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Targeting the regulation of androgen receptor
signaling by the heat shock protein 90 cochaperone
FKBP52 in prostate cancer cells
Johanny Tonos De Leona, Aki Iwaib, Clementine Feauc, Yenni Garciaa, Heather A. Balsigera, Cheryl L. Storera,
Raquel M. Suroa, Kristine M. Garzaa, Sunmin Leed, Yeong Sang Kimd, Yu Chene, Yang-Min Ningf, Daniel L. Riggsg,
Robert J. Fletterickh, R. Kiplin Guyc, Jane B. Trepeld, Leonard M. Neckersb, and Marc B. Coxa,1
aDepartment of Biological Sciences and Border Biomedical Research Center, University of Texas at El Paso, El Paso, TX 79968; bUrologic Oncology Branch,
National Cancer Institute, Bethesda, MD 20892; cDepartment of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, Memphis, TN
38105; dMedical Oncology Branch, National Cancer Institute, Bethesda, MD 20892; eDepartment of Molecular Medicine, University of South Florida,
College of Medicine, Tampa, FL 33612; fCenter for Drug Evaluation and Research, US Food and Drug Administration, Silver Spring, MD 20993; gMayo
Clinic Arizona, Scottsdale, AZ 85259; and hDepartment of Biochemistry and Biophysics, University of California, San Francisco, CA 94158
Edited* by Sue Wickner, National Cancer Institute, National Institutes of Health, Bethesda, MD, and approved June 9, 2011 (received for review April 4, 2011)
Drugs that target novel surfaces on the androgen receptor (AR)
and/or novel AR regulatory mechanisms are promising alternatives
for the treatment of castrate-resistant prostate cancer. The 52 kDa
FK506 binding protein (FKBP52) is an important positive regulator
of AR in cellular and whole animal models and represents an
attractive target for the treatment of prostate cancer. We used a
modified receptor-mediated reporter assay in yeast to screen a
diversified natural compound library for inhibitors of FKBP52-
enhanced AR function. The lead compound, termed MJC13, inhibits
AR function by preventing hormone-dependent dissociation of
the Hsp90-FKBP52-AR complex, which results in less hormone-
bound receptor in the nucleus. Assays in early and late stage
human prostate cancer cells demonstrated that MJC13 inhibits
AR-dependent gene expression and androgen-stimulated prostate
cancer cell proliferation.
immunophilin ∣FKBP4 ∣steroid hormone receptor
Androgens are a major stimulator of prostate tumor growth,
and all current therapies act as classic antagonists by compet-
ing with androgens for binding the androgen receptor (AR) hor-
mone binding pocket. This mechanism of action exploits the
dependence of AR on hormone activation, but current treatment
options become ineffective in castrate-resistant prostate cancer
(CRPC), although CRPC remains ligand/AR-dependent. Thus,
drugs that target novel surfaces on AR and/or novel AR regula-
tory mechanisms may provide promising alternatives for the
treatment of CRPC (reviewed in ref. 1).
The maturation of cytoplasmic steroid hormone receptors
(SHR) to a mature hormone binding conformation is a highly
ordered, dynamic process that involves multiple chaperone and
cochaperone components (reviewed in ref. 2), all of which pre-
sent potential opportunities for therapeutic intervention. The
final mature complex in which the receptor is capable of high
affinity hormone binding includes heat shock protein 90 (Hsp90),
a 23 kDa cochaperone (p23), and one of a class of proteins
(termed FKBPs) characterized by their Hsp90-binding tetratrico-
peptide repeat (TPR) domain. The 52 kDa FK506 binding
protein (FKBP52) associates with receptor–Hsp90 complexes by
way of a C-terminal TPR domain and is a specific positive reg-
ulator of AR, glucocorticoid receptor (GR), and progesterone
receptor (PR) signaling (3–5). FKBP52 is required for normal
male sexual differentiation and development in mice as the
fkbp52-deficient mice (52KO) display characteristics of partial
androgen insensitivity syndrome including dysgenic prostate
(4, 6). FKBP proteins are validated targets of immunosuppressive
drugs. FK506 (Tacrolimus) is used clinically to suppress the
immune system following organ transplantation. FK506 binds
within the peptidyl-prolyl isomerase (PPIase) catalytic pocket of
a related family member, FKBP12. The chemical groups of
FK506 that project out from the PPIase pocket allow the
FKBP12-drug complex to bind tightly to and inhibit calcineurin,
which ultimately leads to immunosupression (7). Although
FK506 binding to a similar region in FKBP52 does not result in
immunosuppression, FK506 does inhibit FKBP52-mediated
potentiation of SHR function (3) and the drug inhibits LNCaP
prostate cancer cell proliferation (8), suggesting that interference
with FKBP52 modulation of AR activity may provide a novel
route to develop AR inhibitors with unique characteristics.
FKBP52 association with receptor-Hsp90 complexes results in
the enhancement of hormone binding (3, 9, 10); yet the mechan-
ism by which this occurs is unknown. Although FKBP52 binding
to Hsp90 is required for FKBP52 regulation of AR, whether or
not FKBP52 interacts directly with the receptor within the con-
text of the chaperone complex is unclear. Studies with chimeric
receptor proteins have localized FKBP52-mediated effects to the
receptor ligand binding domain (LBD) (3). The FKBP52 N-term-
inal FK506 binding domain (FK1) is required for receptor reg-
ulation, and functional domain mapping studies demonstrated
that the proline-rich loop overhanging the PPIase catalytic pocket
may serve as an interaction surface (10). Although PPIase enzy-
matic activity is not required for receptor regulation by FKBP52,
FK506 may disrupt receptor potentiation through disruption of
FK1 interactions with the receptor LBD. Based on this model,
we attempted to target the FKBP52 interaction and/or regulatory
site on the AR LBD with FKBP52-specific inhibitors. Such
compounds would not only serve as valuable pharmacological
tools for analysis of FKBP52-receptor interactions, but they also
may represent a promising therapeutic approach to inhibit AR
transcriptional activity.
Results
Identification of Small Molecule Inhibitors of FKBP52-Enhanced AR
Function. A yeast screen of compound library resulted in the
identification of two candidate inhibitors (H7 and H8) that
specifically inhibited FKBP52-enhanced AR-P723S function (the
P723S mutant increases AR sensitivity to FKBP52 potentiation
and was used to increase sensitivity in the assay) (Fig. S1). H8,
Author contributions: J.T.D.L., K.M.G., R.K.G., J.B.T., L.M.N., and M.B.C. designed research;
J.T.D.L., A.I., C.F., Y.G., H.A.B., C.L.S., R.M.S., S.L., Y.S.K., Y.C., Y.-M.N., D.L.R., and M.B.C.
performed research; J.T.D.L., C.F., D.L.R., R.J.F., J.B.T., L.M.N., and M.B.C. analyzed data;
and J.T.D.L., J.B.T., L.M.N., and M.B.C. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1To whom correspondence should be addressed. E-mail: mbcox@utep.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/
doi:10.1073/pnas.1105160108/-/DCSupplemental.
11878–11883 ∣PNAS ∣July 19, 2011 ∣vol. 108 ∣no. 29 www.pnas.org/cgi/doi/10.1073/pnas.1105160108
though functional, was disregarded because it was found to be
specific for FKBP52-enhanced AR-P723S but had no effect on
wild-type AR. In addition to H7, we assessed 28 additional com-
pounds that represented slight chemical modifications of H7 for
effects on FKBP52-enhanced wild-type AR, AR-P723S, and GR
signaling in the yeast assay. The results and chemical structures
for two of the most promising compounds, MJC01 and MJC13,
and the original hit compound H7 are shown in Fig. 1. While H7
produced maximal inhibition at 100 μM (Fig. 1A), MJC01 and
MJC13 displayed increased potency with maximal inhibition
between 5 and 10 μM (Fig. 1 Band C). All of the FKBP52-specific
compounds identified displayed at least some general receptor
inhibition at high doses. MJC01 generally inhibited AR signaling
in the absence of FKBP52 at 100 μM as evidenced by the upward
trend in the dose response curve at that dose (Fig. 1B). In con-
trast, MJC13 was FKBP52-specific up to 100 μM (Fig. 1C),
although it did produce some general receptor inhibition at
significantly higher concentrations. Neither MJC13 nor MJC01
affected inducible reporter expression indicating that the inhibi-
tion observed is not due to general effects on transcription, trans-
lation, or protein stability (Fig. S2). MJC13 was slightly more
specific for FKBP52-enhanced AR signaling as compared to GR
(Fig. 1C). Although MJC01 has a higher potency than H7 it is less
selective for FKBP52 as evidenced by the reduction in FKBP52-
specific inhibition at 100 μM. Thus, MJC13 is the most promising
lead compound displaying little effect on AR in the absence
of FKBP52 and displaying more selectivity for AR than other
compounds tested.
Some of the compounds tested, including MJC01 (Fig. 1B),
differentially affected wild-type AR and AR-P723S. Interestingly,
P723 is within the recently characterized BF3 surface, and the
MJC molecules are structurally similar to the BF3-binding fe-
namic acid derivatives. Fig. 1Ddemonstrates that the BF3-bind-
ing AR inhibitor flufenamic acid displayed no FKBP52-specific
effects. Thus, although structurally similar to the fenamic acids,
our compounds are functionally distinct. However, the structural
similarity to fenamic acids and differential effects on AR and
AR-P723S suggest the AR BF3 surface as the possible target site.
SHR amino acid sequence alignments identified six amino acid
residues (L805, C806, I842, K845, R840, F673) within the AR
LBD that are conserved in the FKBP52-regulated receptors, PR
and GR, but differ in the FKBP52-insensitive mineralocorticoid
receptor (MR). Analysis of the AR LBD crystal structure re-
vealed that these residues comprise a surface region that overlaps
with the recently described AR BF3 surface (11) (Fig. S3). Inter-
estingly, multiple residues on this surface, including C806, R840,
I842, R846, and P723, have been found mutated in prostate
cancer and/or androgen insensitivity syndrome (AIS) patients
(McGill Androgen Receptor Gene Mutations Database, http://
androgendb.mcgill.ca/). In addition, mutation of P723, within
the BF3 surface, results in a receptor that is hypersensitive to
FKBP52 potentiation (4). To assess the impact of the additional
residues on FKBP52 regulation of AR function we mutated each
of the residues and assessed the mutant receptors for their ability
to respond to FKBP52 potentiation in yeast reporter assays. We
identified two additional mutations, F673P and C806Y, which
resulted in AR hypersensitivity to FKBP52 potentiation (Fig. S3).
As highlighted in Fig. S3, F673 contacts P723 within the BF3
surface and C806, although not a surface residue, is buried di-
rectly below p723 and F673. Thus, the BF3 surface, particularly
the region containing F673 and P723, defines a putative FKBP52
interaction and/or regulatory surface.
We did not observe direct interaction between MJC13 and
FKBP52. In addition, none of the compounds tested were able
to compete with DHT for binding the AR LBD or with SRC2-
3 peptide for binding AF2 at relevant concentrations (Fig. S4).
In the absence of data directly demonstrating interaction with
the AR LBD we performed in silico docking simulations to pre-
dict the possible orientation of the molecules on the BF3 surface
(Fig. S5). Both MJC01 and MJC13 make extensive nonpolar
contacts with residues P723, F673, L830, and Y834 on the BF3
surface. The poses resemble that of flufenamic acid in its AR
complex structure (PDB ID code 2PIX). It is clear that the poses
shown are one of many that are possible and these simulations
should be viewed with caution. However, the poses with the
highest docking scores all contained contacts with and/or around
the P723 and F673 residues of AR.
Compounds Effectively Target FKBP52-Enhanced AR Signaling in
Mammalian Cells. The compound library screen and subsequent
structure activity relationship (SAR) analysis were performed
in yeast assays. To assess the effects of the compounds in higher
vertebrate model systems, we first tested the compounds for their
ability to inhibit AR signaling in MDA-kb2 cells (Fig. 2 Aand B).
This cell line contains a stable androgen-responsive luciferase
reporter and serves as a rapid assay for assessing AR inhibition
(12). MDA-kb2 cells were treated with a range of concentrations
of the indicated compounds for 20 hr and assessed for both cell
Fig. 1. Identification of inhibitors specific for FKBP52-regulated AR tran-
scriptional activity in yeast. Yeast reporter strains expressing wild-type AR
in the absence (control, closed circles) or presence (AR, closed squares) of
FKBP52, the AR-P723S point mutant in the absence (control, closed
circles) or presence of FKBP52 (AR-P723S, closed triangles), and wild-type
GR in the absence (control, closed circles) or presence (GR, closed diamonds)
were treated with a range of concentrations of the indicated compounds in
the presence of DHT. H7 (A) is the original hit identified from the library
screens, MJC01 (B) and MJC013 (C) are the current lead compounds, and flu-
fenamic acid (D) is a known AR inhibitor that associates with the BF3 surface.
The structures of the molecules are illustrated above each respective graph.
The data were normalized to show only effects on FKBP52-enhanced AR
function by calculating the percent reduction in the control strain for each
data point and adding that back to each data point for both the control and
FKBP52 tester strains. Thus, a hormesis-like effect, as seen for MJC01 at
100 μM(B), indicates that the receptor in the absence of FKBP52 was inhib-
ited at the particular drug dose used (FKBP52-independent inhibition).
De Leon et al. PNAS ∣July 19, 2011 ∣vol. 108 ∣no. 29 ∣11879
BIOCHEMISTRY
viability and AR-dependent expression of the luciferase reporter.
The compounds displayed no cellular toxicity as assessed by ATP
quantitation (Fig. 2A). However, H7, MJC01, and MJC13 all
inhibited AR-mediated expression of the luciferase reporter with
half maximal inhibitory concentrations (IC50) of 24.56, 1.79, and
3.60 μM, respectively (Fig. 2B).
Mouse embryonic fibroblasts (MEFs) derived from 52KO
mice (5) are the only mammalian system that provides an
FKBP52-negative background in which to test the compounds
for FKBP52-specific effects. Thus, we established AR-mediated
luciferase assays in 52KO MEF cells in the presence or absence
of an FKBP52 expression vector and assessed the compounds
for cellular toxicity and FKBP52-specific inhibition of androgen-
dependent luciferase expression. None of the compounds was
cytotoxic up to the maximum soluble concentration of 250 μM,
as assessed by trypan blue exclusion (Fig. 2C). MJC01 and
MJC13 specifically inhibited FKBP52-enhanced AR-mediated
expression of the luciferase reporter gene with IC50 values of
0.62 and 0.45 μM, respectively (Fig. 2 Dand E). Consistent with
the data obtained in the yeast assays, MJC01 displayed signifi-
cantly higher FKBP52-independent inhibition of AR function
(Fig. 2D) as compared to MJC13. MJC13 also produced general
AR inhibition in this system at concentrations above 50 μM.
To further evaluate compound specificity, the compounds were
assessed for effects on constitutive renilla luciferase expression
in the 52KO MEF cells (Fig. S2). Neither MJC13 nor MJC01
affected the constitutive expression of renilla luciferase.
Western blots using lysates prepared from the cells in Fig. 2E
showed increasing levels of AR and FKBP52 protein that directly
correlated with increasing concentrations of MJC13 (Fig. 2F).
The degree of stabilization varied between experiments and
one of the more dramatic examples is shown in Fig. 2F. Variable
stabilization of Hsp90 and p23 protein levels was also observed
but to a lesser degree than that seen for FKBP52 and AR.
MJC13 Prevents Receptor–Hsp90 Complex Dissociation and Nuclear
Translocation in Cellular Models of Prostate Cancer. The effects of
MJC13 on the stability of AR and associated chaperones is
similar to that observed in the presence of nonhydrolyzable ATP
analogues or sodium molybdate, which prevent hormone-depen-
dent receptor–Hsp90 complex dissociation. To test the effects
of MJC13 on complex formation and/or hormone-dependent
complex dissociation we performed coimmunoprecipitations of
FKBP52, AR and Hsp90 in lysates of several androgen-respon-
sive prostate cancer cell lines grown in the presence or absence
of hormone and MJC13 (Fig. 3). The ability of FKBP52 to bind
the AR-Hsp90 complex was unaffected by drug alone, while
addition of hormone alone resulted in complex dissociation (as
determined by loss of FKBP52, AR, and Hsp90 coprecipitating
together). However, complex dissociation in the presence of
hormone was abrogated by the addition of MJC13 to LNCaP,
LAPC4, and 22Rv1 cells (Fig. 3 A,C, and D, respectively). In
addition, Western immunoblots of fractionated lysates prepared
from LNCaP, LAPC4, and 22Rv1 cells grown in the presence or
absence of hormone and MJC13 revealed that hormone-induced
AR translocation to the nucleus was blocked by MJC13 (Fig. 3 B,
D, and F, respectively). MJC13 inhibition of AR nuclear translo-
cation was overcome by high hormone concentrations in LNCaP
cells (Fig. 3B), which may reflect the lack of receptor dependence
on FKBP52 at high hormone concentrations (3).
MJC13 Blocks AR-Dependent Gene Expression and Proliferation in
Prostate Cancer Cells. The effects of MJC13 on AR-dependent
gene expression were assessed by analysis of prostate specific
antigen (PSA) expression in LNCaP and VCaP cells. We also
assessed the impact of MJC13 on expression of the 51 kDa
FK506 binding protein (FKBP51) in these cell lines. FKBP51 has
emerged as a potential hormone-dependent cancer biomarker
(13) due to its hormone-inducible expression (14). However, un-
like PSA, FKBP51 is a component of SHR–chaperone complexes
(15) and has recently been shown to promote AR function in
LNCaP cells similar to FKBP52 (16). Thus, inhibition of FKBP51
expression by MJC13 may have therapeutic implications. ELISA
analysis of PSA secretion from LNCaP cells (Fig. 4A) and VCaP
cells (Fig. 4B) demonstrated that MJC13 effectively inhibits PSA
secretion from both cell lines. Inhibition of hormone-stimulated
PSA secretion from LNCaP cells was more potent as compared
to hormone-independent secretion from VCaP cells. However,
in the presence of hormone MJC13 inhibited PSA secretion
from VCaP cells to a similar degree as in LNCaP. The effects
of MJC13 on endogenous levels of PSA and FKBP51 in LNCaP
and VCaP cells were assessed by Western immunoblot and
densitometry (Fig. 4 Cand D). Representative blots for FKBP51,
PSA, and the loading control GAPDH are shown (Fig. 4 C
and D,Upper). The normalized and averaged densitometry data
from three independent experiments demonstrate that MJC13
reduced endogenous FKBP51 and PSA expression in a dose-
dependent manner (Fig. 4 Cand D,Lower). We also assessed
endogenous PSA gene expression and expression of the AR-re-
Fig. 2. Effects of the inhibitors on FKBP52-regulated AR function in mam-
malian cells. (Aand B) MDA-kb2 cells expressing a stably transfected AR-
responsive luciferase reporter were treated with 0.2 nM DHT and a range
of concentrations of the indicated compounds and assessed for cell viability
(A) and AR-dependent expression of a luciferase reporter (B). The IC50 values
for the compounds are shown in the legend. (C–E) Luciferase reporter assays
in 52KO mouse embryonic fibroblast cells in the presence or absence of
FKBP52 were performed. Transfected cells were treated with DHT and a
range of concentrations of the indicated compounds and assessed for cell
viability (C) and AR-dependent expression of a luciferase reporter (Dand E).
The IC50 values for MJC01 (D) and MJC13 (E) are shown in the legends. (F)
Lysates were prepared from 52KO MEF cells transfected with AR and FKBP52
expression vectors after treatment with DHT and a range of MJC13 concen-
trations (0, 0.05, 0.1, 0.5, 1, 5, 10, 50, and 100 μM) for 24 hr. Lysates were
immunoblotted for AR, FKBP52, and GAPDH (loading control).
11880 ∣www.pnas.org/cgi/doi/10.1073/pnas.1105160108 De Leon et al.
sponsive gene TMPRSS2 by quantitative real time PCR (Q-PCR)
in LNCaP and 22Rv1 cells. MJC13 effectively abrogated consti-
tutive expression of both AR-driven genes (Fig. 4E,Left). In
contrast, in 22Rv1 cells, MJC13 was only able to block andro-
gen-induced (but not constitutive) PSA expression (Fig. 4E,
Right). These data are consistent with the fact that 22Rv1 cells
express both full-length AR and a constitutively active, truncated
AR lacking the LBD (and so predicted to be insensitive to andro-
gen, FKBP52, and MJC13).
The effect of these compounds on androgen-dependent
prostate cancer cell proliferation was assessed by tritium thymi-
Fig. 3. Effects of MJC13 on AR-Hsp90 complex dissociation and AR nuclear
translocation in early and late stage prostate cancer cells. The effects of
MJC13 on hormone-dependent AR-Hsp90 complex dissociation and AR
nuclear translocation were assessed in LNCaP (Aand B), LAPC4 (Cand D),
and CWR22Rv1 (Eand F) cells by coimmunoprecipitation and Western blot,
respectively. Lysates from cells grown in the presence or absence of the
indicated concentrations of hormone and MJC13 for 24 hr were subjected
to immunoprecipitation with either an antibody directed against FKBP52
(A)orAR(Cand E) and immunoblotted for the indicated proteins. Fetal
bovine serum served as the source of hormone in A. Lysates prepared from
cells treated with the indicated concentrations of ligand and MJC13 for 24 hr
were also fractionated and immunoblotted for AR in both the cytosol and
nucleus (B,D, and F).
Fig. 4. Effects of MJC13 on AR-dependent gene expression in early and late
stage prostate cancer cells. (Aand B) ELISA assays to measure PSA secretion
were performed in LNCaP (A) and VCaP (B) cells. Cells were treated with the
indicated MJC13 concentrations in the presence (A) or absence (B) of DHT for
24 hr, and PSA levels in the media were quantified. (Cand D) Western blots to
measure AR-dependent expression of PSA and FKBP51 were performed in
LNCaP (C) and VCaP (D) cells. Cells were treated with the indicated concen-
trations of MJC13 in the presence (C) or absence (D) of DHT for 24 hr, lysed,
and lysates were electrophoresed and immunoblotted for FKBP51, PSA, and
GAPDH (loading control). The upper panels show representative Western
blots. The lower panels represent averaged densitometry data from at least
three independent experiments. (E) Left panel: PSA and TMPRSS2 gene
expression in LNCaP cells was assessed by Q-PCR. Cells were treated for
24 hr with increasing concentrations of MJC13 in the presence of 10% fetal
bovine serum. Data are displayed as expression relative to that of 18S rRNA;
right panel: R1881-dependent and independent PSA gene expression in
22Rv1 cells was assessed by Q-PCR. Cells (in the presence of charcoal-stripped
serum) were untreated, treated for 24 hr with MJC13 alone, or with 0.5 nM
R1881 in the presence and absence of 30 μM MJC13. Data are displayed as
PSA mRNA expression relative to that of 18S rRNA.
De Leon et al. PNAS ∣July 19, 2011 ∣vol. 108 ∣no. 29 ∣11881
BIOCHEMISTRY
dine incorporation in LNCaP, LAPC4, and 22Rv1 cells (Fig. 5).
MJC13 inhibited androgen-dependent cell proliferation at con-
centrations consistent with those observed to be effective in
reporter assays. For comparison, the effect of a known AR an-
tagonist, bicalutamide, which interacts with the hormone binding
pocket, was assessed. MJC13 was more potent than bicalutamide
in these assays. None of the compounds affected cell proliferation
in the absence of hormone.
Discussion
Functional domain mapping in yeast suggests that the FKBP52
FK1 domain is critical for regulating SHR function through
interaction with the receptor LBD (10). In support of this pro-
posal, we have identified a surface region on the AR LBD that,
when mutated, displays increased functional dependence on
FKBP52 (Fig. S3). This surface directly correlates with the
recently identified BF3 surface (11). Although we do not provide
direct evidence for FKBP52 interaction with BF3, the data pre-
sented here indicate, at the very least, that FKBP52 can indirectly
influence receptor function through this surface. We demonstrate
that FKBP52 regulation of receptor function can be blocked by
small molecules that are predicted to bind the BF3 surface. We
demonstrate that the lead molecule, MJC13, specifically inhibits
FKBP52-enhanced AR activity in both yeast and mammalian
cell lines (Figs. 1 and 2). MJC13 prevents hormone-induced AR-
Hsp90 complex dissociation in the presence of FKBP52, which
ultimately results in less receptor translocation to the nucleus
(Fig. 3). As a consequence, AR-dependent gene expression and
androgen-stimulated proliferation in prostate cancer cell lines are
inhibited (Figs. 4 and 5). These data suggest that FKBP52 regu-
lates AR function through the BF3 surface and that FKBP52-
mediated receptor potentiation can be inhibited by targeting
the BF3 surface with small molecules. FKBP52 potentiation of
AR signaling may be of particular relevance in CRPC, where
androgen levels are markedly reduced but still effectively stimu-
late the AR (13).
Although our data suggest that MJC13 binds the AR BF3 sur-
face, efforts to provide direct evidence of this interaction through
SPR analysis and cocrystallization have not been informative.
These difficulties are common to other molecules known or
thought to bind BF3. Providing direct evidence of BF3 binding
for some of the fenamic acid-derived AR inhibitors has also pro-
ven difficult. In addition to the fact that some of these molecules
only weakly bind to BF3 (11), we postulate that these molecules
can associate weakly with multiple sites on the LBD at high con-
centrations. It is also possible that in the absence of the dynamic
chaperone-assisted folding cycle the BF3 surface on the purified
AR LBD is not in an optimal conformation for MJC13 binding.
Nevertheless, multiple lines of evidence suggest that MJC13
inhibits FKBP52-mediated AR function through binding BF3.
First, we demonstrated that FKBP52 influences at least a portion
of the BF3 surface (Fig. S3), and the molecules identified in our
screen that inhibit FKBP52 regulation of receptor function are
structurally similar to known BF3 binding molecules. Second,
many of the compounds tested in the SAR studies differentially
affected AR-P723S as compared to wild-type AR and a few of
the molecules were specific for AR-P723S (Fig. 1). Thus, muta-
tions within the putative BF3 binding surface accentuate inhibitor
activity. Third, MJC13 effectively blocked hormone-dependent
PSA expression but failed to block hormone-insensitive PSA
expression in 22Rv1 cells (Fig. 4E,Right). Because MJC13 is
predicted to target the BF3 surface of the AR LBD, MJC13
would not be expected to affect the expression of PSA in these
cells resulting from a constitutively active AR protein lacking the
LBD. Finally, in silico docking simulations support the notion
that MJC13 binds BF3 (Fig. S5).
In summary, we have identified a surface region on the AR
LBD that, when mutated, results in a greater functional depen-
dence on FKBP52. This motif overlaps with the recently charac-
terized BF3 surface. We have developed a series of small
molecules that inhibit FKBP52 regulation of AR function. These
agents are predicted to act via binding to the BF3 surface in
the AR LBD. The most promising compound, MJC13, inhibits
hormone-induced AR-chaperone complex dissociation and nu-
clear translocation and effectively blocks AR-dependent gene
expression in cellular models of prostate cancer. Further studies
to characterize the MJC13 binding site, improve compound
efficacy, and improve receptor specificity are needed. MJC13
is a novel example of an inhibitor that specifically targets the reg-
ulation of SHR function by an Hsp90-associated cochaperone
and thus serves as an excellent starting point for development of
FKBP52-specific inhibitors to treat hormone-dependent diseases.
Materials and Methods
Yeast Strains and Assays. β-galactosidase reporter assays (3, 17) were used as a
quantitative indicator of AR activity for the yeast compound library screens
and for the other experiments described in this study. The basic reporter
strains used for wild-type AR, the indicated AR point mutant, and GR assays
were based on a W303a genetic background (MATa leu2-112ura3-1 trp1-1
his3-11, 15 ade2-1 can1-100 GAL SUC2) and all contained a URA3-marked
steroid receptor-mediated β-galactosidase reporter plasmid (pUCΔs-26X, a
kind gift from Brian Freeman, University of Illinois, Urbana-Champaign, IL).
Fig. 5. MJC compounds effectively inhibit androgen-dependent prostate
cancer cell proliferation. Tritium (tritiated thymidine) incorporation assays
were performed on LNCaP (A), LAPC4 (B), and 22Rv1 (C) cells treated with
a range of compound concentrations in the presence (closed symbols) or
absence (open symbols) of 0.5 nM DHT. The known AR antagonist bicaluta-
mide (circles) was included for comparison with MJC13 (squares). All data
are expressed as a percentage with the level of tritiated thymidine incorpora-
tion in the absence of compound for each condition set to 100%.
11882 ∣www.pnas.org/cgi/doi/10.1073/pnas.1105160108 De Leon et al.
Refer to SI Methods for more detailed information on the yeast reporter
assays.
Compound Library Screen. The yeast β-galactosidase reporter assays used to
screen the library of compounds were modified to a 96-well plate format
(see Fig. S1). The parent strain was deleted for the pleiotropic drug resis-
tance 5 (PDR5) gene to prevent the potential transport of compounds
out of the yeast. The control strain contained a wild-type human AR expres-
sion plasmid and an empty plasmid, and the tester strain contained an
AR-P723S expression plasmid and a human FKBP52 expression plasmid. The
assay protocol was designed to identify compounds that specifically reduce
signaling from the tester strain and not the control strain (FKBP52-specific
inhibition). The use of the AR-P723S mutant in these assays increased the
sensitivity of detection as signaling at the hormone doses used in the tester
strain depends entirely on the presence of FKBP52. The DHT concentrations
used correlated with the EC50 values for DHT in both the control and tester
strains. The Diversity Set Library from the Developmental Therapeutics Pro-
gram of the National Cancer Institute (http://dtp.nci.nih.gov) was used for
the screen. This library contains approximately 2,000 structurally character-
ized compounds representative of a diverse chemical space and derived
from a collection of almost 140,000 compounds. Library compounds were
assayed in the control and tester strains at an initial concentration of 50 μM.
See SI Methods for a more detailed description of the compound library
screens.
Mammalian Cell Lines. LNCaP, LAPC4, and VCaP prostate cancer cells all
express endogenous AR and are sensitive to androgens. LNCaP cells are
characterized by the presence of the AR T877A mutation. Both AR alleles
are wild type in LAPC4 cells. VCaP cells are characterized by endogenous
AR gene amplification and, although they can respond to androgens, are also
capable of androgen-independent growth. The 22Rv1 cell line was derived
from a xenograft that was serially propagated in mice after castration-
induced regression and relapse of the parental, androgen-dependent
CWR22 xenograft. The AR mutation occurred during the progression to
androgen independence. Full-length AR in 22Rv1 is characterized by an
in-frame tandem duplication of exon 3 that encodes the second zinc finger
of the AR DNA-binding domain (18, 19). 22Rv1 cells also express a constitu-
tively active truncated form of AR lacking the C-terminal hormone binding
domain. As a result, 22Rv1 cells can both respond to hormone and display
hormone-independent growth. Refer to SI Methods for more detailed
information on the cell lines and standard growth conditions.
Luciferase Reporter Assays. Plasmid transfections and luciferase assays were
performed according to standard procedures. Refer to SI Methods for a more
detailed description of the transfections and reporter assays.
Cellular Toxicity Assays. Cellular toxicity was determined using trypan blue
(Invitrogen) exclusion or ATP measurements (CellTiter-Glo; Promega). For a
more detailed description of the cellular toxicity assays refer to SI Methods.
Cell Proliferation Assays. Cells were plated in a U-shaped 96-well plate at a
density of 3×103cells∕well. After the cells attached they were treated with
inhibitor for 1 h followed by the addition of 500 pM DHT. The wells were
treated with 20 μl of tritiated thymidine (Isotype-3H from Perkin Elmer)
for 18 hr. Cells were lysed using a Cell Harvester (Micro96 Harvester, Skatron
Instruments) and lysates were transferred to filter paper (FilterMAT Cat #
11731, Skatron Instruments) and incubated for 1 hr at room temperature.
Samples were diluted in 3 ml of scintillation fluid (Scinti SAFE Econo F
(LSC Cocktail) SX-22-5, Fisher) and subjected to scintillation counting. All
experimental measurements were performed in triplicate.
ELISA Assays for PSA. ELISA assays were performed according to manufac-
turer’s instructions (Alpha Diagnostic International). Refer to SI Methods
for a more detailed description of the ELISA assays.
Immunoblots and Coimmunoprecipitations. Western blots and coimmuno-
precipitations were performed according to standard procedures. Refer to
SI Methods for a detailed description. The mouse monoclonal antibody
directed against FKBP52 (HI52D) was previously described (20). The mouse
monoclonal antibody directed against Hsp90 (H90-10) was generously pro-
vided by David Toft (Mayo Clinic, Rochester, MN). The polyclonal rabbit anti-
body directed against AR (N-20; Santa Cruz Biotechnology), the mouse
monoclonal antibody directed against glyceraldehyde phosphate dehydro-
genase (6C5; Biodesign International), and alkaline phosphatase-conjugated
anti-rabbit and anti-mouse secondary antibodies (Southern Biotechnology
Associates) were all obtained commercially.
ACKNOWLEDGMENTS. We thank Brian Freeman, Charles Sawyers, Robert
Reiter, David Toft, and Donald Tindall for providing reagents. The authors
are grateful to David Smith and Charles Miller for critically reading the
manuscript. We thank the Border Biomedical Research Center’s [Grant
5G12RR008124, National Center for Research Resources/National Institutes
of Health (NIH)] Biomolecule Analysis Core Facility, Tissue Culture Core Facil-
ity, and the DNA Analysis Core Facility for the use of the instruments. This
project was also supported in part by American Recovery and Reinvestment
Act funds through Grant SC1GM084863 to M.B.C. from the National Institute
of General Medical Sciences, NIH. J.T.D.L. was supported by Research Initia-
tive for Scientific Enhancement (R25GM069621) and National Science Foun-
dation Louis Stokes Alliances for Minority Participation (HRD-0832951)
fellowships. R.K.G. and C.F. were supported by the American Lebanese
Syrian Associated Charities, St. Jude Children’s Research Hospital, the NIH
(DK58080), and the Department of Defense Prostate Cancer Research Pro-
gram (PC060344-W81XWH-07-1-0073). A.I., S.L., Y.S.K., Y.N., J.B.T., and L.M.N.
were supported by funds from the Intramural Research Program of the
National Cancer Institute.
1. Moore TW, Mayne CG, Katzenellenbogen JA (2009) Minireview: Not picking pockets:
Nuclear receptor alternate-site modulators (NRAMs). Mol Endocrinol 24:683–695.
2. Smith DF, Toft DO (2008) Minireview: The intersection of steroid receptors with
molecular chaperones: observations and questions. Mol Endocrinol 22:2229–2240.
3. Riggs DL, et al. (2003) The Hsp90-binding peptidylprolyl isomerase FKBP52 potentiates
glucocorticoid signaling in vivo. EMBO J 22:1158–1167.
4. Cheung-Flynn J, et al. (2005) Physiological role for the cochaperone FKBP52 in andro-
gen receptor signaling. Mol Endocrinol 19:1654–1666.
5. Tranguch S, et al. (2005) Cochaperone immunophilin FKBP52 is critical to uterine
receptivity for embryo implantation. Proc Natl Acad Sci USA 102:14326–14331.
6. Yong W, et al. (2007) Essential role for co-chaperone Fkbp52 but not Fkbp51 in andro-
gen receptor-mediated signaling and physiology. J Biol Chem 282:5026–5036.
7. Hamilton GS, Steiner JP (1998) Immunophilins: Beyond immunosuppression. J Med
Chem 41:5119–5143.
8. Periyasamy S, Hinds T, Jr, Shemshedini L, Shou W, Sanchez ER (2010) FKBP51 and Cyp40
are positive regulators of androgen-dependent prostate cancer cell growth and the
targets of FK506 and cyclosporin A. Oncogene 29:1691–1701.
9. Davies TH, Ning YM, Sanchez ER (2005) Differential control of glucocorticoid receptor
hormone-binding function by tetratricopeptide repeat (TPR) proteins and the immu-
nosuppressive ligand FK506. Biochemistry 44:2030–2038.
10. Riggs DL, et al. (2007) Noncatalytic role of the FKBP52 peptidyl-prolyl isomerase
domain in the regulation of steroid hormone signaling. Mol Cell Biol 27:8658–8669.
11. Estebanez-Perpina E, et al. (2007) A surface on the androgen receptor that allosteri-
cally regulates coactivator binding. Proc Natl Acad Sci USA 104:16074–16079.
12. Wilson VS, Bobseine K, Lambright CR, Gray LE, Jr (2002) A novel cell line, MDA-kb2,
that stably expresses an androgen- and glucocorticoid-responsive reporter for the
detection of hormone receptor agonists and antagonists. Toxicol Sci 66:69–81.
13. Mostaghel EA, et al. (2007) Intraprostatic androgens and androgen-regulated gene
expression persist after testosterone suppression: Therapeutic implications for castra-
tion-resistant prostate cancer. Cancer Res 67:5033–5041.
14. Febbo PG, et al. (2005) Androgen mediated regulation and functional implications
of fkbp51 expression in prostate cancer. J Urol 173:1772–1777.
15. Chen S, Sullivan WP, Toft DO, Smith DF (1998) Differential interactions of p23 and
the TPR-containing proteins Hop, Cyp40, FKBP52 and FKBP51 with Hsp90 mutants.
Cell Stress Chaperones 3:118–129.
16. Ni L, et al. (2010) FKBP51 promotes assembly of the Hsp90 chaperone complex
and regulates androgen receptor signaling in prostate cancer cells. Mol Cell Biol
30:1243–1253.
17. Balsiger HA, Cox MB (2009) Yeast-based reporter assays for the functional character-
ization of cochaperone interactions with steroid hormone receptors. Methods Mol
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18. Sobel RE, Sadar MD (2005) Cell lines used in prostate cancer research: A compendium
of old and new lines—part 1. J Urol 173:342–359.
19. Sobel RE, Sadar MD (2005) Cell lines used in prostate cancer research: A compendium
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De Leon et al. PNAS ∣July 19, 2011 ∣vol. 108 ∣no. 29 ∣11883
BIOCHEMISTRY
Supporting Information
De Leon et al. 10.1073/pnas.1105160108
SI Methods
Yeast Strains and Assays. The AR point mutants were generated
directly in the yeast expression vector by site-directed mutagen-
esis using the QuickChange kit (Stratagene) according to the
manufacturer’s instructions. For hormone-responsive reporter
assays, the indicated strains were cotransformed with three plas-
mids: a hormone-inducible β-galactosidase reporter plasmid, a
LEU2-marked high-copy number plasmid constitutively expres-
sing the indicated steroid hormone receptor from a glyceralde-
hyde phosphate dehydrogenase (GPD) promoter, and a TRP1-
marked high-copy number plasmid with or without human
FKBP52 where indicated. Transformants were selected and
maintained on synthetic complete dextrose medium lacking ura-
cil, leucine, and tryptophan. All hormones were obtained from
Sigma and were stored as 10 mM stock solutions in ethanol.
Hormone dilutions were setup so that the ethanol vehicle never
exceeded 1% in the yeast cultures. The hormone concentrations
were optimized by performing dose-response curves. Hormone-
induced reporter activity was measured from yeast extracts as
described previously with a single two-hour time point measure-
ment. All assays were performed at least in triplicate and the data
shown are representative of at least three independent experi-
ments that produced consistent results.
Compound Library Screens. Yeast were grown in synthetic complete
medium lacking uracil, leucine, and tryptophan at 30 °C in a
shaking water bath and the optical density at 600 nm (O:D:600 )
was monitored until cultures were in exponential phase growth.
Cultures were then aliquoted into a 96-well plate at 100 μl per
well and the compounds were added. The compounds were
stored in dimethyl sulfoxide (DMSO); the yeast strain used in this
assay can tolerate up to 5% DMSO without significant effects
on assay results. Hormone was added 30 min after compound
addition, and at 2 hrs and 30 min 100 ul of Tropix Gal-Screen
reagent (Applied Biosystems) was added to each well. The plates
were incubated for an additional 1 hr and 30 min at room
temperature and light emission was measured on a microplate
luminometer (Luminoskan Ascent, Thermo Labsystems). Two
compounds were identified (H7 and H8) that inhibited FKBP52-
enhanced AR function but did not affect AR function in the
absence of FKBP52. These compounds were further analyzed
for effects in wild type AR and GR reporter assays as described
above, and H7 was selected for further study. An additional
28 compounds that represented slight structural modifications
of H7 were purchased from the Sigma Rare Chemicals Library
(Sigma-Aldrich) and also assayed.
Mammalian Cell Lines. Mouse embryonic fibroblast cells isolated
from homozygous 52KO embryos (52KO MEF) were previously
described (1). The MDA-kb2, HeLa, 22Rv1, and VCaP cells were
obtained commercially from the American Type Culture Collec-
tion (ATCC). LNCaP cells were obtained from Donald Tindall
(Mayo Clinic, Rochester, MN). LAPC4 cells were obtained from
Charles Sawyers (Memorial Sloan-Kettering Cancer Center, New
York) and Robert Reiter (Geffen School of Medicine, UCLA,
Los Angeles). All cells were maintained in the presence of
10% fetal bovine serum and 5% CO2at 37 °C with the exception
of MDA-kb2 cells. MDA-kb2 cells were grown in the absence of
CO2at 37 °C. 52KO MEF and HeLa cells were maintained in
MEM-EBSS medium with 2 mM L -glutamine, LNCaP and
22Rv1 cells were maintained in RPMI-1640 medium, MDA-
kb2 cells were maintained in L-15 medium, and LAPC4 and
VCaP cells were maintained in DMEM medium. Twenty-four hrs
prior to experiments cells were switched to medium lacking
phenol red and containing charcoal-stripped FBS.
Luciferase Reporter Assays. Plasmid transfections in 52KO MEFs
were performed using lipofectamine 2,000 reagent (Invitrogen)
using standard procedures. The plasmids used for these assays
were: a hormone-responsive firefly luciferase reporter (400 ng
per well), a mammalian expression vector (pCI-neo; Promega)
expressing either the androgen receptor (800 ng per well) or
FKBP52 (800 ng), and a constitutive β-galactosidase expression
plasmid (50 ng) (transfection control). 24 hrs after transfection
the cells were treated with the indicated inhibitor concentrations
for 1 hr followed by treatment with DHT (concentrations used
corresponded to the EC50 for DHT at each condition) for
16 hr. For transfections cells were plated in six-well plates at a
cell density of 2×106cells∕well (approximately 80% confluence)
and plasmid transfections were performed for 4 hr at a DNA (μg)
to lipofectamine ratio of 1∶3in MEM-EBSS lacking fetal bovine
serum. Following transfection and treatment the cells were then
lysed in 150 μl of M-PER mammalian protein extraction reagent
(Thermo Fisher Scientific Inc.) supplemented with Complete
mini EDTA-free protease inhibitors (Roche), and incubated
at room temperature for 10 min. For luciferase activity, 100 μl
of luciferase assay reagent (Promega) was added to 40 μl of cell
lysate in an opaque 96-well plate and light emission was measure
in a luminescence plate reader (Luminoskan Ascent, Thermo
Labsystems). For β-galactosidase activity, 100 μl of Tropix Gal-
screen reagent (Applied Biosystems) was added to 10 μl of cell
lysate in a white 96-well plate and incubated for 2 hr at room
temperature. β-galactosidase activity was measured using a lumi-
nescence plate reader. Differences in transfection efficiency were
normalized by dividing RLU (relative light units) by β-galactosi-
dase activity. The MDA-kb2 cells stably express an androgen-
responsive firefly luciferase reporter construct as previously
described. Thus, no plasmid transfections were necessary in this
cell line. MDA-kb2 cells were treated with 0.2 nM DHT (EC50 for
DHT in this cell line) and the indicated concentrations of inhi-
bitors for 20 hr followed by cell lysis and luminescence measure-
ment using Bright-Glo (Promega) luciferase assay reagent
according to the manufacturer’s instructions. The data shown
are representative of at least three independent assays with each
data point measured in triplicate.
Immunoblots and Coimmunoprecipitations. For the assessment of
AR nuclear translocation cells grown to 50% confluence were
washed in serum-free medium and recultured for 48 hr in fresh
medium followed by addition of MJC13 at the indicated concen-
trations. After an additional 24 hr hormone was added (R1881
or fetal bovine serum) and cells were cultured for an additional
2 hr. Cells were lysed and separated into nuclear and cytosolic
fractions according to established methods. Detection of AR
in the nucleus and the cytosol was performed by Western blotting
after polyacrylamide gel electrophoresis. For Western blots of cell
extracts, cells were lysed 48 hr after transfection with the M-PER
mammalian protein extraction reagent (Thermo Fisher Scientific
Inc.) as described above. To determine protein concentration the
Coomassie Plus protein assay reagent (Thermo Fisher Scientific
Inc.) was used. Typically 20 μg of total cellular protein was separ-
arted on a 10–20% Criterion gel (BioRad) and transferred to
a Polyvinylidene Difluoride membrane. Proteins were visualized
De Leon et al. www.pnas.org/cgi/doi/10.1073/pnas.1105160108 1of6
using an Immuno-star substrate (BioRad) and exposing to
X-ray film.
For coimmunoprecipitations cells were plated in 10 cm dishes
at 10% confluency in RPMI 1640 medium containing 10% char-
coal-stripped fetal bovine. After 24 hr, MJC13 or vehicle was
added to a final concentration of 30 μM. After an additional
24 hr, R1881 was added to a final concentration of 300 pM
and cells were lysed 2 hr later in TMNSV buffer [50 mM
Tris•HCl (pH 7.4), 0.1% Nonidet P-40, 20 mM Na2MoO4,
150 mM NaCl, 2 mM Na3VO4] supplemented with a Complete™
protease inhibitor tablet (Roche). Protein concentrations were
measured with a BCA™assay kit (Thermo Scientific) and
1mlof1mg∕mL cell lysates was prepared. 30 μl of recombinant
Protein G agarose beads were added to each sample and samples
were rotated for 1 hr at 4 °C; 10 μg of immunoprecipitating
antibody was then added to each sample and the samples were
rotated for an additional 2 hr at 4 °C. Beads were washed four
times with TMNSV buffer and boiled in SDS sample buffer
for 5 min. Eluted proteins were subjected to SDS/PAGE, trans-
ferred to nitrocellulose membrane, and blotted with specific
antibody for the protein of interest.
Cellular Toxicity Assays. For trypan blue exclusion, the cells were
plated in six-well plates at a cell density of 2×106cells∕well
and treated with a range of inhibitor concentrations for 24 hr.
The cells were trypsinized and centrifuged for 5 min at 100 ×g.
The cell pellet was resuspended in 1 mL of phosphate buffered
saline and 10 μl of cell suspension, mixed with 10 μl of 0.4% try-
pan blue and incubated for 3 min at room temperature, followed
by counting the number of dye-excluding cells in a hemocyt-
ometer. The quantitation of ATP as a measure of metabolically
active MDA-kb2 cells in the presence of a range of inhibitor con-
centrations was performed using the CellTiter-Glo Luminescent
Cell Viability Assay kit (Promega) according to manufacturer’s
instructions. Cells were treated with the indicated concentrations
of compound for 20 hr prior to evaluation.
ELISA Assays for PSA. Indicated cells were plated at a density of
2×106cells∕well and treated with a range of inhibitor concen-
trations 24 hr after plating, followed by treatment with 500 pM
DHT (LNCaP cells). VCaP cells were not treated with DHT.
Media were collected for assay from each well daily and stored
at −20° C until all treatments were completed. ELISA plates
(Alpha Diagnostic International) previously treated for the de-
tection of human PSA by the manufacturer were used. 25 μlof
sample (conditioned media) or standard was added to each well
with 100 μl of AB-enzyme conjugate and incubated for 30 min at
room temperature. The wells were washed with 300 μl of wash
buffer followed by the addition of 100 uL TMB substrate per well
and an incubation period of 15 min at room temperature. The
reaction was stopped by adding 50 μl of stopping solution to
all wells and absorbance was measured at 450 nm using a Versa
Max microplate spectrophotometer.
Quantitative Real-Time PCR. RNA was isolated using the RNeasy
RNA Isolation kit according to manufacturer’s instructions
(Qiagen). For reverse transcription, 200 ng of total RNA was
used in a reaction mixture containing 1X TaqMan RT buffer
(Applied Biosystems) and Multiscribe Reverse Trasnscriptase.
Reverse transcription was performed for 10 min at 25 °C, 30 min-
utes at 48 °C and 5 min at 95 °C using the PE9700 thermal cycler
(Applied Biosystems). Real-time PCR primers were designed
using Primer Express software (Applied Biosystems). The num-
ber of PCR cycles needed to reach the fluorescence threshold
value is the cycle threshold (Ct). Ct values for the control (18S
rRNA) and PSA gene were determined and relative RNA levels
were calculated by the comparative Ct method as described by
the manufacturer. Experiments were performed in duplicate;
data are shown as PSA expression relative to 18S rRNA.
Scintillation Proximity Binding Assays. The basic protocol for the
scintillation proximity binding assay with AR LBD was previously
described (2). Liquid handling was carried out using an auto-
mated liquid handling system (Biomek FX). To a 384-well Ni-
chelate coated Flashplatefi(PerkinElmer) 50 μlof5μM recom-
binant AR LBD was added in assay buffer (50 mM HEPES,
150 mM Li2SO4, 0.2 mM TCEP, 10% glycerol, 0.01% Triton
X-100, pH 7.2). After a 60-min incubation the protein solution
was discarded, followed by washes with assay buffer. 25 μlof
the indicated serially diluted small molecules in assay buffer
containing 10% DMSO were added into each well followed by
an additional 25 μl of a radioligand solution in assay buffer with
a final assay solution of 5% DMSO. The plates were sealed and
allowed to equilibrate for 5 hr at 4 °C. ½3H-DHT was used at a
final concentration of 20 nM. Radiocounts were measured using a
TopCount Microplate Scintillation and Luminescence Counter
(Packard Instrument Company).
Fluorescence Polarization Assay. The fluorescence polarization
assay was previously described (3). In short, 20 μl of protein mix-
ture (6.25 μM recombinant AR LBD plus DHT and 0.0125 μM
SRC2-3 peptide in dilution buffer; final concentration 50 μM
compound, 4% DMSO) was added to 1.2 μl of respective com-
pound solubilized in DMSO in a 384-well plate (Costar 3710).
Plates were equilibrated for 5 hr before total fluorescence and
fluorescence polarization measurements (excitation 485 nm,
emission 530 nm) were made using an Envision fluorescence
reader (Perkin Elmer).
AR BF3 Docking Simulations. MJC01 and MJC13 were docked into
the BF3 pocket of androgen receptor using DOCK 3.5.54 (4, 5).
Seven AR complex structures (PDB ID codes, 2PIT, 2PIU, 2PIV,
2PIW, 2PIX, 2PKL, and 2QPY) were docked individually to
capture some receptor conformational flexibility. The protein in
the complex structure was used as rigid receptor, with all waters
removed. The original crystal ligand was used to generate match-
ing spheres to place the new compound. Multiple conformations
of the compound were pregenerated and placed in the binding
pocket guided by the matching spheres. Ligand poses were scored
by their interactions with the protein, through a grid-based meth-
od calculating van der Waals, electrostatic interactions and ligand
desolvation energy. The top scoring poses against different recep-
tor conformations were visually examined. The pose selected
for each ligand had one of the best docking scores and also
the best shape complementarity with the protein binding pocket
(PDB ID code 2PIX).
1. Tranguch S, et al. (2005) Cochaperone immunophilin FKBP52 is critical to uterine
receptivity for embryo implantation. Proc Natl Acad Sci USA 102:14326–14331.
2. Feau C, Arnold LA, Kosinski A, Guy RK (2009) A high-throughput ligand competition
binding assay for the androgen receptor and other nuclear receptors. J Biomol Screen
14:43–48.
3. Estebanez-Perpina E, et al. (2007) A surface on the androgen receptor that allosteri-
cally regulates coactivator binding. Proc Natl Acad Sci USA 104:16074–16079.
4. Lorber DM, Shoichet BK (1998) Flexible ligand docking using conformational
ensembles. Protein Sci 7:938–950.
5. Meng EC, Gschwend DA, Blaney JM, Kuntz ID (1993) Orientational sampling and
rigid-body minimization in molecular docking. Proteins 17:266–278.
De Leon et al. www.pnas.org/cgi/doi/10.1073/pnas.1105160108 2of6
Fig. S1. Schema for the yeast-based compound library screen. Overnight saturated cultures of the AR-responsive β-galactosidase reporter strains exogenously
expressing human FKBP52 and AR-P723S (Tester) or wild-type AR alone (control) were diluted back to an OD600 of 0.08 and incubated at 30 °C with shaking
until log phase growth was observed (OD600 of approximately 0.1). The cultures were then aliquoted at 100 μl per well followed by the immediate addition
(time 0) of library compounds at 50 μM. Hormone was added at 30 min followed by the addition of β-galactosidase substrate at 2 hr and 30 min after compound
addition. Luminescence from the wells was measured 4 hr after compound addition. This assay was designed to identify any compound that inhibited FKBP52
regulated receptor activity in the tester strain, but that did not inhibit AR function alone in the absence of FKBP52. The AR alone control strain also controlled
for general toxicity. FK506 was used as positive control.
Fig. S2. The AR inhibitors do not affect general transcription, translation, or protein stability. Galactose-inducible β-galactosidase reporter expression in yeast
(gray bars) and constitutive renilla luciferase reporter expression in fkbp52-deficient mouse embryonic fibroblasts (black bars) was assessed in the presence or
absence of the indicated inhibitors at 100 μM.
De Leon et al. www.pnas.org/cgi/doi/10.1073/pnas.1105160108 3of6
Fig. S3. Mutations in the AR BF3 surface result in increased dependence on FKBP52 for function. (A) The AR LBD crystal structure showing the location of the
mutated residues in relation to the BF3 surface is shown. The BF3 surface is highlighted in green, bound DHT is highlighted in blue, and the F673, P723, and
C806 residues are highlighted in purple, orange and yellow, respectively. (B) Yeast reporter strains for wild type AR or the indicated AR point mutants were
transformed with empty plasmid or plasmid expressing human FKBP52. Yeast were exposed to DHT and assessed for β-galactosidase expression. The data are
plotted as fold change of the activity measured in the presence of FKBP52 over the activity in the absence of FKBP52 and are representative of at least five
independent experiments.
De Leon et al. www.pnas.org/cgi/doi/10.1073/pnas.1105160108 4of6
Fig. S4. The inhibitors do not bind the AR hormone binding pocket nor AF2. (A) A scintillation proximity binding assay using purified AR LBD is shown. Nickel-
coated wells preincubated with 6xhistidine-tagged AR LBD were treated for 5 hr with a range of concentrations of the indicated compounds or cold DHT in the
presence of 20 nM tritiated DHT and assessed for bound radioactivity. (Band C) A fluorescence polarization assay using purified AR LBD is shown. Purified AR
LBD preincubated with the fluorescently labeled SRC2-3 peptide was treated for 5 hr with a range of concentrations of the indicated compo unds or nonlabeled
SRC2-3 peptide and assessed for fluorescence polarization and total fluorescence.
De Leon et al. www.pnas.org/cgi/doi/10.1073/pnas.1105160108 5of6
Fig. S5. AR-inhibitor docking simulations support BF3 as the target site. The docking pose of the lead compounds MJC01 (A) and MJC13 (B), and the crystal
pose of flufenamic acid (C) on the BF3 surface of androgen receptor (PDB ID code 2PIX) is shown. The images in the left panel are slightly rotated in the right
panel to bring L830 and Y834, residues also predicted to interact with the molecules, into view. The poses shown were chosen based on the best docking score
(including electrostatic, van der Waals interactions and ligand desolvation energy) and the best shape complementarity with the binding pocket. The carbon
atoms of the protein and the ligand are shown in cyan and yellow (MJC01), green (MJC13), or light green (flufenamic acid), respectively. Nitrogen, oxygen,
hydrogen and fluorine atoms are shown in blue, red, white, and light blue.
De Leon et al. www.pnas.org/cgi/doi/10.1073/pnas.1105160108 6of6
