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Vol. 63, Issue 5, 1012-1020, May 2003
Novartis Institute for Biomedical Research, Summit, New Jersey (C.H., C.F., P.M., G.K., R.E.C., W.C., R.S., R.S., C.S.); and Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey (K.N.)
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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The steroid compound cyproterone acetate was identified in a
high-throughput screen for glucocorticoid receptor (GR) binding compounds. Cyproterone (Schering AG) is clinically used as an antiandrogen for inoperable prostate cancer, virilizing syndromes in
women, and the inhibition of sex drive in men. Despite its progestin
properties, cyproterone shares a similar pharmacological profile with
the antiprogestin mifepristone (RU486; Roussel Uclaf SA). The binding
affinities of cyproterone and RU486 for the GR and progesterone
receptor were similar (Kd, 15-70 nM). Both
compounds were characterized as competitive antagonists of
dexamethasone without intrinsic transactivating properties in rat
hepatocytes (Ki, 10-30 nM). In osteosarcoma
cells, RU486 revealed a higher potency than cyproterone acetate to
prevent responses to dexamethasone-induced GR transactivation and
NF
B transrepression. Upon administration to Sprague-Dawley rats,
both compounds were found to be orally bioavailable and to inhibit
transactivation of liver GR. Molecular docking of cyproterone acetate
and RU486 into the homology model for the GR ligand binding domain
illustrated overlapping steroid scaffolds in the binding pocket.
However, in contrast to RU486, cyproterone lacks a bulky side chain at
position C11
that has been proposed to trigger active antagonism of
nuclear receptors by displacing the C-terminal helix of the
ligand-binding domain, thereby affecting activation function 2. Cyproterone may therefore inhibit transactivation of the GR by a
molecular mechanism recently described as passive antagonism. New
therapeutic profiles may result from compounds designed to selectively
stabilize the inactive and active conformations of certain nuclear receptors.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Glucocorticoids
are steroid hormones that are essential for normal growth and
development, for liver and immune functions, and for mediating the
stress response. Synthetic derivatives of glucocorticoids, such
as dexamethasone, have immunosuppressive, anti-inflammatory,
osteocatalytic, proteolytic, and hyperglycemic activities and are used
to treat various pathological conditions (Sapolsky et al., 2000
). The
GR is a ligand-activated intracellular transcriptional regulator that
is a member of the nuclear receptor superfamily. In the absence of a
ligand, the GR is retained in the cytoplasm by association with
chaperone proteins. Upon ligand binding, the GR dissociates from
chaperones, dimerizes, and translocates into the nucleus. In the
nucleus, the hormone-bound GR can modulate transcription of target
genes by direct interaction with specific DNA sequences, called
glucocorticoid response elements (GRE) in GR responsive promoters
(Karin, 1998). Alternatively, activated GR can interact with nuclear
factor B (NF-B) or with activator protein 1 (AP-1) to repress
gene expression induced by these proinflammatory transcription factors.
The anti-inflammatory and immune-suppressive properties of
glucocorticoids have been largely attributed to the transrepression of
NF-B and AP-1 function, whereas the hyperglycemic effects have been
ascribed to GRE-mediated transactivation of metabolic enzymes. The
recent elucidation of the GR crystal structure has demonstrated the
relevance of the dimer interface for GR-mediated transactivation
(Bledsoe et al., 2002). In contrast, transrepression of NF-B and
AP-1 function has been shown to be dependent on nuclear translocation
yet independent of dimer formation.
Gene transactivation has been explained by a ligand-induced change in
the nuclear receptor structure that allows the recruitment of a
coactivator. The receptor coactivator complex is believed to acetylate
histones and thus to prepare target gene promotors for transactivation
by decondensation of the corresponding chromatin (McKenna et al., 1999;
Bourguet et al., 2000; Glass and Rosenfeld, 2000). The LBDs in nuclear
receptors contain, at their carboxyl-terminal ends, a helix that
mediates a ligand-controlled AF-2. The LBDs derived from several
nuclear receptors have been crystallized and shown to fold into a
three-layer helical sandwich (Bourguet et al., 1995; Renaud et al.,
1995; Wagner et al., 1995; Williams and Sigler, 1998; Sack et al.,
2001). Ligands bind into a hydrophobic pocket contained in the sandwich
and interact with the AF-2 helix. Ligands with transactivation or
agonistic function position the AF-2 helix in an active conformation
that allows the association of coactivators (Brzozowski et al., 1997).
Antagonists of ligand-dependent transactivation often resemble agonists
in their core scaffold with the exception of an additional extended
bulky side chain. This ligand side chain has been shown to reposition
the AF-2 helix within the nuclear receptor ligand binding groove and
thus to switch affinity of the LBD from coactivator to corepressor
recruitment (Chen and Evans, 1995).
The most widely used antiprogestin, RU486, was originally developed as
an antiglucocorticoid and was subsequently shown to effectively repress
GR-mediated transactivation (Philibert and Teutsch, 1990; Teutsch et
al., 1991). The dimethylaminophenyl moiety of RU486 is believed to
confer antagonist activity via interactions in the 11-pocket of the
progesterone receptor (PR), resulting in the inhibition of
transcriptional activity (Gronemeyer et al., 1992; Cadepond et al.,
1997).
In a search for new GR modulating compounds, we used a high-throughput,
GR-competitive ligand-binding screen and identified a compound of
unique steroidal structure. The compound GP052499 represented
cyproterone acetate and was characterized as a potent GR antagonist
despite the lack of any 11-substitution.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Human Steroid Receptor Binding Assays. GR and PR competitive ligand binding assays (PanVera, Madison, WI) were adapted to a 384-well format for high-throughput screening. Both binding assays required, according to the manufacturer's protocol, the preparation of buffer, receptor, and tracer solutions in addition to the serial dilutions of test compound.
For the GR assay, the binding buffer was prepared by diluting 1 ml of the 10× stock solution [100 mM KPO4 (K2HPO4
80.2% and
KH2PO4 19.8%, pH 7.4),
100 mM Na2MoO4, 1 mM EDTA,
and 20% DMSO] with 7.95 ml H2O, 1 ml of a 10×
stabilizing peptide (1 mM), and 50 µl of 1 M DTT. A 2.2 nM
fluorescent glucosteroid (Fluormone-GS1) solution was prepared in 1×
GR-binding buffer. In the binding reaction, 10 µl of the
Fluormone-GS1 solution was diluted to a final volume of 22 µl,
yielding a 1 nM tracer ligand concentration. The GR preparation was
thawed on ice for at least 1 h before the preparation of an 8.8 nM
GR solution in 1× GR-binding buffer (Srinivasan and Thompson,
1990). In the binding reaction, 10 µl of the GR solution was
diluted to a final volume of 22 µl, yielding a 4.0 nM concentration
of functional receptor. The ratio of 1.0 nM Fluormone-GS1 to 4.0 nM GR
resulted in a receptor saturation of approximately 80%.
For the PR assay, 4.4 nM green fluorescent progesterone ligand
(Fluormone-PL) solution was prepared in binding buffer containing 10%
glycerol, 100 mM potassium phosphate, pH 7.4, 100 µg/ml bovine serum
albumin, and 1 mM DTT. An 88 nM glutathione
S-transferase-PR-ligand binding domain fusion protein
solution was prepared in binding buffer. In the binding reaction, 10 µl of the receptor solution was diluted in a final volume of 22 µl,
yielding a 40 nM receptor concentration that was saturated by
approximately 80% with ligand in the presence of 2 nM Fluormone-PL.
Serial dilutions of steroid test compounds were prepared in 11-fold
excess concentrations with H2O. In the binding
reactions, 2 µl of test compound solution was diluted in a final
volume of 22 µl. The steroid binding assay consisted of pipetting 2 µl of ligand dilution followed by 10 µl of Fluormone-GS1 and 10 µl of GR-solution into a 384-well plate (Nalge Nunc International,
Naperville, IL). Negative and positive controls were included to
determine the fluorescence polarization (FP) window. The plate was
incubated in the dark at room temperature for 1 h before FP
analysis at 485 nm excitation and 530 nm emission using an Analyst II
(Beckman Coulter, Fullerton, CA).
Statistical Evaluation of Binding Curves.
The steroid
concentration that resulted in a half-maximum shift in polarization
equaled the IC50 value, a measure of the relative affinity of ligand for the receptor. The curves were plotted with the
Prism software (GraphPAD, San Diego, CA) using the equation: Y = [mP100% + (mP0% mP100%)]/(1 + 10 [(LogIC50
X) × Hill slope]),
where Y = mP; X = Log [ligand concentration];
mP100% = mP at 100% inhibition;
mP0% = mP at 0% inhibition. The conversion of
IC50 values into
Ki values was calculated with the equation of Cheng and Prusoff (Cheng and Prusoff, 1973):
Ki = IC50/(1 + [fluormone-GS1]/0.2 nM), where Ki
represents the receptor affinity for the competing ligand. The
subscript i was used to indicate that the competitor-inhibited tracer
ligand binding was interpreted as an equilibrium dissociation constant
Kd. It is the concentration of the
competing ligand that will bind to half of the binding sites at
equilibrium in the absence of tracer ligand. Competitor affinities
(Ki values) were determined relative
to tracer affinities (Kd values). The
Kd value for the tracer fluormone-GS1 used was provided by the manufacturer: 0.2 nM at 4°C and 2-h
incubation (PanVera, Madison, WI). To determine experimentally the
Kd value for a FP-labeled tracer, a
saturation titration was performed with a constant amount of
fluormone-GS1 (1 nM) and serial dilutions of GR (0.01-100 nM). The
equilibrium binding curve was then generated by plotting 
-FP [mP]
values against free GR [nM] values.
Cellular Tyrosine Aminotransferase Activity Assay.
Rat
hepatoma H4IIE-C3 cells were obtained from the American Type Culture
Collection (Manassas, VA) and seeded at 40,000 cells per well in a
96-well plate. The cells were cultured at 37°C in a 5%
CO2 atmosphere in 100 µl of low-glucose
Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA)
containing 10% fetal calf serum (Invitrogen) and 500 units/ml
penicillin and streptomycin. After 3 days, the cells were fed with an
additional 100 µl of fresh media. On the following day, media was
removed, and monolayers were treated overnight with varying
concentrations of dexamethasone ± 10 µM concentrations of test
compounds (Schild-format) or varying concentrations of test
compounds ± 10 nM dexamethasone (Titration format) in 200 µl of
fresh media. After 24 h, the compound-containing media were
removed, and the cell monolayers were washed once with phosphate-buffered saline. Monolayers were then treated with 50 µl of
freshly prepared solubilization buffer (125 mM
K2HPO4, pH 7.6, 1.0 mM
EDTA, pH 8.0, 1.0 mM DTT, 0.5% Nonidet P-40) and placed on ice for 10 min. After solubilization, to each well was added: 130 µl
L-tyrosine (8.75 mM stock; 63.125 mg in 32 ml of 125 mM K2HPO4 and 150 µl of 10 N
KOH: final preparation performed immediately before assay by adding 0.6 ml of 125 mM KH2PO4 to 1.0 ml of tyrosine stock), 10 µl of pyridoxyl phosphate (1 mM stock; 4.8 mg in 10 ml of 125 mM
K2HPO4) and 10 µl of
-ketoglutarate (200 mM stock; 368 mg in 10 ml of 125 mM
K2HPO4 and 150 µl of 10 N
KOH). Plates were then incubated for 30 min at 37°C. Reactions were
stopped and final measurable components developed by the addition of 3 µl of 10 N KOH with immediate mixing after addition to each well. Plates were incubated for an additional 30 min at 37°C, transferred to a quartz 96-well plate and then read spectrophotometrically at 340 nm UV.
Statistical Evaluation of the TAT Assay.
The saturation
binding experiment measures steroid concentration-dependent induction
of tyrosine aminotransferase (TAT) activity. The Prism software
(GraphPAD, San Diego, CA) provides a logistic equation describing a
sigmoidal dose-response curve for nonlinear regression analysis:
Y = Amin + (Amax Amin)/(1 + X/Xo)P,
where X represents the concentration of ligand and
Y the specific activity data. With these values, an
activation curve is fitted and analyzed for the
EC50 value (Xo).
Amax represents the plateau value of
maximal effect, whereas Amin is the
value of minimal effect. P represents the Hill slope
variable that controls the slope of the curve.
Reporter Gene Assays. MG63 osteosarcoma cells were obtained from the American Type Culture Collection and transiently transfected with a pGRE-Luciferase reporter plasmid (BD Biosciences Clontech, Palo Alto, CA) at 2 µg of plasmid per million cells using LipofectAMINE 2000 reagent according to the manufacturer's instructions (Invitrogen). After an overnight transfection, the cells were replated into a 96-well plate and allowed to adhere for 8 h. The cells were then treated with test compound or vehicle control (0.1% DMSO) for 1 h before the addition of dexamethasone. On the following day, the induction of luciferase was measured using the Bright-Glo luciferase assay system (Promega, Madison, WI).
Cellular IL-6 Secretion Assay.
MG63 osteosarcoma cells
obtained from American Type Culture Collection were cultured at 37°C
in RPMI 1640 medium and 10% fetal calf serum (Invitrogen) until
reaching confluence. At confluence, the media was changed to RPMI 1640 medium and 2% fetal calf serum (charcoal-stripped). After 3 days, the
cells were treated with test agents or their vehicle (0.1% DMSO) for
1 h before adding interleukin (IL)-1 at 30 ng/ml (BioSource,
Camarillo, CA). After an overnight incubation, the extracellular media
was assayed quantitatively for IL-6 levels using an enzyme immunoassay
kit (Cayman Chemical, Ann Arbor, MI). The IL-1 stimulated cells
produced 400 pg of IL-6 per 5000 cells, whereas unstimulated cells
yielded <10 pg of IL-6 per 5000 cells. The results were normalized to
percentage of inhibition in response to dexamethasone.
In Vivo Rat Treatment and ex Vivo Liver TAT Activity Assay.
Male Sprague-Dawley rats weighing 180 to 220 g were delivered a
week before the study from the Harlan Farms (Harlan Sprague-Dawley, Indianapolis, IN) and housed under standard laboratory conditions outlined by the Animal Care and Use Committee. The rats were fasted for
18 h before the start of the experimental procedure. The rats were
divided into groups of four and dosed orally by gavage with either
vehicle (0.25% carboxymethylcellulose in water containing 20%
ethanol) or vehicle-solubilized test compound (40 or 80 mg/kg). Ten
minutes after the oral gavage procedure, the rats were administered an
i.p. injection of either vehicle (20% ethanol solution) or various
doses of vehicle-solubilized dexamethasone (0.003, 0.01, 0.03, and 0.15 mg/kg). After the injections, the rats were sacrificed at various time
points (1, 3, or 5 h) with isoflurane. The livers were removed and
the periportal regions were immediately frozen in liquid nitrogen and
stored at 70°C. Working on dry ice, a portion of the frozen liver
was placed in 4 ml of cell solubilization buffer (0.125 M
K2HPO4, pH 7.6, 1 mM EDTA,
pH 8.0, 1 mM DTT, 0.5% Nonidet P40) and subjected to mechanical
homogenization. The resultant homogenate was transferred to a 4.7-ml
OptiSeal tube (Beckman Coulter, Fullerton, CA), and cytosolic fractions
were obtained by ultracentrifugation at 40,000 rpm for 1 h.
Supernatant, below the fat plug, was retrieved by needle and syringe
and placed in a clean, sterile tube on ice. All samples were
standardized for protein content at 1.0 µg/µl (Bio-Rad, Hercules,
CA). Subsequently, 50 µl of the standardized cytosolic fraction was
assayed for TAT activity as described previously. After addition of 130 µl of L-tyrosine solution, 10 µl of pyridoxyl phosphate
solution, and 10 µl of -ketoglutarate solution, the samples were
incubated for 30 min at 37°C. The reactions were stopped and the
final measurable components developed by the addition of 3 µl of 10 N
KOH solution with immediate shaking after addition to each well. Plates
were incubated for an additional 30 min at 37°C, transferred to a
quartz 96-well plate and then read spectrophotometrically at 340 nm UV. Data points (n = 4) are presented as mean ± S.D.
Comparisons were made using the Student's unpaired t test.
GR Homology Modeling and Ligand Docking.
A pair-wise
sequence alignment of the human GR-LBD (Swiss-Prot accession number
P04150) and the human PR-LBD (Swiss-Prot accession number P06401) was
carried out using the Smith-Waterman algorithm with a BLOSUM62 scoring
matrix and default gap penalties (gap open, 10; gap extend, 0.5). The
homology model for the GR-LBD was built based on the X-ray crystal
structure for PR-LBD (Protein Data Base code 1A28) using the homology
modeling module in the MOE program (Chemical Computing Group Inc.,
Montreal, ON, Canada). The LMOD (Low MODe conformation search) function
(Kolossvary and Guida, 1996, 1999) in the Macromodel suite version 7.0 (Schrodinger Inc., Portland, OR) was used to dock ligands and refine
structures of GR-LBD homology model.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Steroid Equilibrium Binding to the Human GR and PR.
An in
vitro GR competitive binding assay was established with a baculovirus
derived crude human receptor preparation and a fluorescent
glucocorticoid in a 384-well, high-throughput format (Srinivasan and
Thompson, 1990). Dose-dependent displacement of the fluormone-labeled
glucosteroid with competing compounds was recorded by fluorescence
polarization. In this assay, the reference compounds dexamethasone and
RU486 bound with calculated Kd values of 3 nM (IC50, 10 nM; r2 = 0.99) and 15 nM (IC50, 100 nM;
r2 = 0.98), respectively (Figs.
1 and 2A). The in
vitro measured dexamethasone binding data were consistent with binding
data generated in intact THP-1 monocytic cells using radiolabeled
dexamethasone as a tracer compound (data not shown). The
high-throughput screen of our proprietary compound library led to the
identification of GP052499, a compound that represents the steroid
cyproterone acetate (Neumann, 1994) (Fig. 1). The GR binding data for
cyproterone acetate were fitted to a one binding site equation with a
correlation coefficient of 0.92 that yielded a
Kd value of 45 nM
(IC50, 360 nM) (Fig. 2B).
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Whole-Cell Functional Assays to Distinguish GR Agonistic from GR
Antagonistic Ligand Properties.
To characterize further the
steroids and their functional potentials, a cellular assay for
GR-mediated transcriptional activation was required. The TAT
gene is liver-specific and glucocorticoid-inducible (Diamondstone, 1966). The GR has been shown to bind to the TAT GRE in
vitro and, upon hormone binding, to activate transcription of the TAT
gene in vivo. Diamondstone described an assay for TAT activity that is
based on the conversion of p-hydoxyphenylpyruvic acid to
p-hydroxybenzaldehyde in strong alkali (Diamondstone, 1966).
We adapted the Diamondstone assay concept to a microplate format and
showed a dose-dependent induction of TAT activity by dexamethasone in
the rat hepatoma cell line H4IIE-C3. To optimally characterize the
pharmacological profile of GR binding compounds, the TAT assay was
first executed in the Schild format and then in the titration format.
0.001). Similar to RU486, cyproterone acetate
revealed no ability to intrinsically transactivate the GR (Fig. 3B).
However, unlike RU486, a 10-fold excess of cyproterone acetate over
dexamethasone was insufficient to suppress TAT expression; 100-fold
excess yielded a complete suppression (p 0.001).
Therefore, the antagonistic potency of cyproterone acetate is,
comparison with that of RU486, approximately 1 order of magnitude
lower.
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induced production of IL-6; 0.3 and 30 nM
dexamethasone resulted in 50 and >90% inhibition of cellular IL-6
release, respectively (Fig. 4C). Neither RU486 nor cyproterone acetate
at concentrations up to 10 µM inhibited IL-6 production (data not
shown). However, both compounds dose dependently affected the
inhibitory activity of dexamethasone by shifting the dose-response
curve of dexamethasone to the right (Fig. 4, C and D). Unlike the
antagonism of the transactivational response to dexamethasone, the
highest concentrations of RU486 (100 nM) and cyproterone acetate (10 µM) were only able to partially reverse the transrepressional
activity induced by 100 nM dexamethasone. Similar results were observed
using human dermal and synovial fibroblasts (data not shown).
In Vivo Target Pharmacology.
The ability to measure TAT
activity from partially purified enzyme preparations was subsequently
used for the development of a tissue selective in vivo biomarker for GR
modulation. The procedure to measure TAT activity in cells was adapted
to determine activity in isolated rat liver tissue (Hayashi et al.,
1967; Granner and Tomkins, 1970). The assessment of TAT activity in the
rat livers upon oral administration of a test compound is reflective of
the compound's oral bioavailability and pharmacodynamic activity profile.
0.05). The time-response study was performed by
i.p. administration of dexamethasone at 0.03 mg/kg or vehicle control
and the isolation of liver tissue at various time points (Fig. 5B). One
hour after dexamethasone administration, no significant induction of
hepatic TAT activity was observed. However, 3 and 5 h after
treatment, a significant dexamethasone-induced increase of TAT activity
was recorded. The TAT activity increased 2.1-fold at 3 h
(p 0.01) and 2.5-fold at 5 h (p 0.01) after dexamethasone administration.
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0.05) that was completely prevented by the oral
administration of RU486 (p 0.01). Cyproterone acetate proved to be active in vivo albeit with weaker potency than
RU486. The dexamethasone treatment resulted in a 3.2-fold increase of
TAT activity (p 0.001) that was significantly
suppressed by Cyproterone acetate (p 0.05), albeit to a lesser extent than by RU486. The injections and
gavage treatment resulted in minimal stress with insignificant impact
on TAT activity. Therefore, the assessment of hepatic TAT activity
served as a pharmacological marker for the specific modulation of the
GR in vivo upon oral administration of test compounds.
Pharmacophore Modeling using a GR Homology Model.
A homology
model was created for the LBD of GR based on the X-ray crystal
structure of the progesterone bound LBD of PR (Williams and Sigler,
1998). The GR and PR LBD sequences share 53% identity and 74%
homology. In particular, the pocket regions for hormone binding show
high sequence conservation between PR and GR. Within a shell of 5 Å around the progesterone-bound PR pocket, only five residues are altered
in GR: L715M, V760A, F794Q, L797Q, and M909L (numbers indicate amino
acid residues in PR). In the reported nuclear receptor structures,
steroid hormones with a core template consisting of the A, B, C, and D
rings dock in a common orientation with the A rings oriented toward a
conserved arginine residue from helix 5 and the D rings toward the AF-2
helix. The polar substituents in steroid hormones are mainly located at
position C3 on the A ring and C17 on the D ring. The natural ligands,
progesterone for the PR and cortisol for the GR, differ by the
occurrence of three additional hydroxyl groups in cortisol (Fig. 1).
The L797Q mutation between PR and GR may explain the preferential
binding of cortisol by GR that features at position C17 a hydroxyl moiety.
-amide of Q570. The carbonyl group on
C20 of cyproterone acetate bonded with residue T739 in the GR
binding pocket. These interactions are also recognized by dexamethasone
as shown in the crystal structure of the GR-dexamethasone complex by
Bledsoe et al. (2002). The acetyl group at C17 position on the D
ring of cyproterone, however, did not reveal any recognizable hydrophilic interactions with a nearby residue, such as Q642, despite
the allowance of full conformational docking flexibility.
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-amide of Q570 (Fig. 6B). The dimethylaminophenyl side chain at
position C11 on the C ring of RU486 projected toward the protein
surface to displace the AF-2 helix from its active conformation. This
molecular mode of receptor antagonism has been previously shown for
4-hydroxytamoxifen complexed to the ER (Protein Data Base code 3ERT)
(Shiau et al., 1998). Overlaying cyproterone acetate with RU486 in the
GR binding pocket clearly demonstrated the absence of a protruding
bulky side chain in cyproterone acetate that may cause a reposition of
the AF-2 helix (Fig. 6B). Therefore, based on the molecular docking of
these steroids to the GR homology model, the antagonistic properties of
cyproterone acetate may occur by a molecular mechanism that is
different from the mechanism ascribed to RU486.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In this report, we present a new pharmacological profile of
cyproterone acetate. Cyproterone acetate competitively abolished GR
transactivation by dexamethasone in vitro and in vivo upon oral
administration to rats. The GR activity profile of cyproterone acetate
in our assays was comparable with that of RU486; however, the potency
of cyproterone acetate was inferior to that of RU486. Interestingly,
the apparent potency of both compounds to antagonize dexamethasone-induced GR transactivation was better than their ability
to prevent dexamethasone-mediated transrepression of cellular cytokine
release. This observation may indicate differential kinetics or
mechanisms for GR ligands to control GRE- and NFB-dependent transactivation. Alternatively, this observation may reflect the higher
intrinsic potency of dexamethasone for transrepression than for transactivation.
Cyproterone acetate has orphan drug status in the United States and is
used as antiandrogen in other countries. The steroid has been shown to
compete with dihydrotestosterone for binding to the androgen receptor
and to exhibit progestogenic activities. Indeed, progesterone itself is
a weak antiandrogen. Cyproterone also suppresses the secretion of
gonadotropins and thus interferes with testosterone production
(Barradell and Faulds, 1994; Neumann, 1994). The agent is used for the
treatment of paraphilias and inoperable prostate cancer in men. In
women, cyproterone is used for the treatment of acne or virilizing
syndromes such as hirsutism and combined with estrogens as hormone
replacement therapy and contraception (van Wayjen and van den Ende,
1995; Morin-Papunen et al., 2000; Borissova et al., 2002). A rare
association with severe liver damage and hepatocellular carcinoma,
however, limits the usefulness of cyproterone acetate.
The drug label also indicates a potential change in glucose tolerance and thus a possible interaction with antidiabetic drugs. The metabolic homeostasis is determined by the secretion of glucoregulatory hormones, including insulin, glucagon, epinephrine, growth hormone, and cortisol. Cortisol may increase plasma glucose concentrations by stimulating appetite and hepatic gluconeogenesis. Therefore, inhibition of the GR with cyproterone acetate may affect the peripheral glucose metabolism, the stress response, and the regulation of the hypothalamic pituitary axis.
The recent elucidation of the long-awaited crystal structure of the GR
along with the proposed molecular mechanisms for activation and
repression of nuclear receptors generated a new basis for the
structural interpretation of the biological activities mediated by GR
ligands such as cyproterone acetate. Crystal structures of several
nuclear receptors demonstrated that the selectivity of steroid hormone
binding is achieved by the complementarity of shape and hydrogen
bonding between ligands and the receptor ligand binding pockets. Thus,
the high affinity of dexamethasone for the GR was readily explained by
the extensive network of hydrophobic and hydrophilic interactions
between the ligand and the protein (Bledsoe et al., 2002). Ligands with
the ability to transactivate their receptor have been shown to shift
the conformational equilibrium to an active conformation such that the
AF-2-mediating helix takes a position that increases the affinity of
the LBD for coactivators. Indeed, dexamethasone makes direct contacts
between the C21 hydroxyl group and L753 in the AF-2 helix and between
the C11 hydroxyl group and residue I747 in the loop preceding the
AF-2 helix. These interactions are likely to stabilize the AF-2 helix
in an active conformation and may serve as a molecular basis for
ligand-dependent activation of GR. These hydroxyl groups are conserved
in cortisol yet are lacking in cyproterone acetate that may explain its
inability to transactivate the GR.
The molecular mechanism for "active antagonism" of nuclear
receptors has been explained by the projection of bulky side chains that block the AF-2 activity. RU486 contains such a side chain at
position C11 on the C ring that protrudes out of the binding pocket,
thereby preventing the AF-2 helix from adopting an active conformation
and shifting the LBD affinity from coactivator to corepressor
recruitment. Because cyproterone acetate lacks a bulky side chain
similar to RU486, the observed functional effects of cyproterone
acetate may occur by a different mode of receptor antagonism. Recently,
a similar deviation from the classic nuclear receptor antagonist model
has been made with the ER antagonist compound THC (Shiau et al.,
2002). THC lacks a bulky side chain, and the crystal structure of the
THC-ER complex did not reveal any steric hindrance of the AF-2
helix. In this model, THC antagonized ER by stabilizing
nonproductive conformations of key residues in the ligand-binding
pocket. The authors referred to this molecular mode as "passive
antagonism". Theoretically, cyproterone acetate may induce a similar
mode of passive antagonism by shifting the stability equilibrium in
favor of an inactive receptor conformation. Indeed, we observed a
differential shift in the GR binding affinity for cyproterone and RU486
in presence or absence of a coactivator peptide (supplementary data).
The binding affinity for cyproterone increased in presence of
coactivator peptide in contrast to RU486, the binding affinity of which
decreased. The addition of coactivator peptide had no effect on the
binding affinity of dexamethasone or cortisol. The observed antagonism
of both dexamethasone-induced TAT activation and blockage of IL-6
release by cyproterone may therefore occur by competitive displacement
of dexamethasone from the GR binding pocket to reach a new stability
equilibrium. Competitive displacement by locking a nuclear receptor in
an inactive conformation would also explain the mineralocorticoid and
androgen receptor antagonism by progesterone and drospirenone (Souque
et al., 1995; Singh et al., 2000). By designing compounds that
selectively stabilize inactive conformations in certain nuclear
receptors and active conformations in others, new therapeutic benefits
can be achieved.
The introduction of I628A mutation in the LBD of GR has been shown to
impair receptor dimerization and the ability to mediate transactivation
while retaining the ability to transrepress NF-B activated functions
(Bledsoe et al., 2002). The dimerization defective mutant
GRdim mouse exhibited a similar phenotype
characterized by affected transactivation and unaffected
transrepression abilities (Reichardt et al., 1998; Tronche et al.,
1998). The design of selective GR modulating compounds that inhibit
transactivation while retaining transrepression may therefore occur on
the basis of chemical scaffolds that mediate passive receptor
antagonism and interference in the receptor dimerization properties.
| |
Acknowledgments |
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We thank Carrol Berry, Xin Wang, and Beverly Battle for excellent technical assistance. We are also grateful to Shari Caplan and James Stanton for critically reading the manuscript.
| |
Footnotes |
|---|
Received October 2, 2002; Accepted February 5, 2003
This work was supported by the University of Medicine and Dentistry of New Jersey/Novartis Predoctoral Fellowship Program.
Address correspondence to: Christoph Schumacher, Ph.D., Speedel Holding AG, Hirschgasslein 11, 4051 Basel, Switzerland. E-mail: christoph.schumacher{at}speedelgroup.com
| |
Abbreviations |
|---|
GR, glucocorticoid receptor;
GRE, glucocorticoid response element;
NF-B, nuclear factor B;
AP-1, activator protein 1;
LBD, ligand-binding domain;
AF-2, activation
function 2;
RU486, mifepristone;
PR, progesterone receptor;
DMSO, dimethyl sulfoxide;
DTT, dithiothreitol;
FP, fluorescence polarization;
Fluormone-GS1, fluorescent glucosteroid;
PL, progesterone ligand;
IL, interleukin;
TAT, tyrosine aminotransferase;
THC, (R,R)-5,11-cis-diethyl-5,6,11,12-tetrahydrochrysene-2,8-diol.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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), 15 nM for RU486 (r2 = 0.97)
(
), and 45 nM (r2 = 0.92) for cyproterone acetate
(
). C and D, dose-dependent displacement of fluormone-PL green from
the PR by steroid ligands. The binding equilibrium of fluormone-PL to
the PR preparation was assessed by FP after 2 h. The binding data
were fitted to a one binding site equation that yielded a
Kd of 16 nM (r2 = 0.97) for
progesterone (
), 40 nM (r2 = 0.96) for RU486 (
