JPET Fast Forward. Published on January 24, 2006 as DOI: 10.1124/jpet.105.094334 JPET FastThis articleForward. has not Published been copyedited on and January formatted. 24,The final2006 version as DOI:10.1124/jpet.105.094334 may differ from this version.
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Preclinical pharmacology of a nonsteroidal ligand for androgen receptor mediated imaging of prostate cancer
Downloaded from Jun Yang, Casey E. Bohl, Vipin A. Nair, Suni M. Mustafa, Seoung Soo Hong, Duane D. Miller, and James T. Dalton
jpet.aspetjournals.org
Division of Pharmaceutics, College of Pharmacy, The Ohio State University, Columbus, OH 43210 (JY, CEB, and JTD) and Department of Pharmaceutical Sciences, College of
Pharmacy, The University of Tennessee, Memphis, TN 38163 (VAN, SMM, SSH, and at ASPET Journals on September 25, 2021 DDM)
1
Copyright 2006 by the American Society for Pharmacology and Experimental Therapeutics. JPET Fast Forward. Published on January 24, 2006 as DOI: 10.1124/jpet.105.094334 This article has not been copyedited and formatted. The final version may differ from this version.
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RUNNING TITLE: A Novel SARM for Imaging of Prostate Cancer
CORRESPONDING AUTHOR: James T. Dalton
ADDRESS: 500 West 12th Avenue, L.M. Parks Hall, Room 242, Columbus, OH 43210
TELEPHONE: 614-688-3797; FAX: 614-292-7766
E-MAIL: [email protected] Downloaded from NUMBER OF TEXT PAGES: 15
NUMBER OF TABLES: 1
NUMBER OF FIGURES: 5 jpet.aspetjournals.org
NUMBER OF REFERENCES: 36
NUMBER OF WORDS: Abstract:193; Introduction:550; Discussion:727. at ASPET Journals on September 25, 2021 ABBREVIATIONS: AR, androgen receptor; ER, estrogen receptor; GR, glucocorticoid receptor; MR, mineralocorticoid receptor; PR, progesterone receptor; SARM, selective androgen receptor modulator; BPH, benign prostatic hyperplasia; DMEM, Dulbecco's modified Eagle's medium; MIB, mibolerone; DHT, dihydrotestosterone; T, testosterone;
HAP, hydroxyapatite; SHBG, sex hormone binding globulin.
RECOMMENDED SECTION ASSIGNMENT: Endocrine & diabetes
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ABSTRACT
Proper management of prostate cancer patients is highly dependent on the spread of the disease. High expression levels of the androgen receptor (AR) in prostate tumor offer a target for identifying cancer metastasis. We investigated the use of nonsteroidal AR ligands for receptor-mediated imaging as a diagnostic tool for prostate cancer staging.
Compound S-26 was identified from a series of iodinated ether-linked derivatives of
bicalutamide due to its high AR binding affinity of 3.3 nM (which is similar to Downloaded from testosterone and about 25% of the binding affinity of dihydrotestosterone) in an in vitro competitive binding assay using rat prostate cytosol. Further, S-26 exhibited a greater jpet.aspetjournals.org binding affinity (Ki=4.4 nM) in a whole cell binding assay using COS-7 cells transfected with human AR than testosterone (Ki=32.9 nM) and dihydrotestosterone (Ki=45.4 nM).
We also confirmed that sex hormone binding globulin (SHBG), a plasma protein that at ASPET Journals on September 25, 2021 binds steroids with high affinity, does not bind with S-26. Co-transfection studies with the estrogen, progesterone, and glucocorticoid receptor indicated that S-26 does not cross-react with other members of the steroid hormone receptor family. The nonsteroidal structure, high AR binding affinity, specificity, and lack of binding to SHBG indicate that
S-26 exhibits favorable properties for further development as an imaging agent for prostate cancer.
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INTRODUCTION
Prostate cancer is the most common cancer and remains the second leading cause of death from cancer in American men (Greenlee et al., 2001). Procedures for reliable and accurate staging of prostate cancer are essential, since they greatly contribute to decisions regarding patient treatment and management. Because of the lack of adequate non- invasive diagnostic methods, many prostate cancer patients must undergo surgical staging
to identify lymph node metastases (Kavoussi et al., 1993; Neal and Meis, 1994). The Downloaded from desire and search for non-invasive, more accurate and selective imaging tools for prostate cancer provided the basis for these studies. jpet.aspetjournals.org The androgen receptor (AR) is a member of the nuclear hormone receptor family, which includes the estrogen (ER), glucocorticoid (GR), mineralocorticoid (MR), and progesterone (PR) receptors. Testosterone (T) and dihydrotestosterone (DHT), at ASPET Journals on September 25, 2021 endogenous steroidal ligands for the AR, regulate the growth and response of androgen- sensitive tissues. Upon androgen binding, the AR undergoes a conformational change, binds to specific DNA sequences, and modulates the transcription of target genes. Early immunohistochemical studies on human and rat tissues showed that the expression of AR is largely confined to the male reproductive organs, especially the prostate (Takeda et al.,
1990). AR expression levels in benign prostatic hyperplasia (BPH) and carcinoma samples vary widely (Shain et al., 1983; van Aubel et al., 1985; Bowman et al., 1986;
Habib et al., 1986; Frydenberg et al., 1991). However, Brolin et al. observed a higher proportion of AR-positive cells in BPH and prostate cancer metastases, as compared with normal tissues (Brolin et al., 1992). Early work on radiolabeled androgens demonstrated a selective uptake in the prostate of rats (Tveter and Attramadal, 1968; Symes, 1982;
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Carlson and Katzenellenbogen, 1990). Furthermore, preclinical studies using steroidal
AR ligands in baboons also indicated that AR ligands bind the AR in vivo and were selectively retained in the prostate (Bonasera et al., 1996). The expression of AR in all stages of prostate cancer regardless of tumor sensitivity to hormonal therapy (Sadi et al.,
1991; van der Kwast et al., 1991; Van der Kwast et al., 1996) and the poor pharmacokinetics and specificity of steroidal ligands (Berger et al., 1975; Salman and
Chamness, 1991; Choe et al., 1995; Labaree et al., 1999) are the basis of our approach to Downloaded from prostate cancer imaging. Nonsteroidal high affinity AR ligands containing radioactive iodine provide a rational means for diagnostic imaging and potentially radiotherapy of jpet.aspetjournals.org prostate cancer. The relative lower energy (27 KeV) and longer half-life of iodine-125
(60 days) offers a good radiotracer for imaging research, while iodine-123 (159 KeV with a half life of 13 hours) could be clinically used for diagnosis and iodine-131 (higher at ASPET Journals on September 25, 2021 energy 364 KeV with a half life of 8 days) could be used for receptor-mediated radiation therapy.
Our laboratories continue to explore the preclinical pharmacology of selective androgen receptor modulators (SARMs). We designed and synthesized many nonsteroidal ligands with high binding affinity to the AR. Compared to steroids, nonsteroidal ligands have greater flexibility in structural modifications allowing optimization of physicochemical, pharmacokinetic and pharmacologic properties.
Importantly, we found that iodine could be incorporated into our nonsteroidal compounds and retain high binding affinity to the AR (Bohl et al., 2004; Nair et al., 2004; Nair et al.,
2005). Reported here are the in vitro evaluation and characterization of one of the most promising compounds, S-26, as a potential imaging agent for prostate cancer.
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MATERIALS AND METHODS
Chemicals
Nonsteroidal compounds (Table 1) were synthesized and characterized in our laboratories as previously described (Nair et al., 2004; Nair et al., 2005). The purities of synthesized compounds were confirmed by NMR, elemental analysis and mass
3 3 spectrometry. [17α-methyl- H] Mibolerone ([ H] MIB, 84 Ci/mmol) and unlabeled MIB Downloaded from were purchased from PerkinElmer Life Sciences (Boston, MA). Hydroxyapatite (HAP) was purchased from Bio-Rad Laboratories (Hercules, CA). EcoLite (+) scintillation jpet.aspetjournals.org 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). All other chemicals were purchased from Sigma (St. Louis, MO). at ASPET Journals on September 25, 2021
Cell Culture
Dulbecco's modified Eagle's medium (DMEM), RPMI 1640 medium, trypsin-EDTA, non-essential amino acids and L-glutamine were purchased from Mediatech (Herndon,
VA). Fetal bovine serum (FBS) and LipofectAMINE reagent were purchased from
Invitrogen (Carlsbad, CA). The monkey kidney fibroblast-like CV-1 and its derivative
COS-7 cell lines were obtained from American Type Culture Collection (Rockville,
MD). The cells were grown at 37 °C in a humidified atmosphere with 5 % carbon dioxide.
AR Competitive Binding Assay
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The AR binding affinity of synthesized nonsteroidal compounds was determined using a radioligand competitive binding assay as previously reported (Yin et al., 2003b).
Briefly, an aliquot of AR cytosol preparation (50 µL) was incubated with a saturating concentration (1 nM) of [3H] MIB at 4 °C for 18 h in the absence or presence of increasing concentrations of the compound of interest (10 different concentrations ranging from 10-2 nM to 104 nM). Triamcinolone acetonide (1000 nM) was included in
3 the incubate to block the interaction of [ H] MIB with GR and PR. Nonspecific binding of Downloaded from
[3H] MIB was determined in separate incubates by adding an excess of unlabeled MIB
(1000 nM) to the incubate. After incubation, the protein-bound radioactivity was jpet.aspetjournals.org separated from free radioactivity by HAP precipitation. The bound radioactivity was then extracted from HAP by incubating the HAP pellet with 1 mL of ethanol at room temperature for 1 h. The radioactivity was counted in a Beckman LS6800 liquid at ASPET Journals on September 25, 2021 scintillation counter (Beckman Coulter, Fullerton, CA).
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).
3 The concentration of compound that reduced the specific binding of [ H] 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. WinNonlin was provided by a Pharsight Academic License to The Ohio State University. The apparent equilibrium binding constant (Ki) of the compound of interest was calculated by
3 Ki = Kd × IC50/(Kd + L), where Kd was the equilibrium dissociation constant of [ H] MIB
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(Kd = 0.19 nM; determined in preliminary experiments) and L was the concentration of
[3H] MIB used in the experiment (L=1 nM).
Whole cell binding assay
COS-7 cells were transiently transfected with human AR (plasmid pCMV-hAR was generously provided by Dr. Donald Tindall, Mayo Clinic and Mayo Foundation,
Rochester, MN). Briefly, transfection was conducted in 150 mm diameter dishes using Downloaded from serum-free medium and LipofectAMINE according to the manufacturer's instructions.
The second day after transfection, COS-7 cells were seeded in 24-well plates, and jpet.aspetjournals.org incubated for 1 day in a humidified CO2 (5 %) environment to reach greater than 80 % confluence. The complete DMEM medium was then exchanged to serum-free and phenol red-free DMEM medium. One hour later, the cells were incubated with 1 nM [3H] MIB in at ASPET Journals on September 25, 2021 the absence or presence of the ligand of interest (final concentration ranging from 10-1 nM to 104 nM). After a 4 h incubation at 37 °C, the cells were washed three times with ice-cold PBS and lysed in 400 µL of 1N NaOH. Radioactivity present in an aliquot (100
µL) of the lysate was then counted in a liquid scintillation counter. Cell number was normalized with the protein concentration in each well as determined by the BCA method. Nonspecific binding of [3H] MIB was determined in separate wells by adding an excess of unlabeled MIB (1000 nM) to the incubation medium. The specific binding of
[3H] MIB at each concentration of the compound of interest 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. The equilibrium dissociation constant Kd of [3H] MIB in AR transfected COS-7 was 0.5 nM, which was determined in
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JPET #94334 preliminary experiments. Apparent Ki values were calculated as described above in the
AR competitive binding assay.
Transactivational studies with AR, ER, GR and PR
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 a hormone-
dependent luciferase reporter gene, were examined in transiently transfected CV-1 cells. Downloaded from
To determine the AR transactivational activities, CV-1 cells were maintained in DMEM containing 10 % FBS, 0.1 mM non-essential amino acids and 1 % L-glutamine at 37 oC jpet.aspetjournals.org in a humidified atmosphere containing 5 % CO2. Transient transfections of CV-1 cells were conducted in 150 mm diameter dishes using serum-free medium and
LipofectAMINE according to the manufacturer's instructions. Co-transfection was done at ASPET Journals on September 25, 2021 by adding AR expression vector (plasmid pCMV-hAR was provided by Dr. Donald
Tindall, Mayo Clinic and Mayo Foundation, Rochester, MN), luciferase reporter vector
(pMMTV-Luc was provided by Dr. Ronald Evans, The Salk Institute, San Diego, CA), control β-galactosidase vector (pSV-β-galactosidase was obtained from Promega
Corporation, Madison, WI) and Lipofectamine. At the time of transfection, medium was replaced with transfection medium (DMEM containing 1 % L-glutamine) and the
DNA/Lipofectamine solution. After 3-5 h of transfection, cells were washed once with
DMEM, then recovered in fresh DMEM supplemented with 0.2 % FBS for 10 to 12 h.
After recovery, the transfected cells were distributed into 24-well plates at a density of 8 x 104 cells per well. To start the transactivation studies, the media was aspirated and replaced with 1 ml/well of medium containing the desired concentration of ligands.
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Negative control wells included no drug treatment, while positive control wells included
1 nM DHT only. After 24 h, the media was aspirated, wells were washed twice with 1 ml/well of PBS, and cells were lysed by incubation with 150 µL/well of Reporter Lysis buffer (Promega) for 30 minutes at room temperature. An aliquot (50 µL) of the lysate in each well was sampled to measure luciferase and β-galactosidase activity.
For transactivational studies with ER, PR and GR, the AR plasmid was replaced with
expression vectors for ER, GR (p-hERα, p-hERβ and p-hGR were generously provided by Downloaded from
Dr. Ronald Evans, The Salk Institute, San Diego, CA) and PR (p-hPR was generously provided by Dr. Donald P. McDonnell, Duke University, Durham, NC). Estradiol, jpet.aspetjournals.org progesterone and dexamethasone were used as positive controls, respectively.
Transcriptional activation in each well was calculated as the ratio of luciferase activity to
β
-galactosidase activity to normalize the variance in cell number and transfection at ASPET Journals on September 25, 2021 efficiency. All experiments were performed in triplicate or greater, and data were expressed as the mean ± S.D. in representative experiments.
AR stability and Western blot analysis
COS-7 cells were transiently transfected with AR in 150 mm diameter dishes and distributed in 12-well plates at a density of 2 x 105 cells/well. The second day after transfection, the transfected COS-7 cells were incubated in serum-free, phenol red-free
DMEM medium containing DHT (10 nM), testosterone (10 nM), S-26 (1 nM or 10 nM) or drug-free medium for 10 h. Protein synthesis was blocked by co-incubation with cycloheximide (50 µg/mL) at 37 oC. After incubation, cells were washed with ice-cold
PBS and lysed in 20 mM Tris-HCl, pH 7.4, 1 % deoxycholate, 0.1 % SDS, 150 mM
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NaCl, 1 mM EDTA, 1 % Triton X-100, supplemented with commercially available protease inhibitor cocktail (Sigma). Cell lysates were centrifuged and the supernatant was used for protein measurement and Western blot analysis. The antibodies used were anti-
AR polyclonal antibody N-20, actin polyclonal IgG (Santa Cruz Biotechnology) and anti- rabbit IgG conjugated with horseradish peroxidase (Amersham Biosciences UK limited,
England).
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SHBG protein binding studies
SHBG binding studies were conducted using methods similar to those reported by jpet.aspetjournals.org Winneker et al.(Winneker et al., 1990). Human SHBG was prepared from human serum and precipitated by ammonium sulfate. Triplicate aliquots were incubated for 2 h in ice with [1,2, 3H]-DHT in either the absence or the presence of increasing concentrations (10- at ASPET Journals on September 25, 2021
2 nM to 104 nM) of the compound of interest. At the end of incubation, bound radioactivity was separated by the HAP method and was extracted with ethanol. The radioactivity, representing AR-bound [3H] DHT, was counted in a Beckman LS6800 liquid scintillation counter (Beckman Coulter, Fullerton, CA). Non-specific binding of
[3H] DHT was determined by including an excess (10 µM) of unlabeled DHT in separate incubates.
RESULTS
In vitro AR binding affinity determination
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We designed and synthesized a series of iodinated nonsteroidal AR ligands (Table 1) based on the structure-activity relationship for nonsteroidal AR binding obtained from the literature and previous studies in our laboratories (Tucker et al., 1988; Dalton et al., 1998;
Mukherjee et al., 1999; Kirkovsky et al., 2000; Bohl et al., 2004). The AR binding affinities of these synthetic molecules, reported as apparent Ki values, were determined by a radioligand competitive binding assay using rat prostates as an AR source (Table 1).
Competitive binding studies showed that S-26 has high AR binding affinity with an Downloaded from apparent Ki of 3.3 ± 0.1 nM (i.e., a relative binding affinity [RBA] of 24.9 ± 2.6 % as compared to DHT). We previously reported that the presence of electron-withdrawing jpet.aspetjournals.org groups in the A ring is important for the binding affinity of nonsteroidal ligands, with compounds incorporating a CF3 substituent at the R1 position of the A ring demonstrating high binding affinity to the AR (Marhefka et al., 2004). The R2 position of at ASPET Journals on September 25, 2021
B ring also plays an important role in AR binding affinity (Kim et al., 2005). When comparing compounds with identical substituents at the R2 position of the B ring, the binding affinity of compounds incorporating an iodine atom at position R1 of the A ring was very similar to that observed for compounds incorporating a trifluoromethyl substituent at this position (Table 1). One exception to this observation was the compound incorporating a chloro substituent at R2. In this case, the compound with a
CF3 group at R1 (i.e., S-31) bound the AR with much higher affinity (Table 1) than the iodine containing compound (i.e., S-27).
AR whole cell binding affinity
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As in vivo access to the AR will also be affected by the ability of the compounds to penetrate the cell membrane, we also examined the apparent binding affinity of the compounds in living cells. We performed whole cell binding studies in COS-7 cells transfected with the human AR. To our surprise, the nonsteroidal ligand S-26 demonstrated greater binding affinity and a much smaller Ki value (4.3 ± 0.1 nM) than either DHT (45.4 ± 1.1 nM) or testosterone (32.9 ± 2.6 nM) in living cells (Fig. 1).
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Transactivational studies of AR, ER, GR and PR
We next sought to determine whether S-26 is specific for the AR, as the other jpet.aspetjournals.org hormone receptors share significant homology with the ligand binding domain of the AR.
For these studies, CV-1 monkey kidney cells (a cell line devoid of steroid hormone receptors) were transfected with expression plasmids for estrogen receptor α (ERα), at ASPET Journals on September 25, 2021 estrogen receptor β (ERβ), PR, or GR and their respective hormone-dependent luciferase reporter plasmids. Transfected cells were treated with the known agonist for each receptor (i.e., estradiol for ERα and ERβ, progesterone for PR, and dexamethasone for
GR) and increasing concentrations of S-26. Our preliminary data indicated that DHT elicits maximal AR-mediated transcriptional activation at a concentration of 1 nM, and that higher concentrations were unable to stimulate further increases. We found that S-26 is an AR agonist, albeit with significantly lower potency than DHT. High (1000 nM) concentrations of S-26 were required to stimulate AR-mediated transcriptional activity to the same extent as 1 nM DHT. Importantly, S-26 did not stimulate or inhibit agonist- induced transcriptional activation for ER, PR and GR at biologically relevant concentrations (Fig. 2), although 1000 nM of S-26 exhibit weak PR antagonist activity.
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Separate studies (Fig. 3) showed that S-26 significantly (p<0.01) enhanced the AR- mediated transcriptional activation induced by 0.001 nM DHT, and that the ability of S-
26 to stimulate AR-mediated transcriptional activity was inhibited by bicalutamide (1 or
10 µM), providing further evidence of the specific interaction of S-26 with the AR. These data indicated that S-26 is specific for the AR and does not interact with other steroid hormone receptors.
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Determination of ligand-dependent AR stability
AR stability in the presence of DHT (10 nM), T (10 nM) and S-26 (10 nM) was jpet.aspetjournals.org examined in COS-7 cells transfected with the human AR. After 10 h incubation at 37 oC, the cells were lysed and protein expression examined by Western blot assay. Fig. 4 demonstrates that no decrease in the expression of AR level was observed in presence of at ASPET Journals on September 25, 2021 either DHT, T or S-26. This suggests that S-26 is a high affinity ligand for the AR but does not cause destabilization of AR level in living cells as previous observed for bicalutamide (Waller et al., 2000; Furutani et al., 2002).
SHBG binding
We determined the binding affinity of S-26 and two steroidal androgens (testosterone and DHT) to SHBG, a plasma protein known to avidly bind steroidal androgens
(Avvakumov et al., 2002). By comparison, testosterone bound to SHBG with about 20 % of the binding affinity of DHT. However, S-26 bound very poorly to SHBG, with less than 0.2 % of the binding affinity of DHT (Fig. 5). These data suggest that rats are a valid
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JPET #94334 animal model to predict S-26 biodistribution and pharmacokinetics in humans, as interaction of S-26 with SHBG is unimportant.
Discussion
Obtaining adequate information on the biological and physiological status of tumors is essential for the management and treatment of cancer patients. Targeting the AR with
radioligands provides an approach potentially more sensitive than CT or MRI imaging for Downloaded from prostate cancer staging. The majority of steroidal ligands were shown to be poor candidates for AR-mediated imaging due to their low binding affinities, rapid jpet.aspetjournals.org metabolism, and lack of stability (Berger et al., 1975; Brandes and Katzenellenbogen,
1987; Salman and Chamness, 1991; Liu et al., 1992; Choe et al., 1995; Labaree et al.,
1999). Androgenic steroids bind to other steroidal receptors and SHBG in plasma, at ASPET Journals on September 25, 2021 contributing to their poor target site specificity for imaging. Although SHBG has a high binding affinity to most endogenous and synthesized androgens, it does not bind to the nonsteroidal androgen receptor ligands reported herein. In addition, reactivity with other steroid receptors is less prevalent with nonsteroidal compounds, providing another advantage over steroidal agents.
We developed a new series of ether-linked AR ligands with improved metabolic stability and AR binding affinity as potential imaging agents. Previous literature reports demonstrated that a variety of substitution patterns can be used to modulate AR binding affinity (He et al., 2002; Bohl et al., 2004; Marhefka et al., 2004). For example, multiple substitutions on the B ring (Table 1) are possible with halogens, permitting protection of this ring from oxidative metabolism (Kim et al., 2005). In addition, the para position of
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JPET #94334 the A ring accommodates a variety of hydrogen bond acceptor groups, with nitro and cyano substitution resulting in compounds with greater binding affinity than halogens.
Importantly, the R1 position of the A ring may be modified to include a variety of electronegative substituents, including halogens. S-26 demonstrated that an iodine atom can be effectively incorporated on the A-ring at the R1 position to maintain high AR binding affinity (i.e., Ki of 3.3 ± 0.1 nM, RBA=24.9 ± 2.6 % of that for DHT). The
crystal structure of the trifluoromethyl (at the R1 position) analog of S-26, and the Downloaded from antagonist R-bicalutamide, complexed to the AR demonstrate that the iodine would be accommodated in the same hydrophobic region occupied by the trifluoromethyl group jpet.aspetjournals.org (Bohl et al., 2005a; Bohl et al., 2005b) explaining its favorable interaction with the AR.
Surprisingly, S-26 demonstrates higher binding affinity than T and DHT in a whole cell binding assay with human AR. The reasons for this finding are not likely due to the at ASPET Journals on September 25, 2021 differences in rat and human AR, since the ligand binding domain is 100% identical in sequence (Sack et al., 2001). Further studies are needed to fully understand why DHT has a decreased apparent binding affinity as compared to S-26 in whole cell binding experiments. More importantly, S-26 maintains high binding affinity in whole cell binding studies, demonstrating its ability to efficiently target intracellular AR.
Sex hormone binding globulin (SHBG) is a plasma glycoprotein found in many species that demonstrates high binding affinity for certain estrogens and androgens.
Although it is not expressed in rats, it is present at high concentrations in human serum
(the ultimate milieu in which our imaging agents will be used). SHBG affects the pharmacokinetics of most steroidal ligands by sequestering them in the blood. We determined the binding affinity of S-26 and two steroidal androgens (i.e., testosterone and
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DHT) to SHBG. Our studies showed that S-26 binds very poorly to SHBG, with less than
0.1 % (i.e., 0.02%) of the binding affinity of DHT. By comparison, testosterone bound to
SHBG with about 20 % of the binding affinity of DHT. These data indicate that S-26 does not bind SHBG and that rats are a valid animal model for biodistribution and pharmacokinetics of the compound in humans. Moreover, the nonsteroidal ligands are largely bound to albumin, a low affinity and high capacity plasma binding protein.
Therefore, unlike steroidal analogs, low affinity plasma protein binding to albumin is Downloaded from unlikely to compete significantly with high affinity target tissue binding sites. In addition, we performed in vivo studies with a number of compounds with similar chemical jpet.aspetjournals.org structures demonstrating their favorably pharmacokinetic and pharmacodynamic properties (Yin et al., 2003a; Kearbey et al., 2004; Gao et al., 2005) suggesting that S-26 will be efficacious in vivo. at ASPET Journals on September 25, 2021
We also examined the interaction of S-26 with other members of the nuclear hormone receptor family, as interaction with non-target proteins would interfere with AR-mediated uptake in target tissues. S-26 did not stimulate or inhibit agonist-induced transcriptional activation for any of these nuclear hormone receptors at biologically relevant concentrations (S-26 slightly inhibited PR activation at a concentration of 1,000 nM).
These data indicate that S-26 is specific for the AR and will not interact with non-target nuclear hormone receptors.
Stability is an important factor for a successful receptor mediated imaging studies.
We found that S-26 is very stable in mouse, rat and human plasma at 37 °C up to at least
48 h (data not shown). We also found that AR is very stable in the presence of DHT, T or
S-26. The well-known LNCaP prostate cancer cell line contains a mutated AR. To
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JPET #94334 examine the binding of our nonsteroidal ligands in this cell line, we performed whole cell binding studies in LNCaP cells and the results were similar to what we found in COS-7 cells transfected with wide-type AR (data not shown). In conclusion, the high affinity for the AR in a whole cell binding assay, poor affinity to other steroid receptors and SHBG, and a stabilizing effect on the AR provide incentive for the development of S-26 as an
AR-mediated imaging agent for prostate cancer.
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FOOTNOTES:
Financial support: These studies were supported by grants from Department of Defense, US Army Prostate Cancer Research Program (Grant # PC-001480).
Financial Interest: J. T. Dalton is also an employee of GTx, Inc., Memphis, TN. Downloaded from D. D. Miller is also an employee of GTx, Inc., Memphis, TN.
Address correspondence to:
James T. Dalton, Ph.D., 500 West 12th Avenue, L.M. Parks Hall, Room 242, The Ohio jpet.aspetjournals.org State University, Columbus, OH 43210. E-mail: [email protected] at ASPET Journals on September 25, 2021
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FIGURE LEGENDS
Fig. 1. Whole cell binding studies of DHT, T and S-26. The whole cell binding studies were conducted in AR transfected Cos-7 cells. Different concentrations of compound were coincubated with 1 nM [3H] MIB in serum free, phenol-red free medium for 4 h.
Cells were lysed with 400 µl 1 N NaOH and the radioactivity was counted. The cell number was normalized with BCA by measuring protein levels. The concentration of test
3 compound that displaced the specific binding of H-MIB by 50% (IC50) was obtained by Downloaded from
WinNolin, and the equilibrium binding constant (Ki) was calculated from
3 Ki=Kd*IC50/(Kd+L), where Kd is the equilibrium dissociation constant of H-MIB (0.5 jpet.aspetjournals.org nM as determined in preliminary experiments) and L is the concentration of 3H -MIB used in the experiment (1 nM).
at ASPET Journals on September 25, 2021
Fig. 2. Contransfection studies-lack of S-26 Effects on ERα, ERβ, PR, and GR.
Monkey kidney CV-1 cells were transfected with plasmids for the AR, ER, GR, and PR to characterize the agonist and antagonist activities of steroid receptor ligands. For AR transfection studies, negative control includes neither DHT nor ligands, while positive control included 1 nM DHT (D). In the performance of ER, PR and GR transactivational studies, the AR plasmid was replaced with ER, PR and GR plasmids, respectively.
Estradiol (E), progesterone (P) and dexamethasone (DX) were used as positive controls, respectively. Transcriptional activity was calculated as the Luciferase activity normalized with β-galactosidase activity.
28 JPET Fast Forward. Published on January 24, 2006 as DOI: 10.1124/jpet.105.094334 This article has not been copyedited and formatted. The final version may differ from this version.
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Fig. 3. Cotransfection studies- S-26 enhances DHT activity and is inhibited by bicalutamide. Monkey kidney CV-1 cells were transfected with plasmids for the AR and a hormone-responsive luciferase reporter. Control wells in which no drug was added were included in each experiment. [TOP PANEL] S-26 (10 nM) enhanced the transcriptional activation induced by low concentrations of DHT (i.e., 0.001 nM).
Maximal or non-significant differences in transcriptional activity were observed with
higher concentrations of DHT. [BOTTOM PANEL] Transcriptional activity induced by Downloaded from both DHT (1 nM) and S-26 (10 nM) was inhibited by co-incubation with bicalutamide.
Transcriptional activity was calculated as the Luciferase activity normalized with β- jpet.aspetjournals.org galactosidase activity. * represent statistically significant differences.
Fig. 4. AR stability in the present of DHT, T and S-26. The AR stability in AR at ASPET Journals on September 25, 2021 transfected Cos-7 cells was examined by incubating 10 nM of DHT, T and S-26 (1 and
10 nM) for 10 h at 37 oC. The protein sysnthesis was blocked by coincubation with 50
µg/mL of cycloheximide. The cell lysate was applied to protein measurement and
Western blot assay.
Fig. 5. SHBG protein binding studies of DHT, T and S-26. Human SHBG was purified from human serum. Triplicate aliquots were incubated for 2 h in ice with [1,2,
3H]-DHT in either the absence or the presence of increasing concentrations of DHT or compounds. At the end of incubation, bound 3H-DHT-AR complex was separated by the
HAP method. Non-specific binding of 3H-DHT was determined by including an excess
(10 µM) of unlabelled DHT in incubates.
29 JPET Fast Forward. Published on January 24, 2006 as DOI: 10.1124/jpet.105.094334 This article has not been copyedited and formatted. The final version may differ from this version.
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Table 1. AR binding affinity of iodinated nonsteroidal ligands.
O
NC A N O R2 H B OH H3C
R1
Downloaded from
R1 = R2 = F Cl Br NHC(O)CH3
jpet.aspetjournals.org
I # S-26 S-27 S-28 S-29
Ki (nM) 3.3 ± 0.1 11.3 ± 1.4 22.6 ± 1.7 17.0 ± 0.8 at ASPET Journals on September 25, 2021
CF3 # S-30 S-31 S-32 S-33
Ki (nM) 3.3 ± 0.1 3.4 ± 0.1 ND 12.7 ± 0.03
ND, not determined.
Ki was calculated by Ki = Kd × IC50/(Kd + L), where Kd was the equilibrium
dissociation constant of radiolabeled MIB (Kd = 0.19 nM; determined in
preliminary experiments) and L was the concentration of [3H] MIB used in the
experiment (L=1 nM).
30 JPET Fast Forward. Published on January 24, 2006 as DOI: 10.1124/jpet.105.094334 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 25, 2021 JPET Fast Forward. Published on January 24, 2006 as DOI: 10.1124/jpet.105.094334 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 25, 2021 JPET Fast Forward. Published on January 24, 2006 as DOI: 10.1124/jpet.105.094334 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 25, 2021 JPET Fast Forward. Published on January 24, 2006 as DOI: 10.1124/jpet.105.094334 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 25, 2021 JPET Fast Forward. Published on January 24, 2006 as DOI: 10.1124/jpet.105.094334 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 25, 2021