A surface on the androgen receptor that allosterically
regulates coactivator binding
Eva Este ´banez-Perpin ˜a ´*, Alexander A. Arnold†, Phuong Nguyen‡, Edson Delgado Rodrigues‡, Ellena Mar*,
Raynard Bateman§, Peter Pallai¶, Kevan M. Shokat§, John D. Baxter‡?, R. Kiplin Guy†, Paul Webb‡,
and Robert J. Fletterick*?
*Department of Biochemistry and Biophysics,§Department of Molecular and Cellular Pharmacology, and‡Diabetes Center and Department of Medicine,
University of California, San Francisco, CA 94143;†Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital,
Memphis, TN 38105; and¶Bioblocks, Inc., San Diego, CA 92121
Contributed by John D. Baxter, August 24, 2007 (sent for review May 12, 2007)
Current approaches to inhibit nuclear receptor (NR) activity target
the hormone binding pocket but face limitations. We have pro-
posed that inhibitors, which bind to nuclear receptor surfaces that
mediate assembly of the receptor’s binding partners, might over-
come some of these limitations. The androgen receptor (AR) plays
a central role in prostate cancer, but conventional inhibitors lose
effectiveness as cancer treatments because anti-androgen resis-
tance usually develops. We conducted functional and x-ray screens
to identify compounds that bind the AR surface and block binding
that block coactivator binding in solution with IC50? 50 ?M and
inhibit AF-2 activity in cells were detected: three nonsteroidal
antiinflammatory drugs and the thyroid hormone 3,3?,5-triiodo-
thyroacetic acid. Although visualization of compounds at the AR
surface reveals weak binding at AF-2, the most potent inhibitors
bind preferentially to a previously unknown regulatory surface
cleft termed binding function (BF)-3, which is a known target for
X-ray structural analysis reveals that 3,3?,5-triiodothyroacetic acid
binding to BF-3 remodels the adjacent interaction site AF-2 to
weaken coactivator binding. Mutation of residues that form BF-3
inhibits AR function and AR AF-2 activity. We propose that BF-3 is
a previously unrecognized allosteric regulatory site needed for AR
activity in vivo and a possible pharmaceutical target.
antagonist ? high-throughput screening ? regulatory surface ?
antiandrogens ? nuclear receptors
compounds in development (2). Most NR ligands interact with
the internal ligand binding pocket [binding function (BF) 1] in
the C-terminal ligand binding domain (LBD) core (3). From
the LBD surface, with concomitant effects on coregulator
association and gene expression (3–6). It remains conceivable
that ligands could bind elsewhere. We identified a compound
that binds to the TR dimer interaction surface (7), we and others
identified compounds that bind the NR activation function 2
(AF-2) surface (discussed below), and another group showed
that glucose and oxysterols cooperate in activation of liver X
The strategy of targeting the ligand binding pocket with
pharmaceuticals has limitations. First, ligand size can be limited
by the enclosed nature of the pocket (3). Second, it is difficult to
devise strategies to modulate interaction surfaces that are not
remodeled by ligand or orphan NRs that lack known ligands or
ligand binding cavities. Third, partial agonist or mixed agonist
activities of ligands that bind BF-1 may not be desirable. This is
a particular problem for androgen receptor (AR) and estrogen
receptor antagonists, which are used to treat, respectively,
ineffective and promote tumor growth (9–11).
uclear receptors (NRs) play widespread roles in disease and
are major targets for pharmaceuticals (1), with many new
In principle, pharmaceutical attack at NR surface active sites
could overcome these problems (12), and NR AF-2 is a partic-
ularly attractive drug target (13). AF-2 is formed in response to
agonist binding and binds coregulators, including the steroid
receptor coactivator (SRC) family. Only 6–8 amino acids in
AF-2 are crucial, and these form a hydrophobic cleft that binds
short ?-helical peptides (NR boxes) in target coactivators and
could bind small molecules (BF-2). We identified two com-
pounds that block thyroid hormone receptor AF-2 association
with coactivators in solution and receptor activity in cells (14).
Others identified estrogen receptor-interacting compounds,
some of which block coregulator binding in vitro (14–17).
Strategies that target AR AF-2 could yield new therapeutics
for prostate cancer and other conditions (18–22). AR AF-2-
interacting peptides inhibit androgen response, representing
proof of principle that intervention at this surface is a viable
strategy for inhibition of AR activity in vivo (18). Compounds
that bind AF-2 should inhibit intramolecular association be-
tween the AR LBD and N-terminal domain (23) required for
protein 70 (ARA70) and SRC2 (20). AR AF-2 binds short
?-helical peptides with consensus FXXLF and the more com-
mon NR consensus LXXLL. Our x-ray structures of AR LBD
with representative peptides reveal that AF-2 amino acid side
chains move to create deep pockets that accommodate the bulky
aromatic amino acid side chains and represent attractive targets
for small molecules (19, 20, 24). These conformational changes
nevertheless also make it difficult to rationally design drugs that
bind this protein interaction surface (25).
Author contributions: E.E.-P., A.A.A., J.D.B., R.K.G., P.W., and R.J.F. designed research;
E.E.-P., A.A.A., P.N., E.D.R., E.M., and P.W. performed research; E.E.-P., A.A.A., P.N., E.D.R.,
R.B., P.P., K.M.S., R.K.G., and P.W. contributed new reagents/analytic tools; E.E.-P., A.A.A.,
P.N., J.D.B., R.K.G., P.W., and R.J.F. analyzed data; and E.E.-P., A.A.A., J.D.B., R.K.G., P.W.,
and R.J.F. wrote the paper.
Conflict of interest statement: J.D.B. has proprietary interests in and serves as a consultant
to Karo Bio AB, which has commercial interests in the nuclear receptor field.
Freely available online through the PNAS open access option.
Abbreviations: AF-2, activation function 2; AR, androgen receptor; BF, binding function;
DHT, dihydrotestosterone; FLF, flufenamic acid; FP, fluorescence polarization; K10, 1-tert-
butyl-3-(2,5-dimethyl-benzyl)-1H-pyrazolo[3,4-D]pyrimidin-4-ylamine; LBD, ligand-bind-
ing domain; 2MI, 2-methylindole; NR, nuclear receptor; RB1, 3-((1-tert-butyl-4-amino-1H-
pyrazolo[3,4-D]pyrimidin-3-yl)methyl)phenol; SRC, steroid receptor coactivator; TOL,
tolfenamic acid; Triac, 3,3?,5-triiodothyroacetic acid; T3, triiodothyronine; UCSF, University
of California, San Francisco.
Data deposition: The atomic coordinates and structure factors have been deposited in the
Protein Data Bank, www.pdb.org (PDB ID codes 2PIO, 2PIP, 2PIQ, 2PIR, 2PIT, 2PIU, 2PIV,
2PIW, 2PIX, 2PKL, and 2QPY).
?To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or flett@
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2007 by The National Academy of Sciences of the USA
October 9, 2007 ?
vol. 104 ?
In the current study we identified several AR surface-
interacting compounds that inhibit AR AF-2 activity with com-
plementary functional and structural screens. Similar strategies
were successful for drug development in other settings (25–28)
protein product of the murine double minute gene (mdm2), a
negative regulator of p53 tumor suppressor (29). We find,
unexpectedly, that the most potent compounds bind preferen-
tially to a novel site, BF-3, which allosterically influences co-
regulator association with AF-2, represents a new target for
modulation of AR activity, and may be a previously unknown
AR regulatory surface. Our results emphasize the potential of
x-ray crystallography for detection of regulatory sites on NR
Solution Screening Detects Inhibitors of AR/SRC2 Interactions. We
compounds that bind AR and inhibit coregulator association.
This assay detects binding of AR AF-2 to a 15-aa LXXLL
peptide overlapping the third SRC2 NR box (SRC2–3) in the
presence of the androgen dihydrotestosterone (DHT); SRC2–3
binds with an apparent equilibrium dissociation constant (Kd) of
2.2 ?M and is the tightest AR binding peptide in a library of
LXXLL and FXXLF motifs (20). More than 55,000 compounds
were screened. Most were from the Bay Area Screening Center
library (ChemDiv/ChemBridge), comprising relatively large
compounds (?400 Da) selected for diversity, solubility, and lack
of toxicity. Also included was the collection of ?1,200 off-patent
drugs from Prestwick (Illkirch, France).
We detected four compounds that inhibited AR interactions
with SRC2–3 by 40% or more at a concentration of 50 ?M (Fig.
1A), all from the Prestwick library. These were three nonsteroi-
dal antiinflammatory drugs, flufenamic acid (FLF), tolfenamic
acid (TOL), and meclofenamic acid, (30), and 3,3?,5-
triiodothyroacetic acid (Triac), a low-abundance thyroid hor-
mone (31). These compounds share several features, including
methylene or amine bridged phenyl rings and the carboxylic acid
group. Each inhibited AR interactions with SRC2–3 in a dose-
dependent fashion [Fig. 2 A and B and supporting information
(SI) Table 1], with TOL showing the highest potency (IC50? 47
?M). Competition isotherms exhibited saturation at high con-
centrations, implying the presence of defined binding sites on the
AR LBD and that effects are not caused by nonspecific dena-
turation of AR protein. We later determined that the common
active thyroid hormone triiodothyronine (T3) (31) also inhibits
AR/SRC2–3 interactions by ?24% at 50 ?M (data not shown).
The fact that only five of 55,000 compounds inhibited AR
interactions with SRC2–3 is indicative of high specificity in the
Inhibition of AR Activity. FLF blocked AR LBD binding to a
full-length coactivator. Similar to FP assays, FLF inhibition of
AR LBD interactions with radiolabeled SRC2 in vitro was
detected with 10–50 ?M FLF, and 100 ?M FLF inhibited SRC2
binding as efficiently as excess unlabeled SRC2–3 competitor
peptide (Fig. 2C). Triac activity could not be evaluated in these
assays; it caused AR LBD to precipitate in pull-downs.
All four compounds inhibited AR activity in cultured cells.
FLF (Fig. 2D) and Triac (Fig. 2E) inhibited DHT response with
AR LBD tethered to a reporter with a GAL DNA binding
virus (MMTV) promoter (Fig. 2F). Effects were dose-
obtained with TOL and meclofenamic acid (data not shown) and
in several cell types (data not shown). The nonsteroidal antiin-
flammatory drugs and Triac did not inhibit GAL fusions linked
inhibition of AR LBD/SRC2–3 interaction along with T3. FLF, Triac, and T3were
also identified by x-ray screen. (B) Compounds uniquely identified by x-ray
Small molecules that bind AR. (A) Compounds exceeding 40%
dose–response analysis of TOL (A) and Triac (B) with AR LBD and SRC2–3. (C)
complex plus vehicle (0) and 10 ?g of SRC2–3 peptide or FLF (1, 3, 10, 30, and
100 ?M). (D and E) AF-2 activity. Components are shown in a schematic at the
top. Graphs show luciferase assays (light units ? 104) normalized to ?-galac-
tosidase. Standard errors were derived from sextuplet points. Similar results
a schematic at the top. Data represent a typical experiment with DHT re-
sponse ? 100%.
Inhibition of coregulator binding and AR AF-2 activity. (A and B) FP
Este ´banez-Perpin ˜a ´ et al.PNAS ?
October 9, 2007 ?
vol. 104 ?
no. 41 ?
to activation domains from VP16 and CBP, confirming that
effects are not related to toxicity (data not shown).
X-Ray Screens Reveal AR-Interacting Molecules at AF-2 and a New Site
(BF-3). We also performed structural screens for AR-interacting
compounds (32). AR LBD-DHT crystals were soaked with
individual chemicals in groups of 1–10, and interacting com-
pounds were localized by x-ray diffraction and visual inspection
Prestwick library mentioned above and two libraries of chemical
fragments that are unlikely to bind the AR with high affinity but
were nonetheless chosen for their potential to be linked or
modified to create tighter binding scaffolds. One library, assem-
bled at the University of California, San Francisco (UCSF),
comprises 400 protein kinase inhibitors and related compounds
with characteristics of heterocyclic rings, similar to side chains of
FXXLF motifs. The second is a proprietary library of 200
chemicals with multiple functionalities (BioBlocks).
We found seven drugs at the AR surface. From the Prestwick
screens). We could not assess TOL and meclofenamic acid
binding because these compounds disrupt AR crystals and it was
possible to obtain FLF data sets only with short soaks (15 min).
We also detected four compounds from the UCSF library (Fig.
D]pyrimidin-4-ylamine (K10) and 3-((1-tert-butyl-4-amino-1H-
pyrazolo[3,4-D]pyrimidin-3-yl)methyl)phenol (RB1) are kinase
inhibitors with two aromatic rings; 2-methylindole (2MI) and
indole acetic acid resemble tryptophan indole rings. None of the
UCSF library compounds promotes SRC2–3 dissociation in FP
assays (data not shown), as expected from library design of
probable low-affinity binders.
Unexpectedly, the compounds that displace SRC2–3 from
AF-2, FLF, Triac, T3, and two low-affinity compounds, 2MI and
indole-3-carboxylic acid, bind to a previously unknown site, BF-3
(Fig. 3). This is a hydrophobic cleft at the junction of H1, the
H3–H5 loop, and H9 that is almost as large as AF-2 and exhibits
characteristics of protein interaction surfaces (developed below).
None of the compounds appear at AF-2 in short soaks, but
Triac, T3, 2MI, and kinase inhibitors K10 and RB1 eventually
appeared at this location with soaks of 7–20 h. FLF damages AR
crystals at these times. Slow appearance of small molecules at
AR AF-2 is not related to inaccessibility; crystal soaks with
SRC2–3 peptide revealed electron density corresponding to the
LxxLL motif at AF-2 within 1 h (data not shown).
Together, the studies confirm that it is feasible to detect AR
surface-interacting compounds with x-ray screens, that small
molecules bind AF-2, and, surprisingly, a novel small molecule
binding site, BF-3.
AR Surface-Interacting Compounds Bind Preferentially to BF-3. X-ray
structures suggest that Triac interacts preferentially at BF-3 vs.
yet exhibits stronger, uniformly well defined electron density at
BF-3; the Triac proximal and distal phenyl rings make hydro-
phobic contacts with a large BF-3 surface comprising Pro-723,
Phe-673, and Ile-672 from H1, Gly-724 and Asn-727 from H3–5,
and Phe-826, Glu-829, Glu-837, Arg-840, and Asn-833 from H9
(Fig. 4 A and C). In addition, the distal phenyl ring hydroxyl
group hydrogen-bonds with Asn-727, and the proximal phenyl
ring carboxylate hydrogen-bonds with the oppositely charged
Arg-840 side chain. Weaker association at AF-2 is due to poor
fit (Fig. 4 D and F). The Triac distal ring is well defined and
makes hydrophobic contacts with a deep AF-2 subpocket (S1)
that hosts F1or L1of the signature motif F1XXL4F5/L1XXL4L5,
but the proximal phenyl ring is poorly defined and spans the S2
and S3 subsites that host L4and F5/L5(Fig. 4D). T3displayed
similar binding modes to Triac at both sites (data not shown).
Interactions at BF-3 are well defined for other compounds.
FLF aromatic rings interact tangentially with BF-3 to bury 520
Å2of solvent-exposed surface (Fig. 4 B and C). Whereas 2MI
displays a binding mode similar to the Triac distal phenyl ring
and buries, respectively, 280 Å2and 370 Å2of accessible BF-3
and AF-2 surfaces, it is better resolved at BF-3 (data not shown).
also appears well defined (data not shown). By contrast, K10 and
AF-2 helices 3, 5, and 12, and H1. (B) Space-filling model showing residues in
AF-2 (cyan) and BF-3 (red). (C) As in B, rotated 90° to reveal BF-3.
AF-2 and BF-3. (A) Schematic of AR LBD showing location of DHT, key
www.pnas.org?cgi?doi?10.1073?pnas.0708036104 Este ´banez-Perpin ˜a ´ et al.
RB1 occupy ?580 Å2of solvent-exposed AF-2 surface but
exhibit even weaker electron density than Triac (Fig. 4 E and F
and data not shown). Both compounds engage in hydrophobic
interactions with S1 and S3, with better definition at S1 (Fig. 4F).
Interactions at BF-3 Weaken Coactivator Binding. Comparison of the
AR surface with or without Triac, T3, and FLF reveals structural
alterations. Four BF-3 residues (Arg-840, Asn-727, Phe-826, and
Glu-829) that point out of the pocket into solution point inward
and engage the compound (Fig. 5A). This is accompanied by
large movements of the Arg-726 side chain, close to AF-2, and
repositioning of AF-2 residues Lys-717 and Met-734 (data not
shown). There are also small but significant shifts in secondary
structural elements; residues 720–730 (H3) and 825–847 (H9)
exhibit rmsd of 0.33 and 0.44, respectively. Thus, Triac and FLF
promote structural rearrangements in BF-3 that are propagated
Drug interactions at BF-3 cause coregulator peptides that are
bound to AF-2 to become disordered. In crystals of ternary
complexes of AR LBD-DHT-SRC2–3 (LXXLL) and AR LBD-
DHT-ARA70 (FXXLF), the peptides fold into ?-helices of 15
and 9 amino acids (20), respectively, clearly defined by electron
density (Fig. 5 A Right and B Right). Short Triac incubations
result in loss of electron density in the regions that flank SRC2–3
hydrophobic triads (Fig. 5A) and disruption of Arg-726 interac-
tions with SRC2–3 residues that lie C-terminal to the LxxLL
motif. Triac binding to BF-3 also weakened ARA70 FxxLF
contacts (Fig. 5B); only four residues are visible with Leu?4 and
Phe?5 completely defined (Fig. 5B Right). Arg-726 does not
contact the FXXLF peptide, suggesting that reorganization of
AF-2 itself is important for this effect. Unexpectedly, Arg-840
adopts the inward-facing conformation in this experiment (Fig.
5B). Similar Arg-840 rearrangements are also seen with artificial
FXXLF peptides (19), suggesting that it is a hitherto unappre-
ciated feature of AR interactions with these NR boxes. It is
unlikely that Triac interacts directly with AF-2 to disrupt coac-
tivator binding, because it is not detected at AF-2 at these times
and the electron-rich iodine groups of Triac represent particu-
larly good markers. Control soaks with solvent (DMSO) reveal
no similar effects on coregulator peptide organization (data not
shown). Thus, Triac interactions at BF-3 weaken contacts be-
tween AR and coactivator peptides.
If BF-3 is important for AR action, then BF-3 mutations
should alter AR activity. Mutations at Gln-670, Ile-672, and
Leu-830 are associated with prostate cancer (33–35). Leu-830,
Pro-723, Gly-724, and Arg-840 are mutated in androgen insen-
sitivity syndrome (36) (www.androgendb.mcgill.ca). Targeted
mutagenesis of Asn-727 and Arg-840, which move on Triac
binding, eliminate AR LBD activity (Fig. 5C), similar to inhi-
are in blue, and acidic residues are in red. Shown are close-ups of interactions
with Triac (A) and FLF (B) as yellow stick models. (C) Superimposed Triac
(yellow) plus FLF (dark blue). (D–F) AF-2 lined by Met-734, Lys-720, Glu-897,
and Met-894 with subsites (S1–S3) highlighted by dots. Basic residues are in
blue, acidic residues are in red, and Met is in yellow. D and E show close-ups
Triac interacts with S1 and the area between S2 and S3 whereas RB1 interacts
with S1 and S3.
Interactions at the AR LBD surface. (A–C). BF-3 including Glu-829,
sticks). Arg-840, Phe-826, Asn-727 (BF-3), and Arg-726 (AF-2) adopt different
conformations. Without Triac, Arg-840 points outward and Arg-726 contacts
SRC2–3 (gray). With Triac, Arg-840 contacts ligand and Arg-726 does not
(blue trace). Blue dots indicate regions not visible with Triac; Leu residues are
shown as sticks. (B) As in A, with ARA70 with Triac (blue) and without Triac
(gray). Reorganization is similar to A except that Arg-840 points inward
without Triac. (C) AR AF-2 assay; wild type ? 100%. Results are averages of at
least three different experiments. (D) Transfection with full-length AR active
activity; green, mutations that increase activity.
BF-3 modulates AF-2. (A) Superposition of AR with SRC2–3 with Triac
Este ´banez-Perpin ˜a ´ et al.PNAS ?
October 9, 2007 ?
vol. 104 ?
no. 41 ?
bition obtained with mutations in AF-2 (20). Likewise, muta-
tions at Phe-673, Pro-723, Glu-724, Glu-737, and, possibly,
Arg-726 and Phe-826 reduce activity. Mutations in nearby
residues, Gln-670, Ile-672, Glu-829, and Asn-833, increase AR
AF-2 activity up to 5-fold. Similar results were obtained with
full-length AR at MMTV-LUC; mutations at Phe-673, Pro-723,
and Arg-840 inhibited androgen response (Fig. 5D). The muta-
tions that inhibit AR activity describe a continuous patch that
resembles the BF-3 surface defined by chemical interactions
BF-3 could be present in other NRs. Part of the site, the
H3–H4 loop, is a signature sequence (37). Superposition of
published structures reveals conservation of BF-3 residues in the
steroid subfamily (SI Fig. 6). Mutations in equivalent regions of
estrogen and glucocorticoid receptor are implicated in coacti-
vator binding (38, 39). Collectively, these data provide evidence
for a role of BF-3 in NR action.
We used two screens to identify molecules that inhibit AR
activity by binding the AR LBD surface at important sites. FP
with IC50? 50 ?M in a library of 55,000 compounds (FLF, TOL,
meclofenamic acid, and Triac). T3was identified on the basis of
its similarities to Triac. X-ray screening of three small compound
libraries (Prestwick, UCSF kinase inhibitors, and BioBlocks)
detected seven compounds, including three that were identified
in functional screens (FLF, Triac, and T3) and new compounds
(RB1, K10, 2MI, and indole-3-carboxylic acid).
Our most surprising finding is that the best inhibitors interact
preferentially with a novel surface site (BF-3). Three lines of
evidence suggest that ligand interactions with BF-3 exert indirect
effects on AF-2 to inhibit coregulator binding. First, FLF and
Triac promote reorganization of BF-3 residues (Asn-727, Phe-
826, Glu-829, and Arg-840) and AF-2 residues (Met-734 and
Lys-717) and large-scale repositioning of Arg-726 at the AF-2
boundary. Second, short Triac soaks weaken AR interactions
Third, BF-3 residues are required for optimal AR AF-2 activity
in cell culture. We considered the possibility that compounds
displace SRC2–3 through weak interactions with AF-2. In this
case, rapid binding of compounds to BF-3 may reflect crystal
packing constraints that render BF-3 available for drug inter-
actions. We believe that this is unlikely because a bulky SRC2–3
peptide appears rapidly at AR AF-2 in crystal soaks (data not
shown), but we cannot yet rule out this possibility. The models
are not mutually exclusive; BF-3-dependent effects could com-
plement weak binding of compounds to AF-2.
The natural role of BF-3 in vivo is unknown, but our data,
coupled with natural mutations, suggest that the site is impor-
tant. Mutations at Gln-670, Ile-672, and Leu-830 enhance AR
action in prostate cancer (33, 35, 40), and mutations at Gln-670
and Ile-672 enhance AR AF-2 activity (Fig. 5C). BF-3 is a target
for androgen insensitivity syndrome mutations at Ile-672, Leu-
830, Arg-840, and Asn-727, with mutations in the latter two
diminishing SRC2 binding in vitro although neither contacts
coregulator (41). Finally, mutations in BF-3 of other NRs are
implicated in coactivator binding (38, 42). BF-3 could bind
regulatory proteins or other AR domains and could, for exam-
ple, communicate information about DNA binding domain
position to the LBD and AF-2 and vice versa.
Regardless of the mechanism by which compounds displace
SRC2–3, the fact that we detect such compounds for ARs and
TRs (14) suggests that functional and structural screens are
viable methods for NR inhibitor development. High-throughput
functional screens detect inhibitors among large libraries of
drug-like compounds, and our experience with ARs and TRs
suggests that ‘‘hits’’ are uncommon but are specific and rarely
screens with large libraries, but this method complements func-
tional screens in three ways. First, x-ray screens provide infor-
interact preferentially with AF-2 S1, so strategies to improve
binding to S2 or S3 would yield higher-affinity compounds.
Second, x-ray screening reveals unexpected sites or interaction
modes; the discovery of BF-3 was a surprise. Third, x-ray screens
are the only known method to identify weakly interacting
compounds that bind the surface with high ligand efficiency and
comprise building blocks for tight binding compounds.
It may be feasible to develop three types of small molecules
that modulate AR and NR activity: classical drugs that bind
BF-1, drugs that bind surface-exposed active sites such as AF-2,
or drugs that bind surface allosteric sites such as BF-3. This
greatly expands the number of NR pharmaceutical targets and
the potential spectrum of responses, and representatives of each
class could even be used together. The fact that three leads are
off-patent aspirin derivatives approved for human use (30), and
that others are thyroid hormones with known actions in humans,
raises the possibility that such compounds could be used for
prostate cancer treatment. It is unlikely that natural T3or Triac
concentrations approach levels required to bind the AR surface
in vivo (31), but it is intriguing to speculate that AR surface
interactions contribute to documented inhibitory effects of
nonsteroidal antiinflammatory drugs on growth and survival of
prostate cancer cells (43). We propose that well designed
compounds engaging AF-2 or BF-3 will modulate coregulator
recruitment in physiological settings, including cancer, and that
combined functional/x-ray screening is a useful strategy for
identification of ligands that act at NR surfaces.
Materials and Methods
Peptide Synthesis. Five milligrams of SRC2–3 (CKENALLRYL-
LDKDD) was dissolved in 1 ml of PBS and added to 50 mg of
5-iodoacetamidofluorescein in 1 ml of DMF. After 3 h at room
temperature, 0.5 ml of ethanethiol was added and peptide was
purified by HPLC [XTerra C18 column: A, water (0.05% TFA);
B, CH3CN (0.05% TFA), linear gradient 0100% over 25 min;
Mass analysis (MALDI-TOF) showed one species at 2,195.8 (m/z).
Protein Expression. AR LBD (residues 663–919) was expressed in
Escherichia coli in the presence of DHT and purified by using
published protocols (20). Functionality was determined by
SRC2–3 binding in FP assays (20); Kdfor SRC2–3 binding was
Solution Screening. Plates (384 wells; Costar 3710) were prepared
with 4 ?l of compound (5 mM in DMSO) plus 80 ?l of dilution
EDTA/0.01% Nonidet P-40/10% glycerol/10.5% DMSO) by
using a WellMate (Matrix). Five microliters from the dilution
plates was transferred to 384-well assay plates followed by 20 ?l
of protein mixture (6.25 ?M AR plus DHT and 0.0125 ?M
peptide in dilution buffer; final concentration 50 ?M compound,
4% DMSO). FP was measured after 2 h (excitation ? 485 nm,
emission ? 530 nm) on an AD plate reader (Molecular Devices).
Longer incubation times led to inhibition of FP in negative
controls (DMSO only), possibly reflecting AR instability. For
dose–response, compounds were diluted from 5,000 to 2.44 ?M
in DMSO into a 96-well plate (Costar 3365). Twenty microliters
of mixture was added to 1.2 ?l of compounds in 384-well plates
(Costar 3710), yielding a final concentration of 300 to 0.146 ?M,
and equilibrated for 5 h before FP. Data were analyzed by using
SigmaPlot 8.0 (SPSS, Chicago, IL), and Kdvalues were obtained
by fitting data to y ? minimum ? (maximum ? minimum)/1 ?
(x/Kd) Hill slope.
www.pnas.org?cgi?doi?10.1073?pnas.0708036104Este ´banez-Perpin ˜a ´ et al.
Library Assembly for X-Ray Screens. Three libraries with different Download full-text
characteristics were used. A commercial library of 1,120 FDA-
approved drugs was from Prestwick. Compounds with protein
kinase inhibitor characteristics or multiple heterocyclic rings
were from UCSF. Two hundred small compounds ?200 Da with
druglike character, designed as building blocks for larger mol-
ecules, were from BioBlocks.
X-Ray Screening. Compounds were dissolved in DMSO at 10–20
mM and soaked with AR:DHT crystals on 96-well plates.
compounds. Increasing 0.2 ?l units of compound are added, and
crystals are monitored. If crystals survive, another 0.2 ?l is
added until crystals show fatigue. Fresh crystals plus maximum
tolerated chemical volume were used for cryo treatment and
Crystallization, Structure Determination, and Refinement. Approxi-
mately 80 crystals were flash-cooled in liquid N2for analysis at
the Lawrence Berkeley National Laboratory Advanced Light
Source (beamline 8.3.1) in each trip. Only data sets from crystals
that diffract ?2.5 Å were collected. All compounds that cause
AR LBD crystals to diffract poorly were checked afterward, and
soaks with lower concentration were performed. Data sets from
35 crystals were measured per 8-h shift and indexed and merged
by using ELVES. Molecular replacement solutions were ob-
tained by using rotation and translation functions from CNS
software. Model building used QUANTA (Accelrys Software)
monitored by using free R factor. Visual inspection of electron
densities using QUANTA allows identification of interacting
compounds. A composite omit map was also calculated absent
5% of the molecule to remove model bias. Calculation of
electron density and crystallographic refinement was performed
cycles of model building, conjugate gradient minimization, and
simulated annealing resulted in structures with good stereo-
chemistry. A Ramachandran plot shows that most residues fall
into favored regions (SI Table 2).
Pull-Downs and Transfections. Vectors, expression and labeling,
and assay procedures were previously described (20). New
mutants were made by QuikChange Site-Directed Mutagenesis
We thank Pascal Egea, Elena Sablin, Anang Shelat, and Arnold T.
Hagler for discussions; James Holton and George Meigs for assistance
for K10. This work was supported by the Prostate Cancer Foundation
(R.J.F. and P.W.); National Institutes of Health Grants DK58080 (to
R.J.F. and R.K.G.), DK41482 (to J.D.B.), and DK51281 (to J.D.B.);
Specialized Programs of Research Excellence/National Cancer Institute
Grant CA89520 (to E.E.-P.); Department of Defense Grants W81XWH-
05-1-0545 (to R.J.F.) and PC030607 (to P.W.); the UCSF Prostate
Cancer Program (P.W.); and a Herbert Boyer Postdoctoral Fellowship
1. Laudet V, Gronemeyer H (2002) The Nuclear Receptor Facts Book (Academic,
2. Baxter JD, Goede P, Apriletti JW, West BL, Feng W, Mellstrom K, Fletterick
RJ, Wagner RL, Kushner PJ, Ribeiro RC, et al. (2002) Endocrinology 143:517–
3. Weatherman RV, Fletterick RJ, Scanlan TS (1999) Annu Rev Biochem
4. Nettles KW, Greene GL (2005) Annu Rev Physiol 67:309–333.
5. Rosenfeld MG, Lunyak VV, Glass CK (2006) Genes Dev 20:1405–1428.
6. Lonard DM, O’Malley BW (2006) Cell 125:411–414.
7. Borngraeber S, Budny MJ, Chiellini G, Cunha-Lima ST, Togashi M, Webb P,
Baxter JD, Scanlan TS, Fletterick RJ (2003) Proc Natl Acad Sci USA
E (2007) Nature 445:219–223.
9. Dehm SM, Tindall DJ (2006) J Cell Biochem 99:333–344.
10. Lewis-Wambi JS, Jordan VC (2005) Breast Dis 24:93–105.
11. Scher HI, Sawyers CL (2005) J Clin Oncol 23:8253–8261.
12. Darimont BD (2003) Chem Biol 10:675–676.
13. Feng W, Ribeiro RC, Wagner RL, Nguyen H, Apriletti JW, Fletterick RJ,
Baxter JD, Kushner PJ, West BL (1998) Science 280:1747–1749.
14. Arnold LA, Estebanez-Perpina E, Togashi M, Jouravel N, Shelat A,
McReynolds AC, Mar E, Nguyen P, Baxter JD, Fletterick RJ, et al. (2005) J Biol
15. Arnold LA, Estebanez-Perpina E, Togashi M, Shelat A, Ocasio CA,
McReynolds AC, Nguyen P, Baxter JD, Fletterick RJ, Webb P, Guy RK (2006)
Sci STKE 2006:pl3.
16. Wang Y, Chirgadze NY, Briggs SL, Khan S, Jensen EV, Burris TP (2006) Proc
Natl Acad Sci USA 103:9908–9911.
17. Rodriguez AL, Tamrazi A, Collins ML, Katzenellenbogen JA (2004) J Med
18. Chang CY, Abdo J, Hartney T, McDonnell DP (2005) Mol Endocrinol
19. Hur E, Pfaff SJ, Payne ES, Gron H, Buehrer BM, Fletterick RJ (2004) PLoS
20. Estebanez-Perpina E, Moore JM, Mar E, Delgado-Rodrigues E, Nguyen P,
Baxter JD, Buehrer BM, Webb P, Fletterick RJ, Guy RK (2005) J Biol Chem
21. Chang CY, McDonnell DP (2005) Trends Pharmacol Sci 26:225–228.
22. Heinlein CA, Chang C (2004) Endocr Rev 25:276–308.
23. He B, Wilson EM (2002) Mol Genet Metab 75:293–298.
24. He B, Gampe RT, Jr, Kole AJ, Hnat AT, Stanley TB, An G, Stewart EL,
Kalman RI, Minges JT, Wilson EM (2004) Mol Cell 16:425–438.
25. Wells J, Arkin M, Braisted A, DeLano W, McDowell B, Oslob J, Raimundo
B, Randal M (2003) Ernst Schering Res Found Workshop 19–27.
26. Arkin MR, Wells JA (2004) Nat Rev Drug Discovery 3:301–317.
27. Berg T (2003) Angew Chem Int Ed Engl 42:2462–2481.
28. Toogood PL (2002) Curr Opin Chem Biol 6:472–478.
29. Vassilev LT, Vu BT, Graves B, Carvajal D, Podlaski F, Filipovic Z, Kong N,
Kammlott U, Lukacs C, Klein C, et al. (2004) Science 303:844–848.
30. Flower R (2003) Nat Rev Drug Discovery 2:179–191.
31. Braverman LE, Utiger RD (2000) (Lippincott Williams & Wilkins, Philadel-
32. Nienaber VL, Richardson PL, Klinghofer V, Bouska JJ, Giranda VL, Greer J
(2000) Nature 18:1105–1108.
33. Buchanan G, Yang M, Harris JM, Nahm HS, Han G, Moore N, Bentel JM,
Matusik RJ, Horsfall DJ, Marshall VR, et al. (2001) Mol Endocrinol 15:46–56.
34. Buchanan G, Greenberg NM, Scher HI, Harris JM, Marshall VR, Tilley WD
(2001) Clin Cancer Res 7:1273–1281.
35. Shi XB, Ma AH, Xia L, Kung HJ, de Vere White RW (2002) Cancer Res
36. McPhaul MJ (2002) Mol Cell Endocrinol 198:61–67.
37. Brelivet Y, Kammerer S, Rochel N, Poch O, Moras D (2004) EMBO Rep
38. Tanenbaum DM, Wang Y, Williams SP, Sigler PB (1998) Proc Natl Acad Sci
40. Chavez B, Vilchis F, Zenteno JC, Larrea F, Kofman-Alfaro S (2001) Clin Genet
41. Lim J, Ghadessy FJ, Abdullah AA, Pinsky L, Trifiro M, Yong EL (2000) Mol
43. Andrews P, Krygier S, Djakiew D (2002) Cancer Chemother Pharmacol
Este ´banez-Perpin ˜a ´ et al.PNAS ?
October 9, 2007 ?
vol. 104 ?
no. 41 ?