Published on: Mar 3, 2016
Transcripts - Nat_Chem_Biol_GPR30_2006
Virtual and biomolecular screening converge on
a selective agonist for GPR30
Cristian G Bologa1,7, Chetana M Revankar2,3,7, Susan M Young3, Bruce S Edwards3,4, Jeffrey B Arterburn5,
Alexander S Kiselyov6, Matthew A Parker6, Sergey E Tkachenko6, Nikolay P Savchuck6, Larry A Sklar3,4,
Tudor I Oprea1 & Eric R Prossnitz2,3
Estrogen is a hormone critical in the development, normal
physiology and pathophysiology1 of numerous human tissues2.
The effects of estrogen have traditionally been solely ascribed
to estrogen receptor a (ERa) and more recently ERb, members
of the soluble, nuclear ligand–activated family of transcription
factors3. We have recently shown that the seven-
transmembrane G protein–coupled receptor GPR30 binds
estrogen with high afﬁnity and resides in the endoplasmic
reticulum, where it activates multiple intracellular signaling
pathways4. To differentiate between the functions of ERa or
ERb and GPR30, we used a combination of virtual and
biomolecular screening to isolate compounds that selectively
bind to GPR30. Here we describe the identiﬁcation of the ﬁrst
GPR30-speciﬁc agonist, G-1 (1), capable of activating GPR30
in a complex environment of classical and new estrogen
receptors. The development of compounds speciﬁc to estrogen
receptor family members provides the opportunity to increase
our understanding of these receptors and their contribution to
With our recent description of an intracellular transmembrane estro-
gen receptor that initiates multiple signaling pathways4, some in
common with the traditional estrogen receptors, it is clear that
dissecting GPR30-speciﬁc cellular and physiological responses is
essential to understanding the fundamental mechanisms of estrogen
action. To this end, we sought to identify compounds that show highly
selective binding to GPR30 as compared with the classical estrogen
receptors (ERa and ERb). As GPR30 is known to bind many of the
same ligands as classical estrogen receptors (for example, 17b-estradiol
(2), 4-hydroxytamoxifen (3), ICI182,780 (4))4–6, though with differing
cellular effects (for example, ICI182,780 is an ER antagonist but a
GPR30 agonist), we screened a library of diverse chemical compounds
to identify GPR30-speciﬁc ligands.
To minimize the biomolecular screening required, we ﬁrst used
virtual screening7, which has recently become increasingly recognized
as a complement to bioactivity screening8. Virtual screening aims to
sift through vast numbers of structures to rapidly identify those of
interest for biological screening. Its experimental counterpart, high-
throughput screening, is also aimed at sifting through large numbers
of compounds, often based on single-point, single-experiment results.
Both procedures rely on the ability to process a large number of
structures or compounds9. We therefore began by virtually screening a
library of 10,000 molecules (preoptimized to be enriched in G
protein–coupled receptor (GPCR)-binding ligands based on the con-
cept of the GPCR-privileged substructure10) using a combination of
2D- and 3D-similarity approaches. Our combined similarity score
attributed 40% weight to 2D ﬁngerprints, 40% to shape-based
similarities and 20% to pharmacophore-based similarity. Given this
composite score, the top 100 ranked molecules were selected for
To accomplish the biomolecular screening, we used a ﬂuorescently
labeled estrogen derivative (an Alexa633 conjugate of 17a-[4-
aminomethyl-phenylethynyl]-estra-1,3,5(10)-triene-3,17b-diol (5), E2-
Alexa633) that we have recently developed and shown to bind GPR30
as well as ERa and ERb (ref. 4). Competitive ligand binding was
assessed in transiently transfected COS7 cells, which do not show
detectable speciﬁc binding of the ﬂuorescent estrogen in the absence of
exogenous receptors. Binding was measured with a high-throughput
ﬂow cytometric platform (HyperCyt) capable of sampling rates of
approximately 1 per s from microplate wells11,12. To maximize the
bound ﬂuorescent signal, a GPR30-GFP fusion protein was expressed
and cells were gated for high levels of green (FL1) ﬂuorescence (that is,
Biomolecular screening was carried out with 17b-estradiol as a
positive control to block speciﬁc binding of E2-Alexa633 to GPR30.
Calculation of the Z¢ factor, a measure of screening assay quality that
reﬂects both assay signal dynamic range and data variation of the
signal13, yielded a value of 0.5–0.7, indicating the robustness of the
assay. From the primary screen performed at a compound concentra-
tion of 10 mM, three compounds resulted in inhibition of ﬂuorescent
estrogen binding greater than 60%. These compounds, as well as
numerous intermediate-quality hits, were retested manually from the
Received 23 December 2005; accepted 9 February 2006; published online 05 March 2006; doi:10.1038/nchembio775
1Division of Biocomputing, 2Department of Cell Biology & Physiology, 3Cancer Research and Treatment Center and 4Department of Pathology, University of New Mexico
Health Sciences Center, Albuquerque, New Mexico 87131, USA. 5Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces, New Mexico
88003, USA. 6Chemical Diversity Labs Inc., 11558 Sorrento Valley Road, San Diego, California 92121, USA. 7These authors contributed equally to this work.
Correspondence should be addressed to E.R.P. (EProssnitz@salud.unm.edu) or T.I.O. (TOprea@salud.unm.edu).
NATURE CHEMICAL BIOLOGY ADVANCE ONLINE PUBLICATION 1
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master plate for their ability to inhibit binding of ﬂuorescent estrogen
to GPR30; this revealed that only compound 92, a substituted
dihydroquinoline (Fig. 1a), consistently inhibited binding. This com-
pound will be referred to henceforth as G-1 (GPR30-speciﬁc
compound 1). Its structure, which was subsequently reconﬁrmed
by LC-MS and 1H-NMR, is racemic but diasteromerically pure
To characterize the binding properties of G-1 in greater depth, we
determined the binding afﬁnity to GPR30 and the binding speciﬁcity
with respect to ERa and ERb. Competition binding of the ﬂuorescent
estrogen by 17b-estradiol in GPR30-expressing cells yielded a Ki of
5.7 nM (similar to the value of 6.6 nM we reported previously),
whereas the Ki for G-1 was 11 nM (Fig. 1b). Competition binding in
ERa- and ERb-expressing cells yielded Ki values for 17b-estradiol of
0.30 and 0.38 nM, respectively, with no substantial binding of G-1 at
concentrations up to 1 mM (Fig. 1c,d). The Hill coefﬁcients for the
estrogen-competition binding curves were approximately 1.0 (1.2, 0.9
and 0.9 for GPR30, ERa and ERb, respectively), whereas for G-1, the
Hill coefﬁcient was 0.6, suggesting that there
could be two binding sites of similar but not
identical afﬁnity. These results indicate that
G-1 has not only very high afﬁnity for GPR30
but also great selectivity toward GPR30 as
compared with ERa and ERb.
We next examined the functional cap-
abilities of G-1 versus estrogen with respect to
the rapid mobilization of intracellular cal-
cium. Whereas 17b-estradiol initiates a very
rapid (t1/2 o 2 s) rise in intracellular calcium
concentrations, G-1 produces a slower
(t1/2 B 30 s) but ultimately comparable
calcium increase (Fig. 2a). The speciﬁcity of the response is shown
by the fact that neither 17b-estradiol nor G-1 has an effect on control
COS7 cells (not expressing GPR30) and that 17a-estradiol (6) has no
effect on GPR30-expressing COS7 cells. To conﬁrm the selectivity of
G-1 toward GPR30 as compared with ERa and ERb (as suggested by
the binding assays), we also examined calcium mobilization in COS7
cells expressing either ERa or ERb. In neither case did G-1 elicit a
calcium response (Fig. 2b,c). This is in contrast to 17b-estradiol,
which yields a rapid response with both ERa and ERb. As observed
with GPR30, 17a-estradiol had no effect on ERa or ERb. Dose-
response determinations showed that the mobilization of intracellular
calcium initiated by G-1 in the presence of GPR30 was concentration
dependent, with a half-maximal effector concentration (EC50) of
about 2 nM (Fig. 2d,e), similar to the binding afﬁnity of G-1 for
GPR30, compared with an EC50 for 17b-estradiol of approximately
0.3 nM. In addition to calcium mobilization, we have shown that
stimulation of ERa, ERb and GPR30 with estrogen results in phos-
phatidylinositol 3-kinase (PI3K) activation, resulting in the nuclear
–11 –10 –9 –8
Competitor concentration (log M)
–7 –6 –5
–11–12 –10 –9 –8
Competitor concentration (log M)
–7 –6 –11–12 –10 –9 –8
Competitor concentration (log M)
GPR30 ERα ERβ
100 150 0 50
100 150 0 50 0
Concentration (log M)
–9 –8 –7 –6
40 60 80 100120 140
Time (s) Time (s)
Figure 2 G-1 agonism in the mobilization of intracellular calcium by GPR30. (a–c) We compared the activity of
G-1 with that of 17b-estradiol using indo1-AM–loaded COS7 cells transfected with GPR30-GFP (a), ERa-GFP
(b) or ERb-GFP (c) or with GFP only (a). Ligand addition was performed at 20 s. G-1 and 17b-estradiol were
used at 1 nM; 17a-estradiol was used at 1 mM. The y-axis scales are conserved from a–c. (d) Dose-response
proﬁle of GPR30-GFP–transfected COS7 cells to increasing concentrations of G-1. (e) Comparison of G-1
and 17b-estradiol dose-response proﬁles in GPR30-GFP–transfected COS7 cells. Data in panels a–d are
representative of at least three independent experiments. Data in panel e represent the mean ± s.e.m. of
three separate experiments.
Figure 1 Structure and ligand-binding properties
of G-1. (a) Chemical structure of compound G-1.
(b–d) Ligand-binding afﬁnities of 17b-estradiol
and G-1 for GPR30, ERa and ERb. Results of
competitive ligand-binding assays using 2 nM
E2-Alexa633 and the indicated concentration of
either 17b-estradiol (’) or G-1 (m) in COS7 cells
transfected with either GPR30-GFP (b), ERa-GFP
(c) or ERb-GFP (d). Data indicate the mean ±
s.e.m. of three separate experiments.
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2 ADVANCE ONLINE PUBLICATION NATURE CHEMICAL BIOLOGY
accumulation of phosphatidylinositol 3,4,5-triphosphate (PIP3)
(ref. 4). This is shown by the translocation of a reporter, the pleckstrin
homology (PH) domain of Akt fused to a ﬂuorescent protein, in
response to the de novo production of PIP3 by PI3K (ref. 14). To
conﬁrm our hypothesis that G-1 activates a similar complement of
signaling pathways as estrogen, we examined the activation of PI3K in
receptor-transfected COS7 cells. As previously observed, estrogen
stimulates the nuclear accumulation of PIP3 through all three recep-
tors. However, as observed for calcium signaling, G-1 selectively
activated GPR30 and not ERa or ERb (Fig. 3). In total, these results
indicate that G-1 binds and activates GPR30 with great speciﬁcity over
both ER types.
These results suggest that we should be able to selectively target
GPR30 as opposed to ERs in cells that express both receptors. To test
this, we cotransfected cells with GPR30–monomeric red ﬂuorescent
protein-1 (GFP30-mRFP1) and ERa-GFP, which localize to distinct
subcellular compartments (the nucleus and endoplasmic reticulum,
respectively). The receptors were visualized with E2-Alexa633, and
cells were incubated in the presence or absence of G-1 (Fig. 4a). In the
absence of competitor, ﬂuorescent estrogen staining was seen through-
out the cell (nucleus and endoplasmic reticulum), representing the
sum of the individual compartments. Competition with 17b-estradiol
blocked binding of the E2-Alexa633 to both ERa and GPR30.
However, in the presence of G-1, staining in the endoplasmic
reticulum was selectively lost over that in the nucleus (for both ERa
and ERb). This showed that in the same cell, G-1 selectively binds
GPR30 and not ERs.
Next, we sought to determine whether G-1 activates endogenously
expressed GPR30. We have previously shown that estrogen-mediated
activation of PI3K in SKBr3 breast cancer cells, which are ERa and
ERb negative, occurs exclusively through GPR30. We therefore tested
whether G-1 was able to activate PI3K in SKBr3 cells. We found that
G-1, like estrogen, activated PI3K, resulting in the nuclear accumula-
tion of PIP3 in both SKBr3 cells (that express only GPR30) as well as
in MCF7 cells (which express both GPR30 and classical ERs, with high
levels of ERa and low levels of ERb) (Fig. 4b). This latter result in
MCF7 cells suggests that the estrogen-mediated response is occurring
at least in part through GPR30. To investigate the physiological
PH Merge Merge MergeERα ERβPH PH
a b c
Figure 3 G-1 agonism in PI3K activation by GPR30. We compared the activity of G-1 with that of 17b-estradiol using COS7 cells transfected with Akt-PH-
mRFP1 and either GPR30-GFP (a), ERa-GFP (b) or ERb-GFP (c). 17b-estradiol and G-1 were used at 1 nM and 10 nM, respectively. The white bar in upper
left panel of a denotes 10 mm. Data are representative of three independent experiments.
+ 17β-estradiol + G-1
ER-GFP E2-Alexa 633GPR30-RFP Merge
–13 –12 –11 –10
Concentration (log M)
–9 –8 –7 –6
Figure 4 Selective targeting of GPR30 versus ERa or ERb by G-1. (a) COS7 cells cotransfected with either GPR30-mRFP1 and ERa-GFP or GPR30-mRFP1
and ERb-GFP as indicated and stained with E2-Alexa633 in the absence or presence of excess 17b-estradiol or G-1 (1 mM). Although excess 17b-estradiol
prevents binding of E2-Alexa633 to both ER and GPR30, G-1 selectively competes for binding to GPR30 in cells that also express ERs. (b) SKBr3 breast
cancer cells, which endogenously express only GPR30, and MCF7 cells, which express GPR30 and ERa/b, after transfection with Akt-PH-mRFP1 and
stimulation with either 1 nM 17b-estradiol or 10 nM G-1. White bars denote 10 mm. (c) Cell migration of SKBr3 and MCF7 cells through a Transwell
chamber in response to chemoattractant. Cells in the upper chamber were treated with either 17b-estradiol or G-1 as indicated. Data are representative
of three independent experiments or represent the mean ± s.e.m. of three separate experiments with all P values o 0.05.
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NATURE CHEMICAL BIOLOGY ADVANCE ONLINE PUBLICATION 3
function of GPR30 using G-1, we examined cell migration in response
to estrogen and G-1 (Fig. 4c). Using a Transwell migration apparatus
(Costar), we demonstrated that in response to chemoattractant
(serum, EGF and insulin), both estrogen, as previously documented15,
and G-1 inhibited migration of SKBr3 cells (with half-maximal
inhibitory concentration (IC50) values for estrogen and G-1 of 0.1
and 0.7 nM, respectively) and MCF7 cells (with IC50 values for
estrogen and G-1 of 0.4 and 1.6 nM, respectively). The four- to
seven-fold greater activity of estrogen as compared with G-1 is
consistent with the relative differences between the two compounds
in both binding afﬁnity (Fig. 1) and signaling capacity (Fig. 2e).
Overall, these results conﬁrm that G-1 can selectively bind GPR30 in
the same cell where ERs are present and furthermore that G-1 can
activate endogenously expressed GPR30, resulting in physiologic
responses even in cells expressing multiple estrogen receptor types.
In summary, we have identiﬁed a nonsteroidal, high-afﬁnity, highly
selective agonist of GPR30. This represents the ﬁrst description of a
compound that can distinguish between GPR30 and the classical
estrogen receptors. Superimposition of the structure of 17b-estradiol
with G-1 using rapid overlay of chemical structures (ROCS)16 reveals a
high degree of shape similarity between both enantiomers of G-1 and
17b-estradiol (Fig. 5a). To examine why G-1 shows such high
selectivity for GPR30 over ERs, we performed docking experiments
(with AutoDock (http://www.scripps.edu/mb/olson/doc/autodock/))
of both enantiomers of G-1 (the structure shown in Fig. 1a as well
as its enantiomer) into the ligand-binding pocket of ERa in both its
17b-estradiol- and 4-hydroxytamoxifen-bound crystallographic con-
formations (see Methods for Protein Data Bank accession codes). To
obtain a computational estimate of the binding afﬁnity, both native
ligands were docked into their respective structures as well. Full
ﬂexibility for the ligands was assumed, and no initial positioning of
the ligand into the binding site was performed. The results of the
docking experiment for the enantiomer from Figure 1a are shown in
Figure 5b,c, and the computationally predicted binding afﬁnities for
both enantiomers are summarized in Supplementary Table 1. When
docked into the agonist-bound conformation of ERa (Fig. 5b), both
enantiomers of G-1 are estimated to have an afﬁnity approximately
three orders of magnitude worse than that of 17b-estradiol17. For
example, in the best-docked poses, both G-1 enantiomers do not
present the hydrogen-bonding pattern of
17b-estradiol (that is, no interaction with
Glu353 and His524), and moreover they
show steric clashes with multiple backbone
atoms. The estimated Ki values from Supple-
mentary Table 1 appear to suggest that G-1
might bind ERa as a low-afﬁnity antagonist.
When docked into the 4-hydroxytamoxifen-
bound conformation of ERa (Fig. 5c), both
G-1 enantiomers are estimated to have afﬁ-
nities approximately two orders of magnitude
above that of 4-hydroxytamoxifen. Once
again, however, both enantiomers of G-1 fail
to interact with key hydrogen bond partners,
while showing steric clashes with the receptor
ligand-binding site. Thus, we conclude that
G-1 cannot be positioned to interact opti-
mally with either the agonist- or antagonist-
occupied conformations of ERa, a ﬁnding
consistent with our experimental results.
In conclusion, the overall discovery process
was facilitated by the preliminary step of
virtual or in silico screening combined with technological advances,
including the use of a new ﬂuorescent estrogen derivative in the
binding assay, a high-throughput ﬂow cytometric platform (HyperCyt)
and new assays of estrogen-mediated PI3K activation. The discovery of
the ﬁrst GPR30-speciﬁc agonist that does not bind classical ERs should
facilitate further physiological experiments to deﬁne the role of GPR30
in vivo and open the door to the generation of diagnostics and
therapeutics directed at individual estrogen receptors.
Database processing. A database of 10,000 in-house molecules (CDLDB)
provided by Chemical Diversity Labs, to which 17b-estradiol was added, was
processed according to a procedure detailed elsewhere18. Very brieﬂy, canonical
isomeric SMILES (ref. 19, Daylight Toolkit v4.81, Daylight Chemical Informa-
tion Systems) were derived from input structures. Smi2fp_ascii (Daylight
Chemical Information Systems) and MACCSKeys320 Generator (Mesa Analy-
tics and Computing) software was used to compute Daylight and MDL20
ﬁngerprints, respectively, and 3D structures were derived with the OMEGA
software from OpenEye Scientiﬁc Software.
2D-based similarity. Using 17b-estradiol as a reference point, we computed
similarity coefﬁcients using both Daylight and MDL ﬁngerprints for CDLDB
using Tanimoto’s symmetric distance-between-patterns21 and Tversky’s
asymmetric contrast model22. Tanimoto similarities are symmetric (that is,
when molecule A is compared to molecule B and vice versa, the Tanimoto
coefﬁcients are identical), whereas Tversky similarity is asymmetric (that is,
the two coefﬁcient values differ). In total, six 2D-based similarity coefﬁcients
3D-based similarity. Using the 3D structure of 17b-estradiol as a reference
point, we obtained shape-similarity coefﬁcients using the Tanimoto21 and
Tversky22 formulas using ROCS, rapid overlay of chemical structures16, a
Gaussian-shape-volume-overlap ﬁlter that identiﬁes shapes that match the
query molecule. Another similarity metric, using Euclidian distances in a
nine-dimensional principal component analysis (PCA) space23, was derived
from ALMOND descriptors24. ALMOND encodes hydrophobic, H-bond
donor and H-bond acceptor pharmacophoric elements derived from molecular
interaction ﬁelds computed with GRID25 into a reduced set of variables.
Final similarity score. Our combined similarity score attributed 40% weight to
2D ﬁngerprints, 40% to the shape-based similarities and 20% to pharmaco-
phore-based similarity. Thus, Tanimoto and Tversky (substructure and
a b c
Figure 5 G-1 and 17b-estradiol structural overlap and docking to ERa. (a) Overlay of stick-model
representations of G-1 and 17b-estradiol to maximize spatial overlap by the shape-matching
procedure of ROCS. 17b-estradiol is shown in yellow, G-1 in light gray, oxygen atoms in bright red,
nitrogen in blue and bromine in dark red. (b,c) Docking of G-1 into the 17b-estradiol– (b) and
4-hydroxytamoxifen–bound (c) conformations of ERa (1ERE and 3ERT, respectively). The endogenous
ligand (17b-estradiol in b and 4-hydroxytamoxifen in c) as found in each crystal structure is shown in
red. The optimal shape overlays of G-1 onto 17b-estradiol (as shown in a) and 4-hydroxytamoxifen are
shown in blue. The optimally ERa-docked structures of G-1 based on protein interactions as computed
using AutoDock are shown in green.
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4 ADVANCE ONLINE PUBLICATION NATURE CHEMICAL BIOLOGY
superstructure) coefﬁcients were given a 6.66% contribution each, for both
Daylight and MDL ﬁngerprints, and a 13.33% contribution each for shape. The
Euclidian distance in PCA was given a 20% weight to the ﬁnal score.
Given this composite score, the top 100 ranked molecules were selected for
Ligand overlaying and docking. To overlay the structure of G-1 and 17b-
estradiol or 4-hydroxytamoxifen, we used the shape-matching procedure of
ROCS with full conformational ﬂexibility and the receptor-bound conforma-
tions of 17b-estradiol and 4-hydroxytamoxifen. We performed docking of
ligands into ERa using AutoDock 3.0.5 (ref. 26). We selected the 17b-estradiol
co-crystallized with ERa in its agonist-bound conformation and 4-hydroxyta-
moxifen co-crystallized with ERa in its antagonist-bound conformation. The
structure of G-1 was docked in the ligand-binding site of the two conforma-
tions of ERa, after extracting 17b-estradiol and 4-hydroxytamoxifen, respec-
tively. To get a computational estimate of the binding afﬁnity, both native
ligands were docked into their respective receptors as well. We assumed full
ligand ﬂexibility, and no initial positioning of the ligand into the binding
pocket was performed.
Ligand-binding assays. We performed binding assays as described4. Brieﬂy,
COS7 cells were transiently transfected with either nuclear estrogen receptor
(ERa-GFP or ERb-GFP) or GPR30-GFP and serum starved for 24 h before
assay. For HyperCyt screening, cells (B5 Â 104) were incubated with 10 mM
compound for 20 min in a ﬁnal volume of 10 ml before addition of 10 ml 8 nM
E2-Alexa633 diluted in saponin-based permeabilization buffer. Following
10 min at 37 1C, cells were washed once with 200 ml PBS in 1% BSA, and
2-ml samples were analyzed on a FACS Calibur. Cell Quest time bin–based data
produced by the HyperCyt system were analyzed with IDLQuery software as
described27. We analyzed dose responses manually using the indicated con-
centration of competitor and 2 nM E2-Alexa633. Nonspeciﬁc binding was
determined in presence of 100 nM 17b-estradiol. We carried out labeling of
cells for microscopy as previously described4 using 1 mM 17b-estradiol or G-1
to determine nonspeciﬁc binding. Competition binding studies were analyzed
with Prism 4.0 (GraphPad).
Intracellular calcium mobilization. Transfected COS7 cells (5 Â 106) were
incubated in HBSS containing 5 mM indo1-AM and 0.05% pluronic acid for
30 min at 25 1C. Cells were then washed once with HBSS and resuspended in
HBSS at a density of 107 cells per ml. We determined Ca2+ mobilization
ratiometrically using lex 340 nm and lem 400/490 nm at 37 1C in a spectro-
ﬂuorometer (QM-2000-2, Photon Technology International) equipped with
a magnetic stirrer. The relative 490/400 nm ratio was plotted as a function
PI3K activation. We used the PIP3 binding domain of Akt fused to mRFP1
(PH-mRFP1) to assess PIP3 production by PI3K. COS7 cells cotransfected with
GPR30-GFP or ERa-GFP and PH-mRFP1 were plated on coverslips and
serum starved for 24 h, and then they were stimulated with ligands as
indicated. The cells on coverslips were ﬁxed with 2% PFA in PBS, washed
and mounted in Vectashield (Vector Labs). The cells were analyzed by confocal
microscopy with a Zeiss LSM510.
Cell migration. Migration assays were carried out with 6.5 mm Transwells with
an 8-mm pore size (Costar Corning) as previously described28. The undersur-
face of the Transwell was coated overnight at 4 1C with approximately 50 mg
ml–1 rat-tail collagen and washed with PBS. DMEM/F12 (600 ml) supplemented
with 10% FBS, 10 ng ml–1 of EGF and 10 mg ml–1 of insulin was added to the
lower chamber as chemoattractant. SKBr3 or MCF7 cells (75,000 cells) in
serum-free DMEM/F12 (150 ml) were treated with ethanol (control), 17b-
estradiol or G-1 for 15 min at 37 1C before loading in the upper chamber. After
incubation for 48 h at 37 1C, the remaining cells were wiped from the upper
surface of the membrane with a damp cotton swab. The migrated cells on the
undersurface of the membrane were ﬁxed with 2% paraformaldehyde and
stained with 1% crystal violet. Quantiﬁcation of cells was performed by
counting the number of cells per ﬁeld in ﬁve random ﬁelds per membrane,
and migration was calculated as: % migration ¼ (number of cells in treated/
number of cells in ethanol control) Â 100.
Chemical synthesis and characterization of G-1. The compound G-1
quinolin-8-yl]-ethanone) was synthesized through a previously described
method29: triﬂuoroacetic acid (1.52 g, 13.3 mmol) was added dropwise to a
solution of 4-aminoacetophenone (7) (2.0 g, 14.8 mmol) in acetonitrile. Freshly
distilled cyclopentadiene (8) (3.91 g, 59.2 mmol) was added, followed by
6-bromopiperonal (9) (3.39 g, 14.8 mmol). The mixture was stirred overnight
at 25 1C, and the product was isolated from an aliquot of the mixture by
preparative HPLC (C18, 5–95% CH3CN in H2O gradient, 0.05% triﬂuoroacetic
acid), with a yield of approximately 70%.
Spectroscopic characterization of G-1 yielded the following: 1H NMR
(400 MHz, DMSO-d6): d 7.62 (s, 1H), 7.53 (dd, 1H, J ¼ 1.9 Hz, 8.6 Hz),
7.26 (s, 1H), 7.11 (s, 1H), 6.73 (d, 1H, J ¼ 8.4 Hz), 6.47 (s, 1H), 6.09 (d, 2H,
J ¼ 9.1 Hz), 5.97 (m, 1H), 5.60 (broad d, 1H, J ¼ 5.7 Hz), 4.78 (d, 1H,
J ¼ 3.3 Hz), 4.05 (d, 1H, J ¼ 8.8 Hz), 3.02 (q, 1H, J ¼ 8.4 Hz), 2.41 (s, 3H),
2.36–2.44 (m, 1H), 1.66 (broad dd, 1H, J ¼ 7–9 Hz). 13C NMR (100 MHz,
CD3CN): d 25.76, 31.61, 42.33, 45.50, 56.15, 102.69, 108.44, 112.75, 112.84,
115.49, 124.93, 127.57, 128.48, 130.31, 130.33, 134.19, 134.64, 147.95, 148.04,
150.94, 196.33. HRMS (m/z): calcd. C21H18BrNO3 requires MH+, 412.0543;
found MH+, 412.0539.
G-1 was analyzed by standard LC-MS methods with positive ion detection.
The LC-MS chromatogram showed the correct molecular (MH+) ion as well as
a single peak by both UV (254 nm) and ELSD detection. Only one diaster-
eomer was obtained; examination of the 1H NMR data shows H-4 (4.78 p.p.m.)
with a coupling constant of 3.3 Hz, indicating a cis orientation of the
cyclopentene ring and phenyl group, in agreement with the all-cis stereo-
chemistry determined by other groups for similar imino Diels-Alder
reactions using cyclopentadiene30. Thus G-1 is a racemic but diastereomerically
Accession codes. Protein Data Bank: 17b-estradiol-bound conformation of
ERa, 1ERE; 4-hydroxytamoxifen-bound conformation of ERa, 3ERT.
Note: Supplementary information is available on the Nature Chemical Biology website.
This work was supported by US National Institutes of Health (NIH) grant
AI36357 and a University of New Mexico Cancer Research and Treatment Center
Translational Research Grant to E.R.P., by NIH grant EB00264 to L.A.S., and by
support from the New Mexico Tobacco Settlement fund to C.G.B. and T.I.O.
Additional support was provided by the New Mexico Cancer Research and
Treatment Center (CRTC; NIH 1. P30 CA118100), the New Mexico Molecular
Libraries Screening Center (NIH MH074425) and the New Mexico Center for
Environmental Health Sciences (NIH ES012072). Flow cytometry data and
confocal images in this study were generated in the Flow Cytometry and
Fluorescence Microscopy Facilities, respectively, at the University of New
Mexico Health Sciences Center, which received support from National
Center for Research Resources (NCRR) 1 S10 RR14668, National Science
Foundation MCB9982161, NCRR P20 RR11830, National Cancer Institute
R24 CA88339, NCRR S10 RR19287, NCRR S10 RR016918, the University
of New Mexico Health Sciences Center, and the University of New
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing ﬁnancial interests.
Published online at http://www.nature.com/naturechemicalbiology/
Reprints and permissions information is available online at http://npg.nature.com/
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