442 Vol. 36, No. 3Biol. Pharm. Bull. 36(3) 442–451 (2013)

© 2013 The Pharmaceutical Society of Japan

Regular Article

Mechanism of the Tissue-Specific Action of the Selective Androgen Receptor Modulator S-101479Kazuyuki Furuya,*,a,b Noriko Yamamoto,a Yuki Ohyabu,a Teruyuki Morikyu,a Hirohide Ishige,a Michael Albers,c and Yasuhisa Endob

a Central Research Laboratories, Kaken Pharmaceutical Co., Ltd.; 14 Shinomiya, Minamigawara-cho, Yamashina-ku, Kyoto 607–8042, Japan: b Division of Applied Biology, Kyoto Institute of Technology Graduate School of Science and Technology; Matsugasaki Gosyo Kaido-cho, Sakyo-ku, Kyoto 606–8585, Japan: and c Phenex Pharmaceuticals AG; Donnersbergweg 1, -67059 Ludwigshafen, Germany.Received October 8, 2012; accepted December 20, 2012

Selective androgen receptor modulators (SARMs) comprise a new class of molecules that induce ana-bolic effects with fewer side effects than those of other anabolic agents. We previously reported that the novel SARM S-101479 had a tissue-selective bone anabolic effect with diminished side effects in female animals. However, the mechanism of its tissue selectivity is not well known. In this report, we show that S-101479 increased alkaline phosphatase activity and androgen receptor (AR) transcriptional activity in osteoblastic cell lines in the same manner as the natural androgen ligand dihydrotestosterone (DHT); conversely, stimula-tion of AR dimerization was very low compared with that of DHT (34.4%). S-101479 increased bone mineral content in ovariectomized rats without promoting endometrial proliferation. Yeast two-hybrid interaction as-says revealed that DHT promoted recruitment of numerous cofactors to AR such as TIF2, SRC1, β-catenin, NCoA3, gelsolin and PROX1 in a dose-dependent manner. SARMs induced recruitment of fewer cofactors than DHT; in particular, S-101479 failed to induce recruitment of canonical p160 coactivators such as SRC1, TIF2 and notably NCoA3 but only stimulated binding of AR to gelsolin and PROX1. The results suggest that a full capability of the AR to dimerize and to effectively and unselectively recruit all canonical cofactors is not a prerequisite for transcriptional activity in osteoblastic cells and resulting anabolic effects in bone tis-sues. Instead, few relevant cofactors might be sufficient to promote AR activity in these tissues.

Key words selective androgen receptor modulator; dimerization; cofactor; osteoblast; bone

Androgens are both sex steroid hormones and anabolic hor-mones. The beneficial anabolic effects of androgens on vari-ous tissues such as bone, muscle, and red blood cells are well known.1,2) However, the clinical use of androgens has been limited because of their undesirable virilizing and metabolic actions. Their effect on the prostate gland is an extremely serious side effect because it may increase the risk of pros-tate cancer or benign prostate hyperplasia.3) The side effects of androgens are not lethal in women, but many virilizing effects (e.g., hirsutism, voice change, and acne) have been observed.4,5) Anabolic steroids were developed to reduce the side effects of androgens and to treat osteoporosis. However, their actions were not sufficient to reduce virilizing effects, and they exhibited hepatotoxic activity.6,7) Recently, the ac-tions of non-steroidal selective androgen receptor modulators (SARMs) have been investigated in many laboratories.8–12) We have focused on bone anabolic SARMs to develop anti-osteoporosis agents without serious virilizing effects.

We previously reported S-40503 and S-101479 as non-ste-roidal SARMs.8,13) S-101479 exhibited bone-anabolic activity similar to the natural androgen 5α-dihydrotestosterone (DHT) and reduced side effects on the uterus and clitoral gland in ovariectomized (OVX) rats. S-101479 exhibited beneficial effects in both single or combination treatments with commer-cial anti-osteoporosis drugs such as alendronate, raloxifene, and teriparatide. S-101479 may be a promising drug for treat-ing postmenopausal osteoporosis without serious androgenic side effects. However, the mechanism of its tissue selectivity has not been elucidated.

The androgen receptor (AR) is a member of the nuclear receptor superfamily, and is activated by natural androgens such as testosterone or DHT. Androgen binding promotes conformational changes in the AR resulting in homodimeriza-tion to its activated form and subsequent translocation into the nucleus. In the nucleus, the activated AR interacts with androgen response elements in the promoter regions of andro-gen target genes and modulates transcription.14,15) At that time, the numerous cofactor proteins (coactivators and corepressors) that mediate the transcriptional response should be recruit-ed.16) Although DHT maximally activates the full repertoire of androgen-responsive genes, synthetic AR ligands range from full agonists to complete antagonists depending on the gene and cellular context.17,18) These ligands exert complex activi-ties presumably by differentially modulating the structure of AR cofactor recruitment sites such as the N-terminal-located activation function-1 (AF-1) and the C-terminal-located ac-tivation function-2 (AF-2). The estrogen receptor can recruit different complexes of cofactors to either stimulate or repress the expression of target genes depending on the properties of ligands, the availability of cofactors in a given cell, and chro-matin contexts.19–21) The tissue-selective activities of SARMs may also result from the ligand-dependent, tissue-selective recruitment of cofactors to the AR transcription complex. It has been reported that the tissue selectivity of the SARMs MK-0773 and 2-FPA are inversely correlated with the recruit-ment of the cofactor TIF-2.22)

In this report, we studied the tissue-selective mechanism of S-101479. Firstly, we demonstrated the direct effects of S-101479 on osteoblast differentiation by using MC3T3-E1 cells, a preosteoblastic cell line established from the calvaria

* To whom correspondence should be addressed. e-mail: furuya_kazuyuki@kaken.co.jp

The authors declare no conflict of interest.

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of neonatal mice.23) Next we compared the AR dimerization activities of several SARMs and their AR transcriptional ac-tivity in the human osteoblastic cell line TE-85, which was derived from the sarcoma of a Caucasian female.24) We also evaluated the in vivo tissue selectivity of these agents in OVX rats. Finally, we characterized these SARMs with respect to their influence on AR cofactor recruitment by using the yeast two-hybrid (Y2H)-system.

MATERIAlS AND METHODS

Compounds All reagents were obtained from Na-calai Tesque (Kyoto, Japan) unless stated otherwise. N-(2-((3aS,4S,9bS)-8-cyano-1-formyl-2,3,3a,4,5,9b-hexahydro-1H-py r rolo [3, 2 - c] qu i nol i n - 4 -yl ) -2 -me t hylp ropyl) - 4 ,6 -difluorobenzofuran-2-carboxyamide (S-101479), (E)-N-(2-((3aR,4S,9bS)-8-cyano-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]-quinolin-4-yl)-2-methylpropyl)-3-(pyridin-2-yl) acrylamide (S-49288), BMS-564929,9) and hydroxyflutamide (Flu-OH) were synthesized at Kaken Pharmaceuticals. Bicalutamide was extracted from Casodex tablets (AstraZeneca K.K., Osaka, Japan). DHT was obtained from Wako Pure Chemical In-dustries, ltd. (Osaka, Japan). All compounds were dissolved in dimethyl sulfoxide (DMSO) and diluted with buffer for in vitro assays. S-101479, S-49288, and BMS-564929 were sus-pended in 0.5% methylcellulose #400 (MC) for in vivo oral treatment. DHT was dissolved in DMSO and diluted 10-fold with olive oil for in vivo subcutaneous injection. Chemical structure of compounds (AR agonists and SARMs) were shown in Fig. 1.

Osteoblastic Differentiation Assays Phenol red–free minimum essential medium α (α-MEM) and fetal bovine serum (FBS) were purchased from Gibco (Carlsbad, CA,

U.S.A.). MC3T3-E1, an AR-expressing preosteoblastic cell line, was obtained from Riken (Tsukuba, Japan). MC3T3-E1 cells were cultured in α-MEM supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin (Invitrogen, Carlsbad, CA, U.S.A.), and 10% FBS. MC3T3-E1 cells were seeded at 10000 cells/well (24-well culture plate) in α-MEM supple-mented with 10% charcoal-stripped FBS. On day 4 post-plating, the medium was replaced with the aforementioned medium supplemented with 50 µg/ml ascorbic acid (Wako Pure Chemical Industries, ltd.) and 10 mm β-glycerol phos-phate (Sigma-Aldrich, St. louis, MO, U.S.A.). Then, DHT, S-101479, Flu-OH, or vehicle was added. Seeding of the cul-ture plate was considered as day 0 time point. Medium was replaced every 3–4 d. On day 10, half of the wells were as-sayed for alkaline phosphatase (AlP) activity.25) The cells in each well of the 24-well culture plates were rinsed once with phosphate-buffered saline (PBS). Then, 0.3 ml of 1.33 mg/ml solution of AlP substrate (p-nitrophenyl phosphate, Sigma-Aldrich) in a buffer consisting of 50 mm Tris–HCl saline (pH 9.0) was added per well. After a 30-min incubation at 37°C, the solution was appropriately diluted and transferred to a 96-well culture plate for determination of absorbance at 405 nm on microplate reader (Multiscan Bichromatic, labsystems Oy, Helsinki, Finland). A standard curve generated from a series of dilutions of p-nitrophenol (Sigma-Aldrich) was used to de-termine the concentration of the enzyme reaction product. On day 17, the remaining wells were assayed for mineralization (Ca2+ deposition).26) The presence of mineralized bone nodules in MC3T3-E1 cells was demonstrated using alizarin red stain-ing. Briefly, cells were rinsed with PBS, fixed for 30–60 min in 70% ethanol at 4°C, rinsed with distilled water, and stained for calcium deposition by shaking incubation with 40 nm aliza-rin red-S, pH 4.2, for 10 min at room temperature. The plates

Fig. 1. Chemical Structure of Test CompoundsDihydrotestosterone (DHT) is a natural ligand for the androgen receptor (AR). Stanozolol is a synthetic anabolic steroid derived from DHT. BMS-564929 is selective

androgen receptor modulator (SARM) that was developed by Bristol-Myers Squibb. S-49288 and S-101479 are bone anabolic SARMs that were discovered at Kaken Phar-maceuticals.

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were rinsed five times with distilled water. Experiments were performed in triplicate.

Reporter Assays Phenol red–free Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Sigma-Aldrich. Fetal bovine serum was purchased from Gibco. TE-85 human osteosarcoma cells were obtained from American Type Culture Collection (Manassas, VA, U.S.A.). Professor Kato (Tokyo University, Tokyo, Japan) kindly provided human an-drogen receptor (hAR) cDNA. The hAR expression plasmid was constructed by inserting hAR cDNA into the pBK-CMV (Clontech, Mountain View, CA, U.S.A.) multicloning site. TE-85 cells were stably transfected by the calcium phosphate coprecipitation procedure with a reporter construct, MMTV-luc, containing the mouse mammary tumor virus long termi-nal repeat linked to luciferase and the hAR expression plas-mid. TE-85 cells were cultured in DMEM supplemented with 10% charcoal-stripped FBS. Cells were seeded with or without the test compounds and incubated for 48 h before the measure-ment of luciferase activity. Experiments were performed in triplicate.

Mammalian Two-Hybrid Assays The AR dimerization assay, which measures the AR–AR interaction, was evaluated by a checkmate™ mammalian two-hybrid system (Promega, Fitchburg, WI, U.S.A.) in 293T cells (American Type Culture Collection). The Gal4-DNA binding domain was fused with the full-length (Fl) hAR (pGal-hAR vector), and the VP16A activation domain was fused to the Fl hAR (pVP16-hAR vector). The luciferase reporter plasmid with five copies of GAl-4 binding sites (pG5luc vector) was stably transfected by the calcium phosphate coprecipitation procedure into 293T cells with the pGal-hAR and pVP16-hAR vectors. AR di-merization activity was evaluated as a ligand-dependent dose response. The measurement procedure of AR dimerization as-says was the same as that of the reporter assays.

Animal Studies In the experiments described below, 12-week-old female Sprague-Dawley (SD) rats were used. These rats were bilaterally OVX or sham-operated under ether anesthesia. After surgery, all rats were maintained without treatment for four weeks to permit bone loss before the be-ginning of bone anabolic therapy (as in established osteopo-rosis). During this period, standard chow was replaced with a modified American Institute of Nutrition diet containing 0.4% calcium and 0.5% phosphate (Oriental Yeast Co., Tokyo, Japan) to continue bone loss more progressively. Thereafter, OVX animals were regrouped into experimental groups be-fore the start of 8-week treatments. Each group comprised 10 animals. Initial body weights were equalized among treatment groups. The vehicle (0.5% MC) was administered to sham rats. S-49288, S-101479, BMS-564929, and vehicle were orally administered once daily in a volume of 5 ml/kg BW. The vehicle (10% DMSO, 90% olive oil) and DHT were injected subcutaneously once daily in a volume of 1 ml/kg BW. At the end of the experiment, the rats were euthanized by exsangui-nation from the abdominal aorta. After the 8-week treatment, the uteri were excised and weighed immediately. Right fe-murs were dissected free of the soft tissue and fixed in 100% ethanol to measure the bone mineral content (BMC). BMC was measured by dual-energy X-ray absorptiometry utilizing a bone mineral analyzer (DCS-600EX-IIIR; Aloka, Tokyo, Japan). Statistical analyses were performed using the SPSS Base 11.0J software program (SPSS, Chicago, Il, U.S.A.). All

data are presented as the mean and standard deviation (S.D.). The difference was evaluated by the Student’s t-test when comparing two groups or by one-way analysis of variance fol-lowed by the Dunnett’s test for multiple groups. p<0.05 was considered statistically significant. The experimental protocol was approved by the Institutional Animal Care and Use Com-mittee at Kaken Pharmaceutical Co., ltd.

Cofactor Profiling The yeast two-hybrid system as applied was based on Clontech’s Matchmaker system. A pGBT9-based vector expressed the AR or cofactors C-termi-nally fused to the DNA binding domain (BD) of the GAl4 transcription factor (referred to as Bait-fusion proteins). A pGAD424-based vector expressed the AR or the cofactor C-terminally fused to the transcriptional activation domain (AD) of the GAl4 transcription factor (referred to as Prey-fusion proteins). The Bait expression plasmids were transformed into yeast strain AH109a and the Prey expression plasmids into strain Y187alpha using a standard lithium acetate/polyethylene glycol (PEG)/heatshock procedure. Yeast strains were grown and mated by mixing equal volumes of the haploid yeast strains in selective medium supplemented with 0.5 volumes of rich medium for 5–7 h. After mating, a small aliquot of the yeast cell suspension was transferred into medium selecting for diploid cells and grown to saturation. This step was re-peated once and the cells were then diluted in medium select-ing for interacting clones. The medium contained a fluorigenic substrate (50 µm 4-methylumbelliferyl-alpha-d-galactoside (Sigma-Aldrich, M-7633)) and the compound to be tested for its influence on the interactions or a vehicle control (DMSO). Fluorescence was measured after a 24- or 48-hours incuba-tion. The experiments were performed in quadruplicate.

The DHT, S-49288, S-101479, BMS-564929, and bicalu-tamide were titrated in eleven 1 : 3 dilution steps from the highest concentration. The yeast cells expressing the interac-tion partners were incubated for 24 h with the compounds, fluorescence was measured, and cells were incubated for an additional 18 h until a second measurement was taken (42 h time point). In Fig. 5 only the time point yielding the larg-est assay windows is depicted. In this assay, the data for the highest doses were neglected. likely for reasons of poor com-pound solubility these data were unreliable.

Area under the curve values (AUCs) were calculated using the GraphPad Prism5 software (GraphPad Software, San Diego, CA, U.S.A.). Prior to calculation compound inde-pendent vehicle values (backgrounds) were subtracted. AUC values below 200 were set to zero because these were not regarded as significant peaks. The calculated AUCs were used to cluster the compounds based on their AR-cofactor profiles.using the “gene expression and functional analysis suite” BABElOMICS4 (http://babelomics.bioinfo.cipf.es; Settings: Method: upgma; Distance: pearson). The algorithm clusters the compounds with focus on the overall pattern of recruit-ment (i.e. the rank order) not with focus on similarities/dif-ferences in absolute values (Red cells indicate high-blue cells low AUC values).

RESUlTS

Osteoblastic Differentiation To demonstrate the effects of S-101479 on osteoblastic differentiation, we examined AlP activity, which is a marker for osteoblastic differentiation and

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Ca2+ deposition (mineralization) in MC3T3-E1 cells. DHT increased AlP activity in a concentration dependent man-ner compared with the effects of the vehicle control on day 10 post-plating. S-101479 also increased AlP activity in a concentration dependent manner compared with the effects of the vehicle control. The anti-androgen Flu-OH (1000 nm) significantly inhibited the ALP activity of S-101479 except at a concentration of 1000 nm S-101479 (Fig. 2A). To identify nodule formation after AlP activity was increased, MC3T3-E1 cells were stained with alizarin red on day 17. The vehicle control group did not display any nodule formation. DHT and S-101479 (333 nm) increased the number of nodules (Fig. 2B).

AR Transactivation and AR Dimerization Activity In this experiment, we compared the ability of SARMs and DHT to stimulate AR transcription and dimerization. Chemical structure of compounds were shown in Fig. 1. DHT exhibited a concentration dependent increase in AR transcriptional ac-tivity in the human osteosarcoma cell line TE-85. S-101479 also increased AR transcription in a concentration-dependent manner, and its maximal transactivation relative to that of 1000 nm DHT was 83.0% (Fig. 3). The maximal activity of S-49288 was 64.7%. The anabolic steroid stanozolol and the other SARM BMS-564929, which has a different scaffold as S-101479, exhibited transcriptional activity as strong as that displayed by DHT. In the AR dimerization assay, DHT in-creased luciferase activity in a concentration dependent man-ner. This effect of DHT was not observed when the pGal-hAR or pVP16-hAR vector was transfected into 293T cells with the pG5luc vector (Fig. S1). The maximal activity of stanozolol and BMS-564929 was as strong as their AR transcriptional activity. The maximal activity of S-101479 and S-49288 was approximately 30% of DHT activity.

OVX Study: Dose–Response Study of SARMs To dem-onstrate the difference between the androgenic activity of DHT and SARMs, we administered these agents to OVX rats daily. We evaluated their effects on the bone by BMC. Bone mineral density (BMD) is widely used to evaluate osteoporotic bone, but BMC is a more important parameter than BMD when evaluating androgenic compounds as described previ-ously.13) The BMC of total femurs was decreased significantly by OVX in study 1. DHT increased BMC in a dose-dependent manner compared with the effects of the OVX vehicle (subcu-taneously (s.c.)). BMS-564929 also increased BMC in a dose-dependent manner compared with the effects of the OVX ve-hicle (Fig. 4A). In study 2, we omitted the sham vehicle (s.c.) and OVX vehicle (s.c.). In this study, the BMC of total femurs was decreased significantly by OVX. The positive control DHT increased BMC compared with the effects of the OVX vehicle. S-49288 and S-101479 increased BMC compared with the effects of the OVX vehicle in a dose-dependent manner (Fig. 4B).

The weight of the uterus was decreased by OVX in study 1. DHT and BMS-564929 increased uterine weight in a dose-de-pendent manner compared with the effects of the OVX vehicle (oral or subcutaneous); the effects of the highest doses were the same as that of the sham vehicle (Fig. 4A). In study 2, the weight of the uterus decreased significantly by OVX. The positive control DHT increased uterine weight compared with the effects of the OVX vehicle. S-49288 (3 mg/kg) increased uterine weight, but its effect was extremely different from that of DHT or BMS-564929. S-101479 did not increase uterine weight in this experiment (Fig. 4B) but 10 mg/kg S-101479 inceased uterine weight in another experiment.13)

Cofactor Profiling Firstly, approximately 180 nuclear

Fig. 2. The Alkaline Phosphatase (AlP) Activity (Day 10) and Bone Nodule Formation (Day 17) in MC3T3-E1 CellsMC3T3-E1 cells were seeded in 24-well culture plates. On day 10, half of the wells were assayed for AlP activity (A) as described in Materials and Methods. On day

17, the remaining wells were assayed for mineralization (Ca2+ deposition) (B). Results are means±S.D. (n=3). ** p<0.01 vs. control. # p<0.05 vs. S-101479. ## p<0.01 vs. S-101479. Flu-OH, 1000 nm hydroxyflutamide.

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Fig. 3. The Activity of Compounds in AR Transactivation and AR Dimerization AssaysAR transactivation activities of compounds were examined in osteoblastic cell line TE-85 cells (A) as described in Materials and Methods. luciferase activities are

shown relative to that of 1000 nm of DHT. AR dimerization activities of compounds were examined in 293T cells (B). luciferase activities are shown relative to that of 100 nm of DHT. Results are means±S.D. (n=3).

Fig. 4. Effects of Compounds on Femoral BMC and Uterus Weights in OVX RatsThe dose–response effects of BMS-564929, DHT (A), S-49288 and S-101479 (B) on BMC and uterine weight were examined. Vehicle, BMS-564929, S-49288, and

S-101479 were orally administered once daily for 8 weeks. Vehicle (s.c.) and DHT was injected subcutaneously once daily for 8 weeks. Results are means±S.D. (n=10 rats/group). ** p<0.01 vs. OVX vehicle. * p<0.05 vs. OVX vehicle. BMC, bone mineral content; OVX, ovariectomy; and s.c., subcutaneous.

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receptor cofactor constructs were tested at concentrations of 1 and 10 µm (Table S1, Figs. S2, S3). The influence of the compounds on interactions of cofactors with the full length AR Prey or AR ligand-binding domain (lBD) Bait are readily measurable. However, the full length AR Bait construct exhib-ited strong one-hybrid activity (Fig. S4). From these results, we selected several cofactors for dose–response studies: AR-lBD Bait with TIF-2 (aa548-878), SRC-1 (Fl), β-catenin (Fl), or PROX1 (Fl) as Prey; and AR-Fl Prey with NCoA3 (amino acid boundaries (aa) 391-748), Gelsolin (aa326-782) as Bait. In order to allow a quantitative comparison of compound induced cofactor recruitment we generated eleven point dose response curves for a subset of AR-cofactor interactions (Fig. 5).

By calculating the area under the curve (AUC) for each of the dose response curves from Fig. 5 and other cofactor profiling studies (Fig S5), one yields one value representing the effect of a compound on a given AR-cofactor interaction (Fig. 6A). Running a clustering algorithm on these data al-lows grouping of the compounds with respect to the cofac-tor recruitment patterns they induce. The heat map in Fig. 6B clearly shows the similarity of the SARMs S-101479 and S-49288 which are placed in close vicinity to each other and notably in distance to DHT and BMS-564929. Bicalutamide as an antagonist is placed some distance away from the other compounds, as expected. The full agonist DHT induces re-cruitment of all cofactors tested in this panel. BMS-564929

Fig. 5. Dose Dependent Influence of Test Compounds on Cofactor RecruitmentAR-cofactor interactions were tested in the dose–response mode. In the figure only the time point yielding the largest assay window is depicted. AR-LBD Bait with

TIF-2 (A), SRC-1 (B), β-catenin (C), or PROX1 (F) as Prey; and AR-Fl Prey with NCoA3 (D), or Gelsolin (E) as Bait. Results are means±S.D. (n=4). lBD, ligand bind-ing domain; FL, full-length; and RFU, relative fluorescence unit.

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also acts rather unselective, induces recruitment of most co-factors, interestingly however, with the exception of the p160 cofactors SRC1 and TIF2. Notably, for S-101479 and (to a less-er extent) S-49288 we observe a much more selective cofactor recruitment. Only gelsoline, ARA54 and PROX1 are recruited in presence of S-101479, but not the classical steroid recep-tor coactivators SRC1, TIF2 and NCoA3 and not β-catenin, the orphan receptor DAX1, FKHR, and PRPF6. Noteworthy might be the lack of β-catenin recruitment as well as the lack of or the very poor recruitment of NCoA3 by S-101479 and S-49288, respectively. Both cofactors are clearly recruited in presence of DHT and BMS-564929.

DISCUSSION

The role of androgen/AR signaling in osteoblasts is relative-ly unclear, but the importance of androgens in bone metabo-lism is well recognized. AR is expressed in both osteoblasts and osteocytes.27,28) S-101479 exhibited the same effect as

DHT on osteoblast differentiation, and this effect was inhib-ited by the anti-androgen Flu-OH. S-101479 also displayed AR transcriptional activity in the human osteoblastic cell line TE-85. These data suggested that the effects of S-101479 on osteoblasts represent androgenic actions and not the intrinsic activity of S-101479. Additionally, these findings suggested that S-101479 had direct bone anabolic effects in OVX rats, opposed to an indirect action such as mechanical stimulation via muscle increase.

Conversely, the effects of S-101479 on AR dimerization as evaluated by a mammalian two-hybrid assay were very dif-ferent from those of DHT. The maximal effect of S-101479 was only approximately 30% that of DHT. This response was also observed with S-49288, which possesses the same tetra-hydroquinoline structure as S-101479. In addition, S-101479 could compete with DHT in these dimerization-experiments. 1000 nm S-101479 could completely reverse the dimerization effects of DHT (Fig. S6). This hints that S-101479 occupied AR but its maximal efficacy on stimulating dimerization

Fig. 6. Clustering of Compounds for Their AR-Cofactor Profiles Based on Calculated AUCsAUCs were calculated from the dose response curves (A). AUC values below 200 were set to zero (indicated in italics) because these were not regarded as significant

peaks. Clustering of compounds for their AR-cofactor profiles based on calculated AUCs (B). Red cells indicate high-blue cells low AUC values relative for each com-pound. AUC, area under the curve; β-CAT, β-catenin; b, bait; and p, prey.

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is only marginal as compared to DHT. The other SARM BMS-564929 and anabolic steroid stanozolol displayed ef-ficacies differing from S-101479 or S-49288. Although BMS-564929 and stanozolol required higher concentrations than DHT to induce maximal response, their effects were ap-proximately equal to those of DHT.

In the protein expression process, the homodimerization of AR is an early step, and protein expression is the final step of this process. S-101479 and S-49288 displayed a unique profile; they had strong activity in the last step of protein expression in osteoblastic cell lines despite exhibiting low activity in the early step (AR dimerization). BMS-564929 and stanozolol also exhibited the same profiles as S-101479 and S-49288 at low concentrations, but the effects of BMS-564929 and stanozolol on AR transcription and dimerization reached approximately 100% at high concentrations. ligand-dependent AR dimeriza-tion (interaction between the N- and C-terminal domains) is needed for optimal AR function.29,30) AR point mutations were found in human androgen insensitivity syndrome patients who did not exhibit complete virilization. Many of these mutant ARs are defective in AR dimerization.31,32) Our study revealed that low AR dimerization activity was related to low effects on the uterus in vivo. S-101479 and S-49288 increased BMC to the same extent as DHT, but their effects on the uterus were extremely different in OVX rats. BMS-564929 displayed tissue selectivity at the dose of 0.01 mg/kg. However, higher doses of BMS-564929 increased uterine weight, and tissue selectivity was not observed. Stanozolol also exhibited tissue selectivity at lower doses and similar to BMS-564929, tissue selectivity was not observed at higher doses (Fig. S7). Merck Research laboratories also demonstrated that the interaction between the N-terminal and C-terminal domains of AR af-fected the tissue selectivity of SARMs that had different scaf-folds than S-101479.22) The difference between effects on AR transcription and dimerization activity of SARMs could be an index marker of their tissue selectivity.

Although low AR dimerization activity was adherent with low effects on reproductive organs, the reason why S-101479 and S-49288 could exhibit bone anabolic effects in vitro (os-teoblastic differentiation) and in vivo (BMC increased) was not known. We hypothesized that S-101479 could induce AR tran-scription in osteoblasts without full AR dimerization because of its distinct cofactor profile (cofactor interaction pattern) as compared to DHT. In fact, the cofactor recruitment profile in-duced by S-101479 turned out to be very different from that of DHT. The tissue-selective expression or function of coactiva-tors and corepressors was described earlier as a mechanism of selective nuclear receptor modulation.33) For example, a study comparing the effects of estradiol to the selective estrogen re-ceptor modulators (SERMs) tamoxifen and raloxifene revealed that differences in the selective recruitment of SRC1 directly translated into gene-selective and tissue-selective effects.34) In a SARM study, it was demonstrated that AF-2-mediated TIF2 recruitment was related with tissue selectivity.22) Our data also support these reports. The cofactor profiles of the SARMs studied here were very different from that of DHT. In par-ticular, the profile of S-101479 was very selective, as S-101479 failed to recruit important AR cofactors such as NCoA3 and related p160 cofactors. In this experiment, we detected the cofactors that directly bind to AR; thus, there is a possibility that these cofactors indirectly bind to AR within a cofactor

complex. However, DHT recruited them directly under the same experimental conditions.

We further examined the cofactor profiles of S-101479 in detail. Although the transcriptional activity of S-101479 in yeast cells was not dramatically lower than that of DHT (Fig. S4), the only cofactors recruited in presence of S-101479 were gelsolin and PROX1. However, the bone anabolic activity of S-101479 was equal to or greater than that of DHT. This data indicated that the unbound cofactors are dispensable for the effect on bone. If S-101479 had original activity or a non-genomic action in bone, then it did not need similar cofac-tor recruitment as DHT. However, S-101479 did not display any intrinsic activity in any experiment, and it induced AR transcription (genomic action) in osteoblasts. S-49288 and BMS-564929 also recruited fewer cofactors than DHT. Inter-estingly, β-catenin was not recruited by S-101479 or S-49288. The interrelation of the classical AR and the Wnt/β-catenin signaling pathways has been described in numerous reports. The direct interaction between AR and β-catenin as well as the potential of the protein-complex to colocalize to the nucleus has been demonstrated.35,36) Wnt signaling plays an important role in the development and maintenance of bone tissue. Androgens promote preosteoblastic cell differentiation via effects on the canonical Wnt/β-catenin signaling path-way.37–39) Our results did not indicate that the Wnt/β-catenin signaling pathway was necessary for osteoblastic differentia-tion. However, the direct interaction of β-catenin with AR was not required. Strikingly, the canonical p160 coactivators SRC1, TIF2 and NCoA3 are not or only very marginally recruited in presence of the SARMs S-101479 and S-49288. Notably, BMS-564929 also fails to support SRC1 and TIF2 recruitment but in contrast to the other SARMs clearly, as DHT, promotes NCoA3 binding to AR. It is intriguing to hypothesize, that the ability to recruit NCoA3 is a prerequisite of AR activat-ing agents to stimulate proliferating pathways in the uterus, because both S-101479 and S-49288 fail to increase uterine weight in OVX rats, as opposed to BMS-56929 and DHT. The distinguished role of NCoA3 (AIB1; amplified in breast cancer 1) in cell proliferation and the development and maintenance of several cancers (including endometrial tumors) has been discussed in hundreds of publications.40,41)

In contrast, gelsolin and PROX1 were recruited by all test compounds excluding bicalutamide. Gelsolin is a multifunc-tional actin-binding protein that has been implicated in cell motility, signaling, apoptosis, and carcinogenesis.42,43) Gelsolin regulates the polymerization/depolymerization of actin and the gelsol state alternation of the cytoplasm.44) Gelsolin might be required for cell migration and adhesion. PROX1 is a homeo-box domain 1 transcription factor that plays key roles during the development of diverse organ systems. The use of PROX1 to regulate developmental decisions is conserved through-out evolution. PROX1 was essential for the formation and maintenance of the entire lymphatic vasculature.45) However, there are no reports about PROX1 expression in osteoblasts or osteoclasts. Although we must evaluate the suppression of these cofactors by siRNA, gelsolin and PROX1 may not be key cofactors of bone anabolism. In this study, we could not find a cofactor that was recruited only by SARMs, and many cofactors that were recruited by DHT were not necessary for the bone anabolic effects of androgens. The data suggest that the lower number of recruited cofactors without losing

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the effects on bone is important for the bone selectivity of SARMs. The lower AR dimerization activity could contribute to the reduced number of bound cofactors and in turn reduce the number or severity of side effects in reproductive organs.

Similar to other members of the nuclear receptor superfam-ily, the AR has two regions involved in transcriptional activa-tion: the hormone independent AF-1 in the N-terminal domain and the hormone-dependent AF-2 in the lBD (C-terminal domain).46–48) After AR dimerizaiton by ligand binding, the p160 coactivators such as SRC1, TIF2 and NCoA3 interact with AF-2 in the C-terminal domains, have histone acetyl-transferase activity, and form complexes with p300 and other coactivators with similar chromatin modifying activity.49,50) S-101479 did not recruit these cofactors, but it had strong bone anabolic effect in OVX rats.

From these findings, it looks as if S-101479 does not pro-mote bone anabolic effects via the classical AF-2 involving the canonical p160 cofactors. Our hypothesis is based on the involvement of AF-1. Several reports stress that unlike other steroid receptors, the AF-2 of AR is transcriptionally weak, and that the AF-1 is of high functional importance.48,51,52) Moreover, in studies on the estrogen receptor both transactiva-tion functions, AF-1 and AF-2, depend on promoter context and cell type53,54) and the AF-1 activity is considered to be suppressed by the ligand-unbound lBD but can be restored upon ligand binding.55,56) Maybe in bone tissue S-101479 as well as DHT are able to drive a similar interplay by binding to the AR-lBD and thereby activating AF-1. Putatively for endometrial proliferation the classical AF-2 activation plays the major role.

In this study, we investigated ligand-dependent interactions between the AR and cofactors, but we did not closely look on specific interactions with the AF-1. To clarify the importance of the AF-1 for tissue selective effects of S-101479 our future studies will involve the identification of AF-1-associated co-factors.

Acknowledgments The authors gratefully acknowledge Dr. Shigeaki Kato (Institute of Molecular and Cellular Bio-sciences, the University of Tokyo) for kindly preparing full-length wild-type human AR and Dr. Tsutomu Nakamura for his advice on this manuscript. I received generous support from Mr. Nobuhiro Kimura (CTC laboratory Sysytems Cor-poration).

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