![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 63, Issue 1, 211-223, January 2003
Division of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, The Ohio State University, Columbus, Ohio (D.Y., M.A.P., J.T.D.); and Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee, Memphis, Tennessee (Y.H., S.S.H., C.M., N.S., L.K., D.D.M.)
 |
Abstract |
|---|
 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
|
|---|
The purposes of the present studies were to examine the androgen receptor (AR) binding ability and in vitro functional activity of multiple series of nonsteroidal compounds derived from known antiandrogen pharmacophores and to investigate the structure-activity relationships (SARs) of these nonsteroidal compounds. The AR binding properties of sixty-five nonsteroidal compounds were assessed by a radioligand competitive binding assay with the use of cytosolic AR prepared from rat prostates. The AR agonist and antagonist activities of high-affinity ligands were determined by the ability of the ligand to regulate AR-mediated transcriptional activation in cultured CV-1 cells, using a cotransfection assay. Nonsteroidal compounds with diverse structural features demonstrated a wide range of binding affinity for the AR. Ten compounds, mainly from the bicalutamide-related series, showed a binding affinity superior to the structural pharmacophore from which they were derived. Several SARs regarding nonsteroidal AR binding were revealed from the binding data, including stereoisomeric conformation, steric effect, and electronic effect. The functional activity of high-affinity ligands ranged from antagonist to full agonist for the AR. Several structural features were found to be determinative of agonist and antagonist activities. The nonsteroidal AR agonists identified from the present studies provided a pool of candidates for further development of selective androgen receptor modulators (SARMs) for androgen therapy. Also, these studies uncovered or confirmed numerous important SARs governing AR binding and functional properties by nonsteroidal molecules, which would be valuable in the future structural optimization of SARMs.
| |
Introduction |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
The
role of androgens in the development and maintenance of male sexual
phenotype is mediated by the androgen receptor (AR), a member of the
steroid/thyroid hormone receptor superfamily (Tsai and O'Malley, 1994
;
Zhou et al., 1994). Upon androgen binding, AR undergoes a
conformational change, binds to specific DNA sequences called androgen
response elements, and modulates the transcription of target genes
(Zhou et al., 1994). For decades, AR has been a target of drug
development, aiming at the therapy of diseases caused by altered
androgen levels/responsiveness or the improvement of physical
performance and regulation of male fertility.
Chemicals that modulate the transcriptional activity of AR can be
divided into two structural (steroidal and nonsteroidal) and two
functional (androgenic and antiandrogenic) classes. Steroidal androgens, mainly testosterone and its derivatives, have been used
clinically as replacement therapy for androgen-deficiency (Wu, 1992;
Bagatell and Bremner, 1996). Antiandrogens are used to counteract the
undesirable actions of excessive androgens (e.g., to treat acne,
hirsutism, male-pattern baldness, and androgen-dependent prostatic
hyperplasia and carcinoma) (Neumann, 1982; McLeod, 1993). Nonsteroidal
antiandrogens, such as flutamide (Eulexin), nilutamide (Anandron), and
bicalutamide (Casodex), are often referred to as "pure
antiandrogens" because they bind exclusively to the AR and,
therefore, are devoid of antigonadotropic, antiestrogenic, and
progestational effects. These agents are advantageous over steroidal
antiandrogens (e.g., megestrol acetate, cyproterone acetate) in terms
of specificity, selectivity and pharmacokinetic properties (Neri et
al., 1979; Cockshott et al., 1990; Teutsch et al., 1994).
In recent years, there has been growing interest in the development of
nonsteroidal modulators for steroid hormone receptors as therapeutic
agents. In addition to the above-discussed nonsteroidal antiandrogens,
selective estrogen receptor modulators (SERMs) and nonsteroidal
modulators for progesterone receptor have been successfully developed
(Hamann et al., 1998; Mitlak and Cohen, 1999; Weryha et al., 1999; Zhi
et al., 1998, 2000). These nonsteroidal ligands are known for their
better receptor selectivity than steroidal ligands, and are more
flexible in structural modification for optimal physicochemical,
pharmacokinetic, and pharmacologic properties. More importantly, with
these nonsteroidal ligands, it is possible to achieve tissue-selective
actions and thus to generate agents with diverse activity profiles
meeting specific therapeutic needs. SERMs, for example, are well known
to demonstrate tissue-selectivity (Mitlak and Cohen, 1999; Weryha et
al., 1999). SERMs, such as tamoxifen and raloxifene, are nonsteroidal
estrogen receptor (ER) ligands that manifest distinctive agonist and
antagonist actions in various target tissues. Both tamoxifen and
raloxifene are ER antagonists in breast but agonists in bone. However,
unlike tamoxifen, raloxifene has no ER agonist activity in the uterus.
Whereas nonsteroidal antiandrogens have been used clinically for many
years, nonsteroidal androgens were only recently conceptualized. In the
search for AR affinity ligands, our laboratories discovered a group of
nonsteroidal androgens that are electrophilic derivatives of
bicalutamide and hydroxyflutamide (Dalton et al., 1998). Also, several
analogs of quinoline-based AR antagonists were reported by other
research groups to have AR agonist activity (Edwards et al., 1998,
1999; Hamann et al., 1999; Higuchi et al., 1999; Zhi et al., 1999).
These studies marked the emergence of a novel category of
pharmacological agents with potential applications in androgen therapy.
The discovery of nonsteroidal androgens not only provides an
opportunity to identify agents with superior therapeutic index and
pharmacokinetic profiles to steroidal androgens but also implicates the
possibility to obtain tissue-selective AR modulators (SARMs), the
counterpart of SERMs.
Although nonsteroidal androgens bearing different pharmacophores have been reported, the general structural elements of nonsteroidal ligands that lead to optimal agonist activity remain poorly defined. Systematic studies to explore the structure-activity relationships (SARs) of nonsteroidal ligands for AR binding and agonist activity are of crucial importance for the optimization of chemical structures for maximal functional activity and for the ultimate development of SARMs. Toward a better understanding of the SARs, our laboratories designed and synthesized several series of nonsteroidal compounds that incorporated a variety of structural features known or unknown to influence the ligand-AR interaction and evaluated their biological properties. We present herein the results of our AR binding and in vitro functional activity studies with these synthetic molecules and the SARs revealed by these compounds.
| |
Materials and Methods |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Organic Synthesis
Compounds were prepared in our laboratories and the purities
were greater than 99% (He et al., 2002). The structures of synthesized compounds were confirmed using elemental analyses and spectroscopic data (1H and 13C NMR, mass
spectroscopy, and infrared spectroscopy).
Chemicals and Animals
[17
-methyl-3H]Mibolerone
([3H]MIB, 84 Ci/mmol) and unlabeled MIB were
purchased from PerkinElmer Life Sciences (Boston, MA). Triamicinolone acetonide, phenylmethylsulfonyl fluoride (PMSF), Tris
base, sodium molybdate, dihydrotestosterone (DHT),
o-nitrophenyl-
-D-galactopyranoside, and ATP were purchased from Sigma Chemical Co. (St. Louis, MO). Hydroxyapatite (HAP) was purchased from Bio-Rad Laboratories (Hercules, CA). EcoLite (+) scintillation cocktail was purchased from ICN Research
Products Division (Costa Mesa, CA). Ethyl alcohol (USP grade) was
purchased from AAPER Alcohol and Chemical Company (Shelbyville, KY).
Minimal essential medium (MEM), Dulbecco's modified Eagle's medium
(DMEM), penicillin-streptomycin, trypsin-EDTA, and LipofectAMINE reagent were purchased from Invitrogen (Carlsbad, CA). Fetal
bovine serum (FBS) was obtained from Atlanta Biologicals, Inc.
(Norcross, GA). Adult male Sprague-Dawley rats, weighing approximately
250 g, were purchased from Harlan Biosciences (Indianapolis, IN).
Buffers
Homogenization buffer contained 10 mM Tris, 1.5 mM disodium EDTA, 0.25 M sucrose, 10 mM sodium molybdate, and 1 mM PMSF and was adjusted to pH 7.4. PMSF was prepared as a stock solution of 200 mM in ethanol and added to other components immediately before use. HAP wash buffer contained 50 mM Tris and 1 mM KH2PO4 and was adjusted to pH 7.4.
-Galactosidase assay buffer was 200 mM sodium phosphate buffer, pH
7.3, containing 2 mM MgCl2, 100 mM
-mercaptoethanol, and 1.33 mg/ml
o-nitrophenyl--D-galactopyranoside.
Luciferase assay buffer consisted of 25 mM glycylglycine, 15 mM
MgCl2, 5 mM ATP, and 0.5 mg/ml bovine serum
albumin, and was adjusted to pH 7.8 with 1 M sodium hydroxide.
Preparation of Rat Prostate Cytosolic AR
Cytosolic AR was prepared from ventral prostates of castrated
male Sprague-Dawley rats as described previously (Mukherjee et al.,
1999). Briefly, rats were surgically castrated via a scrotal incision
before the removal of prostates. One day after castration, rats were
anesthetized with a mixture of ketamine/xylazine (87:13; v/v) at 1 ml/kg body weight. Ventral prostates were excised, weighed, and
immersed immediately in ice-cold homogenization buffer. The prostates
were minced with scissors and homogenized (Model Pro 200 homogenizer;
Pro Scientific, Monroe, CT) in homogenization buffer (1:2, w/v). The
homogenate was then centrifuged at 114,000g, 0°C for
1 h in an ultracentrifuge (Model L8-M; Beckman Instruments Inc.,
Palo Alto, CA). The supernatant (cytosol), containing AR proteins, was
collected and stored at 
80°C until use.
AR Competitive Binding Assay
The AR binding affinity of synthesized nonsteroidal compounds
was determined using a radioligand competitive binding assay. An
aliquot of AR cytosol preparation (50 µl) was incubated with a
saturating concentration (1 nM) of [3H]MIB and
1 µM of triamcinolone acetonide at 4°C for 18 h in the absence
or presence of increasing concentrations of the compound of interest
(10 different concentrations ranging from 10
1
nM to 104 nM). MIB is a synthetic high-affinity
ligand for the AR. Triamcinolone acetonide (1 µM) was included in the
incubate to block the interaction of [3H]MIB
with glucocorticoid and progesterone receptors. Nonspecific binding of
[3H]MIB was determined separately by adding an
excess of unlabeled MIB (1000 nM) to the incubate. After incubation,
the protein-bound radioactivity was separated from free radioactivity
by HAP precipitation. HAP was prepared as a slurry in 50 mM Tris, pH
7.2 (1:15, w/v) after being washed twice with HAP wash buffer (1:15,
w/v). An aliquot (500 µl) of HAP slurry was added to the incubate and
gently agitated for 15 min at 4°C. The mixture was centrifuged at
2000g, 4°C for 1 min, and the HAP pellet was obtained and
washed three times with 1 ml of 50 mM Tris, pH 7.2. The bound
radioactivity was then extracted from HAP by incubating the HAP pellet
with 1 ml of ethanol at room temperature for 1 h. After
centrifugation at 2500g for 1 min, 0.8 ml of the ethanolic supernatant
was added to 5 ml of scintillation cocktail. The radioactivity was
counted in a Beckman LS6800 liquid scintillation counter (Beckman
Coulter, Fullerton, CA).
Cell Culture
Monkey kidney fibroblast-like CV-1 cells were obtained from American Type Culture Collection (Manassas, VA). The cells were grown at 37°C in a humidified atmosphere with 5% carbon dioxide, and maintained in minimal essential medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin.
Cotransfection and Enzyme Assays
The in vitro functional activities of nonsteroidal ligands, as
assessed by the ability of each ligand to induce or repress AR-mediated
transcriptional activation of the luciferase reporter gene, were
examined in transiently transfected CV-1 cells. One day before
transfection, CV-1 cells were seeded into DMEM supplemented with 10%
fetal bovine serum at a density of 2 × 105
cells/well in 12-well tissue culture plates. For the following steps,
DMEM without phenol red was used. Transient transfection of plated
cells was carried out in serum-free medium using LipofectAMINE according to the manufacturer's instructions. Cells in each well were
transfected with 50 ng of a human AR expression construct (pCMVhAR;
generously provided by Dr. Donald J. Tindall, Mayo Clinic and Mayo
Foundation, Rochester, MN), 1 µg of an androgen-dependent luciferase
reporter construct (pMMTV-Luc; generously provided by Dr. Ronald Evans
at The Salk Institute, San Diego, CA), and 1 µg of a
-galactosidase expression construct (pSV--galactosidase; Promega
Corporation, Madison, WI) for constitutive expression of
-galactosidase using 6 µl of LipofectAMINE. After 10 h of transfection, cells were washed once with DMEM, and then recovered in
fresh DMEM supplemented with 0.2% FBS for 10 to 12 h before the
start of treatments.
To determine the AR agonist activity, the transfected cells were
treated with increasing concentrations of the ligand of interest. To
determine the AR antagonist activity, the cells were treated simultaneously with increasing concentrations of the ligand of interest
and 1 nM DHT. Controls, where cells were treated with 1 nM DHT alone or
vehicle alone, were included in each experiment. To measure any
AR-independent effect, a parallel experiment was also performed in
which cells cotransfected with pMMTV-Luc and pSV--galactosidase only
were treated with 500 nM of the ligand of interest. All drugs were
initially dissolved in 100% ethanol, and then serially diluted to the
desired concentration using DMEM containing 0.2% FBS. The volume of
ethanol in the final solutions was 
0.05%. The final concentrations
of the compound of interest were 1, 10, 100, and 500 nM. Drug
treatments continued for 48 h, during which all drug-containing
solutions were replaced at a 24-h interval to minimize the effect of
possible chemical degradation.
After treatment, the cells were washed twice with phosphate-buffered
saline and lysed with 200 µl/well of 1× Reporter Lysis Buffer
(Promega Corporation) at room temperature for 30 min. Cell lysates were
placed in 1.5 ml of polypropylene centrifuge tubes and centrifuged at
12,000g and 4°C for 2 min. For -galactosidase assays,
an aliquot (50 µl) of supernatant from each cell extract was
transferred to a 96-well plate and incubated with 50 µl of -galactosidase assay buffer at 37°C for 3 h. An aliquot (150 µl) of 1 M Na2CO3 was
then added to each incubate to stop the enzyme reaction, and absorbance
at 414 nm was measured using a 96-well plate reader (Titertek Multiskan
MCC/340; Labsystems Inc., Franklin, MA). For luciferase assays, an
aliquot (100 µl) of supernatant from each cell extract was added with
an equal volume of luciferase assay buffer. Luminescence was then
measured in an automated luminometer (AutoLumat LB953; PerkinElmer
Wallac Inc., Gaithersburg, MD) immediately after injecting 100 µl of
1 mM beetle luciferin to each sample.
Data Analyses
AR Binding.
The specific binding of
[3H]MIB at each concentration of the compound
of interest (B) was obtained after subtracting the nonspecific binding
of [3H]MIB, and expressed as the percentage of
the specific binding in the absence of the compound of interest
(B0). The concentration of compound that reduced
the specific binding of [3H]MIB
(B0) by 50% (IC50) was
determined by computer-fitting the data to the following equation using
WinNonlin (Pharsight Corporation, Mountain View, CA): B = B0 × [1 C/(IC50 + C)], where
C was the concentration of the compound of interest.
), and L was
the concentration of [3H]MIB used in the
experiment (1 nM). Statistical analyses were performed using single
factor analysis of variance, with p values less than 0.05 being considered as statistically significant differences.
Transcriptional Activation.
Transcriptional activation in
each well was calculated as the ratio of luciferase activity to
-galactosidase activity to normalize the variance in cell number and
transfection efficiency. Transcriptional activation induced by each
ligand of interest, in the absence or presence of 1 nM DHT, was
expressed as a percentage of that induced by 1 nM DHT. The efficacy of
agonist activity for a ligand of interest was the maximal percentage of
transcriptional activation induced by that ligand in the absence of
DHT. All experiments were performed in triplicate or greater, and data
were expressed as the mean ± S.D. in representative experiments.
| |
Results |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Androgen Receptor Binding of Nonsteroidal Compounds.
We
designed and synthesized several series of nonsteroidal compounds,
based on the SARs for nonsteroidal AR binding obtained from the
literature and previous studies in our laboratories (Glen et al., 1986;
Tucker et al., 1988; Morris et al., 1991; Mukherjee et al., 1996, 1999;
Dalton et al., 1998; Kirkovsky et al., 2000). Bicalutamide,
hydroxyflutamide, or nilutamide pharmacophores (Fig. 1) were investigated. The AR binding
affinities of these synthetic molecules, reported as
Ki values, were determined by a
radioligand competitive binding assay using rat prostates as an AR
source. A representative binding displacement curve (with
R-5) is shown in Fig. 2. The
lower the Ki value, the greater the
receptor binding affinity.
|
|
).
Thus, all compounds in this series were prepared as pure optical
isomers (R or S). Consistent with our previous
finding, all R-isomers showed at least 4-fold higher AR
binding affinity (lower Ki values)
than their corresponding S-isomers. Because the
S-isomers are essentially of no use for our purposes, the
following discussion of SARs for binding is focused entirely on
R-isomers. In this series, R-5, R-12,
and R-13 exhibited higher AR binding affinity than the lead
compound (R)-bicalutamide (R-1; p < 0.05). AR binding affinity in this series of compounds was
influenced by the X-linkage group and B-ring substituents. When the
para-substitution in the aromatic B-ring was an amino group,
the sulfone (X = SO2, R-4) showed
greater affinity than the sulfide (X = S, R-2), whereas
the sulfoxide (X = SO, R-3) had no binding affinity.
However, when the nitrogen molecule in the para-amino group
was further substituted, sulfides demonstrated higher binding than
sulfones (compare R-5 with R-7, and
R-12 with R-13). In compounds with the same
X-linkage, the B-ring substituent clearly played an important role in
AR binding. The overall effect seemed to be determined by a delicate
balance of factors, including nature, size, and position of the
substituent. Introduction of a chloroacetamido group at the
para- position (R-12 and R-13)
resulted in the highest binding affinity in each of the sulfide and
sulfone sets of compounds (compare R-12 with R-5,
R-8, R-10, and R-9; R-13
with R-1, R-4, R-6, and
R-16), indicating a beneficial role of an electrophilic
group at this position in AR binding. However, the beneficial effect of
an electrophilic group might also be offset by the negative effect of
its bulky size on receptor binding. For instance, R-10,
which bears a bromoacetamido group at the para-position,
showed lower binding affinity than R-5 (with a
para-acetamido substitution) and R-8 (with a
para-propionamido substitution), probably because of the
bulkiness of the bromoacetamido moiety. However, it is also important
to note that the bromoacetamido group is less electrophilic than the
chloroacetamido group. Excluding those compounds that bear strong
electrophilic substituents and R-4 (with an amino group),
there was a general observation that an increase in the size of the
substituent above a yet undefined limit decreased the binding affinity.
This effect can be illustrated by comparison of the binding affinities
of R-5, R-6, R-8, and R-9
in the sulfides and R-7 and R-16 in the sulfones.
In addition, as shown by comparisons of R-10 with
R-11 and R-13 with R-14, para-substitution of the B-ring was superior to
meta-substitution in terms of AR binding. Tucker et al.
(1988) proposed that the tertiary hydroxy group was involved in direct
interaction with the AR. This hypothesis was corroborated by our
observation that introduction of an acetyl group to that hydroxy moiety
in R-17 abolished the receptor binding of R-9.
|
). We tested whether the same SAR could be extended to our
bicalutamide derivatives. Thus, the second series of bicalutamide
derivatives, with a para-nitro group in the A-ring and
various substituents in the para-position of the B-ring or a
modified X-linkage group, was evaluated for their AR binding affinity
(Table 2). Confirming the previous
results, these compounds showed enantioselective binding. All
R-isomers had receptor affinity at least 9-fold higher than
their corresponding S-isomers. As far as the
R-isomers were concerned, the introduction of a nitro group
in the A-ring generally improved, or at least maintained, the binding
affinity compared with analogs bearing the cyano moiety at this
position (compare R-18 with R-2, R-19
with R-5, R-20 with R-7, and
R-24 with R-13). A single exception to this
trend, where the nitro substitution slightly decreased the receptor
binding (compare R-23 with R-12), was noted.
Among the present series, a total of five R-isomers
(R-19, R-21, R-22, R-23,
and R-24) were superior to R-bicalutamide in
terms of AR binding (p < 0.05). Sulfides in most cases
showed at least a 2-fold higher binding affinity than corresponding
sulfones (compare R-19 with R-20, R-21
with R-22, and R-23 with R-24).
However, this relationship was reversed when the B-ring substituent was
a NHSO2CH3 group, where the
binding affinity of R-26 (sulfone) was 3-fold higher than
that of R-25 (sulfide). These results further indicated that the B-ring substituents largely determined the effect of the X-linkage on AR binding. A trifluoroacetamido, chloroacetamido, or acetamido substituent at the para-position of B-ring led to superior
AR binding, regardless of the X-linkage.
|
showed that hydroxyflutamide analogs require
strong hydrogen bond donor ability of the tertiary hydroxy group for
efficient AR binding. To examine the effect of an increased hydrogen
bond donor ability of the tertiary hydroxy group on the binding
affinity of our bicalutamide analogs, we synthesized and evaluated a
group of compounds bearing a trifluoromethyl group connected to the
chiral carbon (Table 3). These compounds
were prepared as racemic mixtures of R- and
S-isomers. Among this series, compounds 29, 30, and 31 demonstrated very high binding affinities for the AR, each similar to
the binding affinity of the corresponding R-isomer in the
previous series, in which a methyl group was connected to the chiral
carbon (i.e., R-19, R-23, and R-24,
respectively). Considering that the S-isomers generally have
poor receptor binding, the binding affinities of pure
R-isomers in these racemic compounds would thus be expected
to be even higher. Although a definitive conclusion can be made only by
directly comparing binding affinities of parallel pure isomers, it
seemed that the replacement of methyl group with trifluoromethyl group
improved AR binding affinity. Moreover, the introduction of an
electron-withdrawing nitro group into the B-ring (compounds 27 and 28)
was apparently not beneficial for AR binding in this series.
|
). We thus further evaluated the effects of various combinations of
electron-withdrawing substitutions in the aniline ring on the binding
affinity (compounds 34 to 39). As shown in Table 4, different substitution patterns led to compounds with a wide range of binding affinities. However, none of the newly synthesized compounds showed higher binding than ligands 32 and 33. Nitro substitution in the 2-position of the aniline ring resulted in decreased AR binding (compare 34 with 35). The 3,5-dinitro substitution (compound 37) was
superior to the 2,4-dinitro substitution (compound 35) in terms of AR
binding. The binding affinity was improved with the presence of
electron-withdrawing groups in the para- and
meta-positions of the aniline ring (compare 33 with 34 and
32-33 with 38-39). We also determined the binding affinities of a group
of compounds (compounds 40-44) that differed from hydroxyflutamide in
the aromatic ring substituents. None of these compounds bound to the
AR, suggesting that bisubstitution in this ring was essential for high
AR binding affinity.
|
; Hamann et al., 1999;
Higuchi et al., 1999; Zhi et al., 1999). Because the quinolinone
skeleton is structurally different from the previously discussed
pharmacophores, we were interested to determine whether hybrids of
these two sets of lead structures bound to the AR and elicited agonist
activity. Meanwhile, Ekena et al. (1998) showed that a proton-accepting
group in the ligand is important for AR binding. We hypothesized,
therefore, that replacement of the NH in the aromatic ring system
(carbostyril ring, Fig. 3B) of quinolinones with an oxygen molecule, a
proton acceptor in hydrogen bonding, might improve the receptor
binding. The change from NH to oxygen would convert the carbostyril
ring to a coumarin ring (Fig. 3C). This rationale led to our design and
synthesis of compounds bearing the coumarin ring and segments from
bicalutamide, hydroxyflutamide, or nilutamide analogs. To our surprise,
the introduction of a coumarin ring into the bicalutamide, hydroxyflutamide, or nilutamide derivatives was detrimental to AR
binding affinity. As shown in Table 5, in
the series of bicalutamide analogs whose aromatic A-ring was replaced
with a coumarin ring, the highest binding affinity was observed for
compound 47 (Ki = 80 ± 16 nM).
The replacement of aromatic A-ring with coumarin resulted in decreased
binding affinities (compare compounds 47, 51, and 52 with
R-1, R-2, and R-5, respectively), and
the relative magnitudes of reduction were related to the
para-substituent in the B-ring. Particularly, the largest
drop in binding was observed when there was a para-acetamido
group in the B-ring (compare 52 with R-5). Data in Table
6 showed that compounds combining a coumarin ring with segments from hydroxyflutamide or nilutamide derivatives also exhibited poor AR binding. Most of the compounds in
the latter series (series B) had no affinity for the AR.
|
|
|
In Vitro Functional Activity of Nonsteroidal AR Ligands.
Previous experiments in our laboratories determined that the potency of
DHT (i.e., the lowest DHT concentration that produced the maximal
transcriptional activation of the AR) was 1 nM (Dalton et al., 1998).
Therefore, in the present study, the transcriptional activation induced
by this concentration of DHT was set as 100%, and used as the
reference for quantifying the agonist and antagonist activity of other
testing ligands.
) and may be
related to squelching of intracellular factors, such as coactivators
and some transcriptional factors, in the cellular system under
excessive ligand stimulation. Similar results were observed with
R-19 and R-23 (Fig.
5B).
|
|
previously showed that hydroxyflutamide analogs
with a para-nitro group in the anilide ring possess greater AR agonist activity than those with a para-cyano
substituent. In AR binding studies, we also observed that the change
from a para-cyano to a para-nitro in the A-ring
of bicalutamide derivatives in most cases improved AR binding affinity.
Here, we further examined the effect of this structural change on
functional activities of bicalutamide derivatives. Shown in Fig. 5 are
the agonist (A) and antagonist (B) activities of a series of
bicalutamide derivatives with a para-nitro group in the
A-ring. Similar to the observation with ligands shown in Fig. 4,
R-18, with a para-amino group in the B-ring,
functioned as an AR antagonist, whereas N-alkylamido substituents at this position conferred agonist activity to other ligands in this series. Among the four agonists, the change of a
sulfide to a sulfone in the X-linkage converted full AR agonists to
partial agonists, as shown by a comparison of R-19 with
R-20 and R-23 with R-24. Unlike those
ligands with a para-cyano in the A-ring, the presence of an
electrophilic group in the para-position of the B-ring
slightly decreased the agonist activity in this series of ligands
(compare R-19 with R-23, and R-20 with
R-24). An interseries comparison of ligands in Fig. 5 with
ligands in Fig. 4 indicated that the replacement of the
para-cyano with a para-nitro in the A-ring
increased AR agonist activity whenever the binding was improved
(compare R-19 with R-5, and R-20 with R-7). However, the agonist activity decreased when there was
no notable enhancement in AR binding (compare R-24 with
R-13).
Surprisingly, although the change from sulfide to sulfone led to
decreased agonist activity in the previous series of ligands, it
switched a full agonist (R-21) to an antagonist
(R-22) when the para-substituent in the B-ring
was a trifluoroacetamido group (Fig. 6).
This change in functional activity was accompanied by a more than
2-fold decrease in binding affinity. R-22 differs from
R-20, a partial agonist, only in the
para-substituent in the B-ring. Despite a 1.5-fold higher
binding affinity than R-20, R-22 functioned as an
antagonist, again demonstrating that higher AR binding affinity does
not necessarily predict greater agonist activity. The functional
activities of another pair of sulfide and sulfone ligands,
R-25 and R-26, are shown in Fig.
7. R-25 showed a minimal level
of agonist activity (15% at 500 nM), but antagonist activity at higher
concentrations. R-26 showed no agonist activity at
concentrations up to 500 nM and a weak antagonist activity. The change
of the carbonyl link of the B-ring substituent in R-21 and
R-22 to a sulfonyl link in R-25 and
R-26 not only impaired the AR binding but also reduced the
activities (compare agonist activity of R-21 with
R-25 and antagonist activity of R-22 with
R-26).
|
|
|
| |
Discussion |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
By modifying the structures of known nonsteroidal AR ligands, we
obtained several series of compounds that covered a wide range of
binding affinity for the AR. These novel compounds were mainly based
upon three structurally related antiandrogen pharmacophores: bicalutamide, hydroxyflutamide, and nilutamide. Earlier studies with
these chemical scaffolds revealed several structural elements that are
important for nonsteroidal AR binding and antiandrogen activity (Glen
et al., 1986; Tucker et al., 1988; Morris et al., 1991). For compounds
based on the bicalutamide and hydroxyflutamide pharmacophores, Glen et
al. (1986) and Tucker et al. (1988) showed that an electron-deficient
aromatic ring separated from the tertiary carbinol by an amide link was
required for AR binding. Substitutions at the 3- and 4- positions of
the anilide ring with electron-withdrawing groups, particularly with
cyano or nitro as the 4-substituent and trifluoromethyl or chloro as
the 3-substituent, improved AR binding affinity. Physicochemical
studies also showed that a coplanar geometry between the amide bond and
the tertiary hydroxyl group, along with electron-withdrawing groups in
the anilide ring, enhanced the hydrogen bond donor ability of the
tertiary hydroxyl group; hydrogen bonding is regarded as another
crucial factor for receptor binding (Tucker et al., 1988; Morris et
al., 1991). Results from present binding studies with novel
nonsteroidal compounds were consistent with findings from these SAR
studies. Besides, the present binding data revealed new AR binding SARs
for the linkage group and the aromatic B-ring substitutions of the
bicalutamide pharmacophore. A number of our novel compounds, mainly
from the bicalutamide-related series, exhibited an AR binding affinity higher than their lead molecules. For the bicalutamide-related series,
the AR binding was generally enhanced by: 1) R-isomer; 2) an
electrophilic para-substituent in the aromatic B-ring; 3) a
nitro group in the para-position of A-ring; and 4) a
trifluoromethyl group linked to the chiral carbon. The AR binding of
hydroxyflutamide-related series was improved in the presence of
electrowithdrawing substituents at the para-and
meta-positions of the aniline ring. The introduction of a
coumarin ring decreased the AR binding of bicalutamide,
hydroxyflutamide, and nilutamide analogs.
The recently reported crystal structure of metribolone (R1881)-bound
human AR ligand binding domain (LBD) showed that a total of 18 amino
acid residues, scattering sparsely over two regions (amino acids 700 to
788, and 875 to 896) in the domain, interact directly with the ligand
(Matias et al., 2000). These amino acids may form a ligand-binding
pocket to accommodate the ligand. Most of these residues are
hydrophobic in nature and interact mainly with the hydrophobic moieties
in the ligand molecule, whereas a few residues are hydrophilic and may
form hydrogen bonds with polar atoms in the ligand (Matias et al.,
2000). Moreover, in vitro mutagenesis studies demonstrated that the
last 12 carboxyl-terminal amino acid residues in the receptor are also
required for ligand binding, because truncation of these residues
abolishes the binding of ligands to the human AR (Jenster et al., 1991;
Kuil et al., 1995). With the present nonsteroidal ligands, we found
that the AR binding is influenced by structural properties such as
stereoisomeric conformation, steric effect, and electronic effect in
the molecule. These findings indicated indirectly that accessibility to
the binding pocket, and interaction with amino acid residues in the pocket through hydrophobic interaction and hydrogen binding are also
critical for nonsteroidal AR binding. It would be very interesting to
determine whether these nonsteroidal ligands bind to the same binding
pocket and amino acids in the LBD as those steroid ligands.
The AR agonist and antagonist activities of identified high-affinity AR ligands were determined in vitro using the cotransfection assay. These nonsteroidal ligands demonstrated a range of functional activity profiles, including antagonists, partial agonists, and full agonists. No general correlation was observed between the receptor binding affinity and functional activities with these ligands. In general, agonist activity was most often observed in bicalutamide derivatives with a sulfide linkage and an N-alkylamido substituent in the para-position of B-ring. The change from sulfide to sulfone in the linkage position resulted in attenuated agonist activity or even switched a full agonist to an antagonist.
The present functional activity data demonstrated that minor structural
differences in these nonsteroidal molecules could greatly alter the
nature of receptor-ligand interaction and lead to completely different
pharmacological responses (i.e., agonist or antagonist activities). The
divergent event triggering the differentiated activities of these
structurally related molecules is most likely the ligand-induced
conformational change in the receptor, an essential step in steroid
hormone receptor signaling pathways. Compelling evidence shows that AR
agonists and antagonists induce distinct conformational changes in the
receptor, so that the receptor interacts differently with other
transcriptional factors and/or coactivator/corepressors in the
subsequent signaling pathway (Kallio et al., 1994; Kuil and Mulder,
1994, 1995; Kuil et al., 1995). It is possible, as was suggested for
other steroid receptors (Brzozowski et al., 1997), that the AR may
respond to agonists and antagonists by positioning helix H12, one of
the 12 helices contained in the secondary structure of its LBD, to either seal (in the case of agonists) or open (in the case of antagonists) the ligand binding pocket. The full spectrum of functional activities displayed by our series of nonsteroidal ligands suggests the
potential value of these compounds as tools in studying ligand-AR interactions at the molecular level.
Very importantly, the identification of numerous potent and efficacious full AR agonists by in vitro assay represents another major progress in our efforts toward defining the SAR necessary for AR agonist activity of bicalutamide-based nonsteroidal ligands. These SARs provided valuable guides for the eventual structural optimization for AR agonist activity and development of a new generation of tissue-selective nonsteroidal androgens, SARMs. With obvious advantages over steroid androgens, SARMs have great therapeutic implications in that they could not only be used as superior alternatives to steroidal androgens in androgen replacement therapy for male hypogonadism but also expand the scope of androgen therapy to treat wasting syndromes in critically ill patients, to prevent aging-related disorders, and to regulate male fertility.
In summary, the present studies examined the AR binding and functional activities of a group of novel nonsteroidal compounds with in vitro systems. From these studies, high-affinity AR ligands with diverse activity profiles were discovered, and SARs for AR binding and transcriptional activation were identified. Because in vitro cotransfection studies cannot account for the myriad of factors that govern in vivo activity, later studies in our laboratories have focused on characterization of the in vivo functional activity of selected full agonists. These studies and others detailing the preclinical pharmacology of SARMs are the topics of forthcoming reports from our laboratories.
| |
Footnotes |
|---|
Received June 25, 2002; Accepted October 14, 2002
These studies were supported by grants from the National Institute of Child Health and Human Development (R15-HD35329), National Institute of Diabetes and Digestive and Kidney Diseases (R01-DK59800), National Cancer Institute (R29-CA68096), the St. Francis of Assisi Foundation, and the Harriet S. Van Vleet Professorship in Pharmacy.
Address correspondence to: James T. Dalton, 500 West 12th Avenue, L.M. Parks Hall, Room 242, The Ohio State University, Columbus, OH 43210. E-mail: dalton.1{at}osu.edu
| |
Abbreviations |
|---|
AR, androgen receptor; SAR, structure-activity relationship; SARM, selective androgen receptor modulator; SERM, selective estrogen receptor modulator; ER, estrogen receptor; MIB, mibolerone; PMSF, phenylmethylsulfonyl fluoride; DHT, dihydrotestosterone; FBS, fetal bovine serum; HAP, hydroxyapatite; LBD, ligand binding domain.
| |
References |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
uses and abuses.
N Engl J Med
334:
707-714: estrogen versus androgen discrimination.
J Biol Chem
273:
693-699This article has been cited by other articles:
|
|
 |
|
  A. Jones, J. Chen, D. J. Hwang, D. D. Miller, and J. T. Dalton Preclinical Characterization of a (S)-N-(4-Cyano-3-Trifluoromethyl-Phenyl)-3-(3-Fluoro, 4-Chlorophenoxy)-2-Hydroxy-2-Methyl-Propanamide: A Selective Androgen Receptor Modulator for Hormonal Male Contraception Endocrinology, January 1, 2009; 150(1): 385 - 395. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Narayanan, C. C. Coss, M. Yepuru, J. D. Kearbey, D. D. Miller, and J. T. Dalton Steroidal Androgens and Nonsteroidal, Tissue-Selective Androgen Receptor Modulator, S-22, Regulate Androgen Receptor Function through Distinct Genomic and Nongenomic Signaling Pathways Mol. Endocrinol., November 1, 2008; 22(11): 2448 - 2465. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Chen, K. C. Ahn, N. A. Gee, M. I. Ahmed, A. J. Duleba, L. Zhao, S. J. Gee, B. D. Hammock, and B. L. Lasley Triclocarban Enhances Testosterone Action: A New Type of Endocrine Disruptor? Endocrinology, March 1, 2008; 149(3): 1173 - 1179. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. H. Bisson, A. V. Cheltsov, N. Bruey-Sedano, B. Lin, J. Chen, N. Goldberger, L. T. May, A. Christopoulos, J. T. Dalton, P. M. Sexton, et al. Discovery of antiandrogen activity of nonsteroidal scaffolds of marketed drugs PNAS, July 17, 2007; 104(29): 11927 - 11932. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. E. Bohl, Z. Wu, D. D. Miller, C. E. Bell, and J. T. Dalton Crystal Structure of the T877A Human Androgen Receptor Ligand-binding Domain Complexed to Cyproterone Acetate Provides Insight for Ligand-induced Conformational Changes and Structure-based Drug Design J. Biol. Chem., May 4, 2007; 282(18): 13648 - 13655. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Yang, C. E. Bohl, V. A. Nair, S. M. Mustafa, S. S. Hong, D. D. Miller, and J. T. Dalton Preclinical Pharmacology of a Nonsteroidal Ligand for Androgen Receptor-Mediated Imaging of Prostate Cancer J. Pharmacol. Exp. Ther., April 1, 2006; 317(1): 402 - 408. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Wu, Z. Wu, J. Yang, V. A. Nair, D. D. Miller, and J. T. Dalton PHARMACOKINETICS AND METABOLISM OF A SELECTIVE ANDROGEN RECEPTOR MODULATOR IN RATS: IMPLICATION OF MOLECULAR PROPERTIES AND INTENSIVE METABOLIC PROFILE TO INVESTIGATE IDEAL PHARMACOKINETIC CHARACTERISTICS OF A PROPANAMIDE IN PRECLINICAL STUDY Drug Metab. Dispos., March 1, 2006; 34(3): 483 - 494. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Gao, Z. Wu, C. E. Bohl, J. Yang, D. D. Miller, and J. T. Dalton CHARACTERIZATION OF THE IN VITRO METABOLISM OF SELECTIVE ANDROGEN RECEPTOR MODULATOR USING HUMAN, RAT, AND DOG LIVER ENZYME PREPARATIONS Drug Metab. Dispos., February 1, 2006; 34(2): 243 - 253. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Chen, D. J. Hwang, K. Chung, C. E. Bohl, S. J. Fisher, D. D. Miller, and J. T. Dalton In Vitro and in Vivo Structure-Activity Relationships of Novel Androgen Receptor Ligands with Multiple Substituents in the B-Ring Endocrinology, December 1, 2005; 146(12): 5444 - 5454. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. E. Bohl, D. D. Miller, J. Chen, C. E. Bell, and J. T. Dalton Structural Basis for Accommodation of Nonsteroidal Ligands in the Androgen Receptor J. Biol. Chem., November 11, 2005; 280(45): 37747 - 37754. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Perakyla, M. Malinen, K.-H. Herzig, and C. Carlberg Gene Regulatory Potential of Nonsteroidal Vitamin D Receptor Ligands Mol. Endocrinol., August 1, 2005; 19(8): 2060 - 2073. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Chen, J. Kim, and J. T. Dalton Discovery and Therapeutic Promise of Selective Androgen Receptor Modulators Mol. Interv., June 1, 2005; 5(3): 173 - 188. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. E. Bohl, W. Gao, D. D. Miller, C. E. Bell, and J. T. Dalton Structural basis for antagonism and resistance of bicalutamide in prostate cancer PNAS, April 26, 2005; 102(17): 6201 - 6206. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Chen, D. J. Hwang, C. E. Bohl, D. D. Miller, and J. T. Dalton A Selective Androgen Receptor Modulator for Hormonal Male Contraception J. Pharmacol. Exp. Ther., February 1, 2005; 312(2): 546 - 553. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. R. Brown Nonsteroidal Selective Androgen Receptors Modulators (SARMs): Designer Androgens with Flexible Structures Provide Clinical Promise Endocrinology, December 1, 2004; 145(12): 5417 - 5419. [Full Text] [PDF]
|
|
|
|
|
|
W. Gao, J. D. Kearbey, V. A. Nair, K. Chung, A. F. Parlow, D. D. Miller, and J. T. Dalton Comparison of the Pharmacological Effects of a Novel Selective Androgen Receptor Modulator, the 5{alpha}-Reductase Inhibitor Finasteride, and the Antiandrogen Hydroxyflutamide in Intact Rats: New Approach for Benign Prostate Hyperplasia Endocrinology, December 1, 2004; 145(12): 5420 - 5428. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Yin, H. Xu, Y. He, L. I. Kirkovsky, D. D. Miller, and J. T. Dalton Pharmacology, Pharmacokinetics, and Metabolism of Acetothiolutamide, a Novel Nonsteroidal Agonist for the Androgen Receptor J. Pharmacol. Exp. Ther., March 1, 2003; 304(3): 1323 - 1333. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Yin, W. Gao, J. D. Kearbey, H. Xu, K. Chung, Y. He, C. A. Marhefka, K. A. Veverka, D. D. Miller, and J. T. Dalton Pharmacodynamics of Selective Androgen Receptor Modulators J. Pharmacol. Exp. Ther., March 1, 2003; 304(3): 1334 - 1340. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
) represents the mean ± S.D. in a
representative experiment of triplicate measurements. The smooth line
is the fitted curve by the inhibitory effect sigmoid
Emax model.
