries to select the
energetically most favorable complex.
Computational methods
Molecular modeling: To develop binding models of the ERa coacti-
vator binding inhibitors described herein and elucidate initial struc-
ture–activity relationships, we employed the crystal structure of
the human estrogen receptor a (hERa) ligand binding domain
(LBD) bound to the agonist diethylstilbestrol (DES) and a peptide
derived from the NR-box II region of the coactivator GRIP1 (2 ꢁ res-
olution; PDB: 3ERD). The peptide was deleted and flexible docking
of the ligands to the coactivator site was performed with the in-
duced-fit docking module of Schrçdinger Suite (2008).[15]
Desmond molecular dynamics (MD) simulations and determination
of numbers of displaced waters: MD simulations were performed
using the Schrodinger Desmond module developed by D. E. Shaw
Research.[31] The above-described crystal structure (PDB: 3ERD) pre-
pared for induced-fit docking by the protein preparation wizard
was chosen for a Desmond simulation as well. The coactivator pep-
tide was removed and the protein was solvated in an orthorhom-
bic SPC water box with a outer boundary of 10 ꢁ from the protein.
The system was initially subjected to OPLS-2000 optimization to
relax the system to the nearest local energy minimum. Trajectory
data were recorded every 1.2 ps for a total of 1 ns simulation at
300 K and a pressure of 1.0 bar using an NPT ensemble class. In ad-
dition, seven sodium cations were added to neutralize the system,
and the Ewald method was used to affect efficient and accurate
long-range electrostatics.
Since the quality of pose prediction depends strongly on reasona-
ble starting structures, the protein LBD was prepared in a form
suitable for docking prior to ligand docking, subsequent MM-GBSA
calculations and MD simulation were then performed using the
“Protein Preparation Wizard” in Maestro (v. 9.0.211, 2009). Thus, co-
activator peptide and tightly bound water molecules were deleted,
bond orders were assigned, hydrogen atoms were added, protein
termini were capped with ACE (N-acetyl) and NMA (N-methyl
amide), structures were fixed, and labeling was systematized. In ad-
dition, since several residues with missing side chains were detect-
ed within and near loops, these were automatically added and
conformations for them were predicted using the Prime side-chain
prediction module (Schrçdinger) and the OPLS-2000 protein opti-
mized all-atom force field.[29]
The last structure of the 1 ns simulation was taken for analysis of
the water molecules in the binding site since its energy is very sim-
ilar to that of the average energy value over the nearly isoenerget-
ic 1 ns time course. To guarantee that the ERa protein had equili-
brated faithfully, the 1 ns structure was superposed with the start-
ing complex to show that both the protein backbone and the DES
agonist are in essentially identical spatial locations. After the MD
treatment, the crystal structure of the coactivator peptide and the
docking poses of 1b, 1e and 4o were individually placed in the
binding site water pool by superposing the respective protein
complex backbones with that of the solvated and simulated apo-
protein. Only water molecules 2 ꢁ or less from any atoms of coacti-
vator peptide, 1b, 1e and 4o were considered to be ligand over-
lapped and, therefore, candidates for extrusion from the binding
site upon inhibitor binding.
For the prediction of the first residue, the side-chain rotamer li-
brary is searched to find the rotamer with the lowest predicted
energy, while keeping all other side chains fixed. Then the next res-
idue is considered, and so forth, until all residues have been treat-
ed. Once the process is complete, the steps are repeated until no
residues change rotamer states, that is, rotamer convergence.
Once this is achieved, structure optimization is run on all of the
side-chain atoms, while keeping backbone atoms fixed. Finally, re-
strained minimization of the protein is carried out by the IMPREF
utility in the protein preparation wizard, which performs protein
refinement with OPLS-2000 until heavy-atom positions deviate
from the crystal structure with an root mean square deviation
(RMSD) of no more than 0.3 ꢁ.
Supporting Information
Frequently, ligands are docked flexibly into the binding site of a
rigid protein receptor. However, in many situations this yields mis-
leading results as many proteins experience side-chain or back-
bone movements upon ligand binding. Small-molecule blockade
of protein–protein interactions as in the present case is particularly
susceptible to such effects. Therefore, although more time con-
suming, we incorporated both ligand and protein flexibility in all
docking exercises in an effort to maximize accuracy. As a result,
binding site residues, including Ile358, Lys362, Leu372, Glu380,
Glu542, and Met543, experienced movements to accommodate
docked blockers. In particular, Lys362 was displaced up to 4 ꢁ
towards solvent during the course of induced fitting of various
inhibitors.
Synthetic procedures, elemental analyses, additional docking ex-
periments and graphics illustrating the water displacement by
Leu690, Leu693, Leu694 and Ile689 of the SRC are available as Sup-
porting Information on the WWW under http://dx.doi.org/10.1002/
cmdc.201000507.
Abbreviations
Coactivator binding inhibitor (CBI); diethylstilbestrol (DES); N,N-di-
methylformamide (DMF); dimethyl sulfoxide (DMSO); estradiol (E2);
estrogen receptor (ER); 1-ethyl-3-(3-dimethylaminopropyl) carbodi-
imide hydrochloride (EDCI); 1-hydroxybenzotriazole (HOBt); hy-
droxytamoxifen (HT); high-throughput screening (HTS); ligand
binding domain (LBD); nuclear receptor interaction box (NR box);
structure–activity relationship (SAR); steroid receptor coactivator
(SRC); time-resolved fluorescence resonance energy transfer (TR-
FRET).
QikProp calculations: To explore a potential basis for the low CBI
IC50 values in the reporter gene assay, the Caco-2 cell permeabili-
ties of three key ligands (1b, 1g, 4o), agonist DES and antagonist
HT were predicted with the QikProp facility.[16]
Results: 1b, 745; 1g, 488; 4o, 572; DES, 907; HT, 624; where pre-
dicted cell permeability <25 is poor and >500 is excellent.
Acknowledgements
MM-GBSA energy evaluation: To identify the optimal binding poses
generated by docking, MM-GBSA calculations were performed
within Prime following induced-fit docking. The method is capable
of identifying experimental protein–ligand complexes (i.e., the
We are grateful to the US National Institutes of Health (NIH) for
support of the work (US Public Health service grant HG003918 to
ChemMedChem 2011, 6, 654 – 666
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www.chemmedchem.org
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Received: November 19, 2010
Revised: January 13, 2011
Published online on March 1, 2011
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