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SERMs and SARMs: Detection of their activities with yeast based bioassays
Journal of Steroid Biochemistry & Molecular Biology 118 (2010) 85–92
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SERMs and SARMs: Detection of their activities with yeast based bioassays Toine F.H. Bovee a,∗ , Mario Thevis b , Astrid R.M. Hamers a , Ad A.C.M. Peijnenburg a , Michel W.F. Nielen a,c , Willem G.E.J. Schoonen d a
RIKILT-Institute of Food Safety, Department of Safety & Health, Bornsesteeg 45, 6708 PD Wageningen, The Netherlands Center for Preventive Doping Research – Institute of Biochemistry, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933 Cologne, Germany c Wageningen University, Laboratory of Organic Chemistry, Dreijenplein 8, 6703 HB Wageningen, The Netherlands d Schering-Plough Research Institute, Department of Toxicology and Drug Disposition, P.O. Box 20, 5340 BH Oss, The Netherlands b
a r t i c l e
i n f o
Article history: Received 11 June 2009 Received in revised form 21 October 2009 Accepted 22 October 2009 Keywords: Agonists Antagonists In vitro bioassays Androgenic activity Estrogenic activity Metabolism
a b s t r a c t Selective estrogen receptor modulators (SERMs) and selective androgen receptor modulators (SARMs) are compounds that activate their cognate receptor in particular target tissues without affecting other organs. Many of these compounds will find their use in therapeutic treatments. However, they also will have a high potential for misuse in veterinary practice and the sporting world. Here we demonstrate that yeast estrogen and androgen bioassays can be used to detect SERMs and SARMs, and are also useful screening tools to investigate their mode of action. Six steroidal 11-substituents of E2 (SERMs) and some arylpropionamide- and quinoline-based SARMs were tested. In addition, 7 compounds previously tested on AR agonism and determined as inactive in the yeast androgen bioassay, while QSAR modelling revealed strong binding to the human androgen receptor, are now shown to act as AR antagonists. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction Compounds with hormonal activities are widely used in medical practice. Human estrogen receptor ␣ (hER␣) agonists are used for birth control and the treatment of osteoporosis in postmenopausal women [1]. Human androgen receptor (hAR) agonists can be of interest in case of delayed puberty, libido loss, impotence, or muscle weakness [2]. The decrease in the endogenous production of hormones like 17-testosterone (T), 17-estradiol (E2), growth hormone, insulin, and insulin like growth factor 1 is the major cause of these health problems [2,3]. Hormone replacement therapies (HRT) with either estrogens or androgens however may cause severe side effects and more specific drugs are needed [4–6]. Antiestrogens and anti-androgens, in turn, are very important in the treatment of certain tumours. The pure anti-estrogens ICI 182,780 (fulvestrant) and RU 58668 are successfully used to treat breast cancer patients. The pure anti-androgens, flutamide and bicalutamide (Casodex), are proven to be useful in curing prostate cancer patients [7,8]. Selective estrogen receptor modulators (SERMs) and selective androgen receptor modulators (SARMs) are compounds that activate their cognate receptor in particular target tissues without affecting other organs. Such new drugs can be very beneficial alternatives for HRT. Tamoxifen (TAM), chlomiphene citrate, and
∗ Corresponding author. Tel.: +31 317 480391; fax: +31 317 417717. E-mail address: [email protected] (T.F.H. Bovee). 0960-0760/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2009.10.007
raloxifen are the best known SERMs with respect to breast specific anti-estrogens [9,10]. Unfortunately, TAM acts as an ER agonist on the uterus and prolonged use is associated with the development of endometrial cancer [11]. SARMs like bicalutamide (Casodex), flutamide and nilutamide are known as non-steroidal anti-androgens and used to treat benign prostate hyperplasia and prostate cancer [12,13]. The search for new SERMs and SARMs as alternatives for HRT or breast and prostate cancer treatment is one of the major topics within the pharmaceutical industry. A new generation of SARMs is being developed for androgen replacement. These new SARMs should reduce the unwanted side effects occurring with particular synthetic 17␣-substituted derivatives of T, as these latter compounds induce liver cholestasis, prostate hypertrophy, and acne [14–16]. Ideally these new SARMs must act as full agonists, like T, in anabolic target tissues, e.g. muscle and bone, but should demonstrate only partial or pure AR antagonistic activities on prostate tissue. This can partly be obtained by the inactivation or inhibition of 5␣-reductase [15]. However, especially these SARMs also will have a high potential for misuse in veterinary practice and the sporting world. Screening methods to detect these new compounds are required to ensure food safety and fair play. Previously we described the development of yeast based estrogen and androgen bioassays. These bioassays were fully validated for screening animal feed and calf urine samples for the presence of natural and synthetic steroids [17]. Both bioassays were shown to be highly specific for their cognate (ant)agonistic compounds, which made them also suited to determine the (anti)estrogenic and
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Fig. 1. Chemical name and structures of the 11-substituents towards 17-estradiol.
(anti)androgenic properties of plant-derived compounds [18,19]. The aim of the present study was to determine whether new SERMs and SARMs can also be detected with respect to their specific hormonal activities in these yeast transcription activation assays. The 6 SERMs tested were derived from the steroid E2 and contained 11-substituents with a variation in the carbon length from 4 to 6 atoms. The 6 non-steroidal SARMs tested were 3 arylpropionamide and 3 quinoline-based compounds. These SERMs and SARMs are commercially not available, have never been tested in yeast based bioassays before and little information, especially for the nonsteroidal SARMs, is available. The ability of the yeast androgen bioassay to detect the known non-steroidal pure anti-androgens bicalutamide, flutamide, hydroxyflutamide, and nilutamide was therefore investigated first, while the steroidal AR agonists oxandrolone, desoxymethyltestosterone (DMT), and fluoxymesterone and the steroidal AR antagonist cyproterone acetate (CPA) were tested in order to further prove the specificity of the yeast androgen bioassay. Finally, 7 compounds, previously tested in the yeast androgen bioassay and being inactive as AR agonists, were now investigated on their anti-androgenic properties, as our earlier QSAR modelling results revealed strong binding affinities towards hAR [20].
gen biosensor were grown overnight at 30 ◦ C with vigorous orbital shaking. At the late log phase, the culture was diluted in selective MM/L medium till an OD value at 630 nm between 0.04 and 0.06 was reached. For exposure, aliquots of 200 l of the diluted yeast culture were pipetted into each well of a 96-well plate and 2 l of a stock solution in DMSO was added to test the agonistic properties of the compounds. To test the anti-estrogenic and anti-androgenic properties of compounds, 1 l amounts of the compound’s stock solutions were co-exposed with 1 l of E2 or T stock solutions, respectively. Two doses of both E2 and T were used, giving a half-maximal response or a near maximal response in the yeast estrogen and androgen bioassay, respectively. DMSO, E2, and T controls were included in each experiment and each sample concentration was assayed in triplicate. Exposure was performed for 24 h at 30 ◦ C and orbital shaking with 125 rpm. Fluorescence and OD were measured at 0 and 24 h directly in a SynergyTM HT Multi-Detection Microplate Reader (BioTek Instruments Inc., USA) using excitation at 485 nm and emission at 530 nm. The fluorescent signal was corrected with the signals obtained the DMSO control. Densities of the yeast culture were determined by measuring the OD at 630 nm to check whether the yeast cells were growing, in order to determine whether a sample concentration was toxic for the yeast cells.
2. Materials and methods 2.3. Chinese hamster ovary (CHO) cell ER˛ transactivation assay 2.1. Chemicals Dehydroepiandrosterone (DHEA), dexamethasone, 17estradiol (E2), flutamide and nilutamide were obtained from Sigma. Cyproterone, epiandrosterone (5␣-androstane-3-ol17-one), epietiocholanolone (5-androstane-3-ol-17-one), 17␣-ethinylestradiol (EE), 17-testosterone (T), and 5␣androstane-3-17␣-diol were obtained from Steraloids, dimethyl sulfoxide (DMSO) from Merck, and 16-stanozolol from NMI (Australia). The six 11-substituted E2 compounds (see Fig. 1 for their structures), fluoxymesterone, oxandrolone, hydroxyflutamide, bicalutamide (Casodex), and cyproterone acetate (CPA) were obtained from Schering-Plough (Oss, The Netherlands). The 3 arylpropionamide and 3 quinoline-based SARM compounds were synthesised at the Centre for Preventive Doping Research – Institute of Biochemistry, German Sport University (Cologne, Germany) and were previously described in detail [15,21]. Their structures are given in Fig. 5. Also the desoxymethyltestosterone (DMT) was obtained from the German Sport University and kindly provided by Prof. W. Schänzer. Chemicals to prepare the growth media and the preparation of the growth media for yeast cells were as described previously [19]. 2.2. Yeast estrogen and androgen bioassays with fluorescence measurement The estrogenic, anti-estrogenic, androgenic, and antiandrogenic properties of the compounds were tested as described previously [19]. In short, cultures of the yeast estrogen and andro-
For transactivation studies the stably transfected CHO cells were used according to the procedures described by Schoonen et al. [22]. These CHO cells were cultured in DMEM/HAM F12 medium supplemented with 5% defined bovine calf serum from Hyclone (UT, USA) at 37 ◦ C in Roux flasks (175 cm3 ) and flushed with 5% CO2 in air until pH 7.2–7.4 was reached. Complete medium was refreshed every 2–3 days. These CHO cells express luciferase upon exposure to estrogens. 3. Results The structures of six 11-substituents of E2 are presented in Fig. 1 and the responses of these SERMs when tested in the yeast estrogen bioassay, expressing human estrogen receptor ␣ (hER␣) and yeast enhanced green fluorescence protein (yEGFP) in response to estrogens, are shown in Fig. 2. All six 11-substituted compounds show an agonistic response and five are active as real partial anti-estrogens. E2 with the 11-hydroxypentyl substituent gave only an agonistic effect (90% of E2 being 100%), while a decrease from agonistic activity towards 22% was found in the sequence of 11-3-cyclopropylidenepropyl (65%), 11pentenyl (65%), 11-pentyl (53%), 11-butyl (37%), and 11-n-yl carbon chain (22%). The 11-hydroxypentyl compound is with respect to potency the weakest estrogen receptor agonist, demonstrated by its EC50 of 153 nM, followed directly by the 11-butyl compound. Than with a 10-fold higher potency the 11-pentyl and 11-pentenyl estrogens were found, while the 11-carbon chain and 11-3-cyclopropylidenepropyl were with EC50 values
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Fig. 2. Responses of 11-substituents of estradiol in the yeast estrogen bioassay. (a–f) The responses of six 11-substituents of E2 alone () and by co-administration of 1 nM E2 () or 3 nM E2 (). In addition (a) the response of E2 (). Fluorescence signals are the mean of a triplicate with SD.
of, respectively 7 and 8 nM the most potent agonists. As already described above E2 with the 11-hydroxypentyl substituent did not show antagonism, even at the level of 3000 nM, while the 11-butyl E2 was the next weakest anti-estrogen. Than the ranking of the four other compounds for anti-estrogenic activity was from 75 to 17 nM as follows: 11-pentenyl, 11-pentyl, 11-3cyclopropylidenepropyl, and 11-n-yl carbon chain. Comparing the dose–response curves for these substituents demonstrates that an increasing chain length on the 11 side chain, from
butyl to n-yl, increases the antagonistic potency and more or less also the agonistic potency of the compound in yeast. Table 1 shows a summary of these yeast results as well as of the transactivation data as obtained with a CHO cell line. In the CHO cells the 11-butyl (70%) and 11-3-cyclopropylidenepropyl (64%) substituted compounds were the strongest partial agonists, followed by an increasing weaker effect from 55% to 14% from 11-pentenyl, 11-pentyl, 11-hydroxypentyl, and 11-nyl carbon chain. With respect to antagonistic activities in CHO
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Table 1 The estrogenic and anti-estrogenic properties of E2 with various 11-substituents in yeast and CHO assays. Compounds
Yeast hER␣ a
E2 +11-Butyl +11-Pentyl +11-n-yl +11-Pentenyl +11-HO-pentyl +11-3-Cyclopropylidenepropyl
CHO hER␣ a
b
b
Agonist (nM)
Antagonist (nM)
Ratio (ago/ant)
Efficacy (%)
Agonist (pM)
Antagonist (pM)
Ratio (ago/ant)
Efficacy (%)
1 63 12 7 11 153 8
>1000 600 60 17 75 >3000 50
0.001 0.11 0.20 0.41 0.15 0.05 0.16
100 37 53 22 65 90 65
50 200 227 53 93 36 42
>1000 2100 2830 3230 2700 1270 5400
0.001 0.095 0.080 0.016 0.034 0.028 0.008
100 70 46 14 55 15 64
a The EC50 and IC50 values for agonistic and antagonistic human estrogen receptor ␣ activities, respectively of E2 and E2 with various 11-substituents in yeast and CHO assays. b The ratio for agonism and antagonism as well as the overall agonist efficacy is given for these compounds compared to the maximal response caused by E2 (E2 = 100%).
Fig. 3. Anti-androgenic responses in the yeast androgen bioassay of 7 compounds that show strong binding to the hAR according to a QSAR model. The AR antagonist activities of EE, DHEA, 5␣-androstane-3-ol-17-one (5␣-3-ol-17-one), 5-androstane-3-ol-17-one (5-3-ol-17-one), 5␣-androstane-3,17␣-diol (5␣3,17␣-diol), dexamethasone (Dex) and 16-hydroxystanozolol (16-OH-Stan) are examined by co-administration of 70 nM T (). In addition, the agonistic response of T () is shown. Fluorescence signals are the mean of a triplicate with SD.
cells these compounds were active in the range of 1.27 to 5.4 nM. Fig. 3 shows the anti-androgenic responses of 7 compounds that were not active as AR agonists in the yeast androgen bioassay [20]. The data in Fig. 3 show that 6 of these 7 com-
pounds, namely 5␣-androstane-3-ol-17-one, 5-androstane3-ol-17-one, 5␣-androstane-3,17␣-diol, 17␣-ethinylestradiol (EE), dehydroepiandrosterone (DHEA), and 16-hydroxystanozolol (16-OHStan) showed a clear antagonistic response in the yeast androgen bioassay. Both 5␣-androstane-3-ol-17-one and 16hydroxystanozolol also showed a weak agonistic response (data not shown). Only dexamethasone (Dex) was almost inactive in yeast and only showed a small anti-androgenic response at very high concentrations (mM). The strength and specificity of the yeast androgen bioassay was further demonstrated by the analysis of the steroidal AR agonists desoxymethyltestosterone (DMT), fluoxymesterone, and oxandrolone, the partial steroidal AR antagonist CPA, and the non-steroidal AR antagonists bicalutamide, flutamide, hydroxyflutamide (HF), and nilutamide. In Fig. 4a it is shown that the steroidal AR agonists DMT, fluoxymesterone, and oxandrolone indeed acted as AR agonists in the yeast androgen bioassay, being, respectively about half as potent as T, 100-fold, and 4-fold less potent than T. These three steroidal agonists were not able to inhibit a response caused by T (data not shown). On the other hand, Fig. 4b shows that the partial steroidal AR antagonist CPA acted as an antagonist, almost completely inhibiting (80%) the response caused by 70 nM T. CPA also showed an agonistic response of 5% (data not shown) and is thus also classified in yeast as a partial AR antagonist, which is in line with responses as obtained in the human U2OS-based AR receptor and hAR-MMTV-LUC CHO cell lines [23,24]. Moreover, epi-oxandrolone was not active as a pure AR agonist (data not shown), but acted as a pure AR antagonist in the yeast androgen bioassay (see Fig. 4b), once more demonstrating the importance of
Fig. 4. Responses of known AR agonists and antagonists in the yeast androgen bioassay. (a) The androgenic responses of the known steroidal AR agonists desoxymethyltestosterone (DMT), fluoxymesterone, and oxandrolone. This agonistic response is shown as % of the maximal response by T. (b) The anti-androgenic responses of several compounds tested by co-administration of 70 nM T. Fluorescence signals are the mean of a triplicate with SD.
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Fig. 5. Chemical structures of SARMs. The given structures of the arylpropionamide-derived SARMs are APA1, APA2, and APA3 and of the 2-quinoline-based SARMs are Q1, Q4, and Q2. In addition the structure of the major metabolite M11 of APA3 is given.
the hydroxyl group in the 17 position for agonistic activity (see oxandrolone in Fig. 4a) [20,25]. With respect to the non-steroidal AR antagonists flutamide, hydroxyflutamide (HF), and nilutamide, Fig. 4b shows that they acted as pure AR antagonists in the yeast androgen bioassay and completely inhibited the response induced with 70 nM T. These compounds showed no agonistic activities. Bicalutamide, however, was completely inactive in yeast and unable to show its antiandrogenic properties. Fig. 5 shows the structures of the three arylpropionamidebased compounds and the three quinoline-based compounds, together with the structures of the non-steroidal AR antagonists bicalutamide, flutamide, nilutamide, and M11. The M11 compound is the proposed metabolite of the arylpropionamide-based SARMs and bicalutamide. Fig. 6 shows the responses of the active arylpropionamide-based and quinoline-based SARMs as obtained in the yeast androgen bioassay. All three arylpropionamide-based compounds, APA1, APA3 and APA4 are inactive as an agonist in the yeast androgen bioassay (data of the latter not shown), but Fig. 6 shows that APA1 and APA3 have clear antagonistic properties at high concentrations. From the three quinoline-based SARMs tested, Q1, Q2 and Q4, Q1 acts as a weak agonist and Q2 as a weak antagonist, while Q4 turns out to be inactive in the yeast androgen bioassay (data of the latter not shown). As expected, when tested in the yeast estrogen assay all these 6 SARMs turned out to be inactive as an agonist and antagonist (data not shown).
4. Discussion In general agonistic and additive effects of compounds can best be observed in combination with a half-maximal stimulating dose of E2 or T. Antagonistic effects, on the other hand, can best be observed with a near maximal stimulating dose of E2 or T. However, the results in the present study show that it is crucial to include also a combination with the half-maximal stimulating dose of T in order to elicit the very weak antagonistic properties of the SARMs APA1, APA3, and Q2. All six 11-substituents of E2 showed clear estrogenic or antiestrogenic responses in the yeast estrogen bioassay and even for some of the compounds combined estrogenic and anti-estrogenic SERM properties can be identified in these yeast cells. Comparison of the yeast transactivation data with those of the CHO cells learned that the ranking of the compounds with respect to agonistic potency was slightly different. The largest difference is observed with the 11-hydroxypentyl substituent, while the 11n-yl and 11-3-cyclopropylidenepropyl substituted compounds are the most potent estrogen receptor agonists in both assay types and show similar efficacies. The relative low agonistic and antagonistic potencies of the 11hydroxypentyl substituent in the yeast estrogen bioassay point into the direction that very polar compounds might have difficulties to diffuse through the yeast cell wall or membrane or are more actively transported from the cytoplasm towards the exter-
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Fig. 6. Responses of two arylpropionamide-based and two quinoline-based SARMs in the yeast androgen bioassay. SARMs were tested alone () and by co-administration of 70 nM T () or 1 M T (). In addition (c) the response of T (). Fluorescence signals are the mean of a triplicate with SD.
nal environment. However, other compounds with at least three hydroxyl groups, like genistein, estriol (E3) and ␣-zearalanol, and that are even more polar than 11-hydroxypentyl-E2, were all very potent in the yeast estrogen bioassay and showed relative estrogenic potencies that were exactly in line with those determined in Ishikawa, U2-OS, and MCF-7 mammalian cells [18,19,23,26,27]. Even the in vivo Allen Doisy test showed similar relative activities for genistein, estriol and estradiol [23]. With respect to the antagonistic activity, the ranking of the compounds was different with both assay types and the ratio of the EC50 and IC50 values between agonistic and antagonistic activities in the CHO cells therefore showed a larger difference than those determined in yeast cells. There are several possible explanations for observed differences between different assay types, as differences in co-repressor or co-activator recruitment, metabolism, and even crosstalk can cause large discrepancies [28]. However, in general receptor systems based on different cell types show similar results and once the differences in responses as obtained with different in vitro cell systems are understood, it will help to elicit a compound’s mode of action and predict its real in vivo activity [23,28]. The responses of the known steroidal AR agonists DMT, fluoxymesterone, and oxandrolone, the known partial steroidal AR antagonist CPA and the known pure non-steroidal AR antagonists flutamide, hydroxyflutamide, and nilutamide in the yeast androgen bioassay were all assessed as predicted. Showing that this bioassay can specifically detect these drugs. Moreover, cyproterone was not active as an AR agonist or antagonist in the yeast (data not shown), which correlates well with the general idea that CPA is the active metabolite of cyproterone and the finding that the 17␣-
acetate group of CPA induces movement of the Leu-701 side chain of the hAR, resulting in a disconnection of the loop between helices 11 and 12 [29]. This probably explains the antagonistic properties of CPA and the inactivity of cyproterone on the hAR. Only bicalutamide was completely inactive in yeast and unable to show its known anti-androgenic properties. We have no straightforward explanation why bicalutamide is inactive in the yeast androgen bioassay. Although the permeability of yeast cells is sometimes an issue, the molecular weight of bicalutamide is with 430.37 g/mol not a likely obstacle. As it has been demonstrated that the isolated yeast cell wall is permeable to solutes with an average Einstein–Stokes hydrodynamic radius less than 0.8 nm and an average molecular weight of 620 g/mol [30]. The wall of living yeast cells can be even more permeable, allowing much larger molecules into the cell [31]. The plasma membrane is a lipid bi-layer that might provide a relatively impermeable barrier to hydrophilic compounds, but this lipid bi-layer is also part of mammalian cell membranes [32]. So far, effects in yeast cells that point into the direction that small (<600 g/mol) relatively polar or a-polar compounds might have difficulties to diffuse through the yeast cell wall or membrane in comparison to mammalian cell systems, have never been observed for these kind of hormonal compounds. Moreover, bicalutamide’s structure analogues APA1 and APA3 showed a clear anti-androgenic effect in the yeast androgen bioassay. Six out of seven compounds, namely 5␣-androstane-3-ol-17one, 5-androstane-3-ol-17-one, 5␣-androstane-3,17␣-diol, 17␣-ethinylestradiol (EE), dehydroepiandrosterone (DHEA), and 16-hydroxystanozolol (16-OHStan), which were previously shown to be without agonistic potential in the yeast androgen
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bioassay, although QSAR modelling indicated a strong binding to the hAR [20], are now shown to possess clear anti-androgenic properties in the yeast androgen bioassay. The synthetic steroid EE is known to posses anti-androgenic properties and the results for the other 5 AR antagonists in yeast might thus point into directions of possible in vivo anti-androgenic activities of these compounds too, as we have demonstrated before that the agonistic effect of TAM in the yeast estrogen assay was not an artefact, but correlated well with the agonistic effect of this compound on endometrial cells [19]. The latter is clinically important as the agonistic effect of TAM is known to be responsible for the increased chance of developing endometrial cancer [11]. The compounds 5␣-androstane-3-ol-17-one and 16hydroxystanozolol also showed a weak androgenic response and thus can be classified as partial AR antagonists. Moreover, both DHEA and 5␣-androstane-3,17␣-diol were also inactive as AR agonists in the U2OS-AR cell line and 5␣-androstane-3-ol-17-one was only slightly active as an AR agonist in this bone cell line, and thus confirm our observations [33]. Dexamethasone (Dex) was hardly active in the yeast androgen bioassay and only showed a weak antagonistic response at huge concentrations (mM). As it is not likely that Dex is a strong AR agonist or antagonist [20,23,33], the strong binding of Dex to the hAR as predicted by the QSAR model might need some improvement by fine-tuning of the free energy calculations. Two out of the three arylpropionamide-based compounds showed clear antagonistic properties in the yeast androgen bioassay at relatively high concentrations (APA1 and APA3) and from the three quinoline-based compounds, Q1 acts as a weak AR agonist and Q2 acts as a weak AR antagonist. These outcomes are hard to interpret, not only because these compounds were not tested before, but especially since the mechanism of action by which SARMs mediate tissue selective anabolic, androgenic, and anti-androgenic effects via the AR is not yet resolved [16,34,35]. Although, the three arylpropionamide-based compounds APA1, APA2, and APA3 are mainly described as androgens, they are structurally related towards the pure anti-androgen bicalutamide. Also the three quinoline-based compounds Q1, Q2, and Q4 are mainly described as androgens, but their structure analogue nilutamide acts as a pure androgen receptor antagonist. Nilutamide was also tested as a pure AR antagonist in this study. It is not unlikely that these SARMs, just as the case with the well known SERMs TAM, RAL, and chlomiphene citrate, will show different characteristics, agonistic or antagonistic, with different cell based systems [19,28]. Formation of active metabolites often represents an important mechanism for observed differences between different in vitro bioassays and between in vitro bioassays and the real in vivo situation [28]. The pharmacological activity of the nonsteroidal AR antagonist flutamide for instance is mainly mediated by its more active metabolite hydroxyflutamide [13,36] and structurally resembles the hydroxylated metabolite M11 of both bicalutamide and the arylpropionamide-based SARMs [37]. This metabolic fate of bicalutamide and the most widely described arylpropionamide-derived SARMs were only recently studied by employing microsomal and S9 human liver enzymes in vitro and in vivo with rat [37,38]. As both flutamide and hydroxyflutamide are potent AR antagonist in yeast and are structurally almost equal to this M11 metabolite of bicalutamide, it can be hypothesised that the in vivo anti-androgenic effect of bicalutamide is circumstanced by its related M11 metabolite. More efforts are needed to get hold of milligram amounts of these M11 metabolites and subsequently to determine whether these metabolites are active compounds in vitro and in vivo, including yeast, and whether they could be responsible for the androgenic, anabolic and anti-androgenic effects of bicalutamide and its structural analogues.
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In conclusion, the data presented in this study demonstrate that yeast estrogen and androgen bioassays are very useful to detect hormone abuse in different areas and valuable tools to assess the mode of action of existing and new compounds. Acknowledgement This project was supported by the Dutch Ministry of Agriculture, Nature and Food Quality (project number 8.72027.01). References [1] H.J. Coelingh Bennink, Are all estrogens the same? Maturitas 47 (2004) 269–275. [2] R. Bross, T. Storer, S. Bashin, Aging and muscle loss, Trends Endocrinol. Metabol. 10 (1999) 194–198. [3] M. Muller, D.E. Grobbee, J.H.H. Thijssen, A.W. Van den Beld, Y.T. Van der Schouw, Sex hormones and male health: effects on components of the frailty syndrome, Trends Endocrinol. Metabol. 14 (2003) 289–296. [4] S.R. Davis, The therapeutic use of androgens in women, J. Steroid Biochem. Mol. Biol. 69 (1999) 177–184. [5] F.J. Lopez, New approaches to the treatment of osteoporosis, Curr. Opin. Chem. Biol. 4 (2000) 383–393. [6] T.J. Doherty, Aging and sarcopenia, J. Appl. Physiol. 95 (2003) 1717–1727. [7] G. Chodak, L. Gomella, H. De Phung, Combined androgen blockade in advanced prostate cancer: looking back to move forward, Clin. Genitourin. Cancer 5 (2007) 371–378. [8] L. Klotz, Maximal androgen blockade for advanced prostate cancer, Best Pract. Res. Clin. Endocrinol. Metab. 22 (2008) 331–340. [9] L. Plouffe Jr., Selective estrogen receptors modulators (SERMs) in clinical practice, J. Soc. Gynecol. Investig. 7 (2000) 38–46. [10] B. Arun, M. Anthony, B. Dunn, The search for the ideal SERM, Expert Opin. Pharmacother. 3 (2002) 681–691. [11] K. Leblanc, E. Sexton, S. Parent, G. Belanger, M.C. Dery, V. Boucher, E. Asselin, Effects of 4-hydroxytamoxifen, raloxifene and ICI 182 780 on survival of uterine cancer cell lines in the presence and absence of exogenous estrogens, Int. J. Oncol. 30 (2007) 477–487. [12] G.J. Kolvenbag, G.R. Blackledge, Worldwide activity and safety of bicalutamide: a summary review, Urology 47 (1996) 70–84. [13] P.F. Schellhammer, R. Sharifi, N.L. Block, M.S. Soloway, P.M. Venner, A.L. Patterson, M.F. Sarosdy, N.J. Vogelzang, C.Y. Chen, G.J.C.M. Kolvenbag, A controlled trial of bicalutamide versus flutamide, each in combination with luteinizing hormone-releasing hormone analogue therapy, in patients with advanced prostate carcinoma, Urology 45 (1995) 745–752. [14] F. Chen, G.A. Rodan, A. Schmidt, Development of selective androgen receptor modulators and their therapeutic applications, Zhonghua Nan Ke Xue 8 (2002) 162–168. [15] M. Thevis, M. Kamber, W. Schänzer, Screening for metabolically stable arylpropionamide-derived selective androgen receptor modulators for doping control purposes, Rapid Commun. Mass Spectrom. 20 (2006) 870–876. [16] J.N. Miner, W. Chang, M.S. Chapman, P.D. Finn, M.H. Hong, F.J. López, K.B. Marschke, J. Rosen, W. Schrader, R. Turner, A. Van Oeveren, H. Viveros, L. Zhi, A. Negro-Vilar, An orally active selective androgen receptor modulator is efficious on bone, muscle, and sex function with reduced impact on prostate, Endocrine 148 (2007) 363–373. [17] T.F.H. Bovee, G. Bor, H.H. Heskamp, J.J.P. Lasaroms, M.B. Sanders, M.W.F. Nielen, Validation and application of a yeast bioassay for screening androgenic activity in calf urine and feed, Anal. Chim. Acta 637 (2009) 225–234. [18] T.F.H. Bovee, J.R. Helsdingen, I.M.C.M. Rietjens, J. Keijer, L.A.P. Hoogenboom, Rapid yeast estrogen bioassays stably expressing human estrogen receptors ␣ and , and green fluorescent protein: a comparison of different compounds with both receptor types, J. Steroid Biochem. Mol. Biol. 91 (2004) 99–109. [19] T.F.H. Bovee, W.G.E.J. Schoonen, A.R.M. Hamers, M. Jorge-Bento, A.A.C.M. Peijnenburg, Screening of synthetic and plant-derived compounds for (anti)estrogenic and (anti)androgenic activities, Anal. Bioanal. Chem. 390 (2008) 1111–1119. [20] T.F.H. Bovee, J.P.M. Lommerse, A.A.C.M. Peijnenburg, E. Antunes-Fernandes, M.W.F. Nielen, A new highly androgen specific yeast biosensor, enabling optimisation of (Q)SAR model approaches, J. Steroid Biochem. Mol. Biol. 108 (2008) 121–131. [21] M. Thevis, M. Kohler, J. Maurer, N. Schlörer, M. Kamber, W. Schänzer, Screening for 2-quinolinone-derived selective androgen receptor agonists in doping control analysis, Rapid Commun. Mass Spectrom. 21 (2007) 3477–3486. [22] W.G.E.J. Schoonen, R. Dijkema, R.J.H. De Ries, J.L. Wagenaars, J.W.H. Joosten, M.E. De Gooyer, G.H. Deckers, H.J. Kloosterboer, Human progesteron receptor A and B isoforms in CHO cells II comparison of binding, transactivation and ED50 values of several synthetic (anti)progestagens in vitro in CHO and MCF-7 cells and in vivo in rabbits and rats, J. Steroid Biochem. Mol. Biol. 64 (1998) 157–170. [23] E. Sonneveld, J.A.C. Riteco, H.J. Jansen, B. Pieterse, A. Brouwer, W.G. Schoonen, B. Van der Burg, Comparison of in vitro and in vivo screening models for androgenic and estrogenic activities, Toxicol. Sci. 89 (2006) 173–187.
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Journal of Steroid Biochemistry and Molecular Biology journal homepage: www.elsevier.com/locate/jsbmb
SERMs and SARMs: Detection of their activities with yeast based bioassays Toine F.H. Bovee a,∗ , Mario Thevis b , Astrid R.M. Hamers a , Ad A.C.M. Peijnenburg a , Michel W.F. Nielen a,c , Willem G.E.J. Schoonen d a
RIKILT-Institute of Food Safety, Department of Safety & Health, Bornsesteeg 45, 6708 PD Wageningen, The Netherlands Center for Preventive Doping Research – Institute of Biochemistry, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933 Cologne, Germany c Wageningen University, Laboratory of Organic Chemistry, Dreijenplein 8, 6703 HB Wageningen, The Netherlands d Schering-Plough Research Institute, Department of Toxicology and Drug Disposition, P.O. Box 20, 5340 BH Oss, The Netherlands b
a r t i c l e
i n f o
Article history: Received 11 June 2009 Received in revised form 21 October 2009 Accepted 22 October 2009 Keywords: Agonists Antagonists In vitro bioassays Androgenic activity Estrogenic activity Metabolism
a b s t r a c t Selective estrogen receptor modulators (SERMs) and selective androgen receptor modulators (SARMs) are compounds that activate their cognate receptor in particular target tissues without affecting other organs. Many of these compounds will find their use in therapeutic treatments. However, they also will have a high potential for misuse in veterinary practice and the sporting world. Here we demonstrate that yeast estrogen and androgen bioassays can be used to detect SERMs and SARMs, and are also useful screening tools to investigate their mode of action. Six steroidal 11-substituents of E2 (SERMs) and some arylpropionamide- and quinoline-based SARMs were tested. In addition, 7 compounds previously tested on AR agonism and determined as inactive in the yeast androgen bioassay, while QSAR modelling revealed strong binding to the human androgen receptor, are now shown to act as AR antagonists. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction Compounds with hormonal activities are widely used in medical practice. Human estrogen receptor ␣ (hER␣) agonists are used for birth control and the treatment of osteoporosis in postmenopausal women [1]. Human androgen receptor (hAR) agonists can be of interest in case of delayed puberty, libido loss, impotence, or muscle weakness [2]. The decrease in the endogenous production of hormones like 17-testosterone (T), 17-estradiol (E2), growth hormone, insulin, and insulin like growth factor 1 is the major cause of these health problems [2,3]. Hormone replacement therapies (HRT) with either estrogens or androgens however may cause severe side effects and more specific drugs are needed [4–6]. Antiestrogens and anti-androgens, in turn, are very important in the treatment of certain tumours. The pure anti-estrogens ICI 182,780 (fulvestrant) and RU 58668 are successfully used to treat breast cancer patients. The pure anti-androgens, flutamide and bicalutamide (Casodex), are proven to be useful in curing prostate cancer patients [7,8]. Selective estrogen receptor modulators (SERMs) and selective androgen receptor modulators (SARMs) are compounds that activate their cognate receptor in particular target tissues without affecting other organs. Such new drugs can be very beneficial alternatives for HRT. Tamoxifen (TAM), chlomiphene citrate, and
∗ Corresponding author. Tel.: +31 317 480391; fax: +31 317 417717. E-mail address: [email protected] (T.F.H. Bovee). 0960-0760/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2009.10.007
raloxifen are the best known SERMs with respect to breast specific anti-estrogens [9,10]. Unfortunately, TAM acts as an ER agonist on the uterus and prolonged use is associated with the development of endometrial cancer [11]. SARMs like bicalutamide (Casodex), flutamide and nilutamide are known as non-steroidal anti-androgens and used to treat benign prostate hyperplasia and prostate cancer [12,13]. The search for new SERMs and SARMs as alternatives for HRT or breast and prostate cancer treatment is one of the major topics within the pharmaceutical industry. A new generation of SARMs is being developed for androgen replacement. These new SARMs should reduce the unwanted side effects occurring with particular synthetic 17␣-substituted derivatives of T, as these latter compounds induce liver cholestasis, prostate hypertrophy, and acne [14–16]. Ideally these new SARMs must act as full agonists, like T, in anabolic target tissues, e.g. muscle and bone, but should demonstrate only partial or pure AR antagonistic activities on prostate tissue. This can partly be obtained by the inactivation or inhibition of 5␣-reductase [15]. However, especially these SARMs also will have a high potential for misuse in veterinary practice and the sporting world. Screening methods to detect these new compounds are required to ensure food safety and fair play. Previously we described the development of yeast based estrogen and androgen bioassays. These bioassays were fully validated for screening animal feed and calf urine samples for the presence of natural and synthetic steroids [17]. Both bioassays were shown to be highly specific for their cognate (ant)agonistic compounds, which made them also suited to determine the (anti)estrogenic and
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Fig. 1. Chemical name and structures of the 11-substituents towards 17-estradiol.
(anti)androgenic properties of plant-derived compounds [18,19]. The aim of the present study was to determine whether new SERMs and SARMs can also be detected with respect to their specific hormonal activities in these yeast transcription activation assays. The 6 SERMs tested were derived from the steroid E2 and contained 11-substituents with a variation in the carbon length from 4 to 6 atoms. The 6 non-steroidal SARMs tested were 3 arylpropionamide and 3 quinoline-based compounds. These SERMs and SARMs are commercially not available, have never been tested in yeast based bioassays before and little information, especially for the nonsteroidal SARMs, is available. The ability of the yeast androgen bioassay to detect the known non-steroidal pure anti-androgens bicalutamide, flutamide, hydroxyflutamide, and nilutamide was therefore investigated first, while the steroidal AR agonists oxandrolone, desoxymethyltestosterone (DMT), and fluoxymesterone and the steroidal AR antagonist cyproterone acetate (CPA) were tested in order to further prove the specificity of the yeast androgen bioassay. Finally, 7 compounds, previously tested in the yeast androgen bioassay and being inactive as AR agonists, were now investigated on their anti-androgenic properties, as our earlier QSAR modelling results revealed strong binding affinities towards hAR [20].
gen biosensor were grown overnight at 30 ◦ C with vigorous orbital shaking. At the late log phase, the culture was diluted in selective MM/L medium till an OD value at 630 nm between 0.04 and 0.06 was reached. For exposure, aliquots of 200 l of the diluted yeast culture were pipetted into each well of a 96-well plate and 2 l of a stock solution in DMSO was added to test the agonistic properties of the compounds. To test the anti-estrogenic and anti-androgenic properties of compounds, 1 l amounts of the compound’s stock solutions were co-exposed with 1 l of E2 or T stock solutions, respectively. Two doses of both E2 and T were used, giving a half-maximal response or a near maximal response in the yeast estrogen and androgen bioassay, respectively. DMSO, E2, and T controls were included in each experiment and each sample concentration was assayed in triplicate. Exposure was performed for 24 h at 30 ◦ C and orbital shaking with 125 rpm. Fluorescence and OD were measured at 0 and 24 h directly in a SynergyTM HT Multi-Detection Microplate Reader (BioTek Instruments Inc., USA) using excitation at 485 nm and emission at 530 nm. The fluorescent signal was corrected with the signals obtained the DMSO control. Densities of the yeast culture were determined by measuring the OD at 630 nm to check whether the yeast cells were growing, in order to determine whether a sample concentration was toxic for the yeast cells.
2. Materials and methods 2.3. Chinese hamster ovary (CHO) cell ER˛ transactivation assay 2.1. Chemicals Dehydroepiandrosterone (DHEA), dexamethasone, 17estradiol (E2), flutamide and nilutamide were obtained from Sigma. Cyproterone, epiandrosterone (5␣-androstane-3-ol17-one), epietiocholanolone (5-androstane-3-ol-17-one), 17␣-ethinylestradiol (EE), 17-testosterone (T), and 5␣androstane-3-17␣-diol were obtained from Steraloids, dimethyl sulfoxide (DMSO) from Merck, and 16-stanozolol from NMI (Australia). The six 11-substituted E2 compounds (see Fig. 1 for their structures), fluoxymesterone, oxandrolone, hydroxyflutamide, bicalutamide (Casodex), and cyproterone acetate (CPA) were obtained from Schering-Plough (Oss, The Netherlands). The 3 arylpropionamide and 3 quinoline-based SARM compounds were synthesised at the Centre for Preventive Doping Research – Institute of Biochemistry, German Sport University (Cologne, Germany) and were previously described in detail [15,21]. Their structures are given in Fig. 5. Also the desoxymethyltestosterone (DMT) was obtained from the German Sport University and kindly provided by Prof. W. Schänzer. Chemicals to prepare the growth media and the preparation of the growth media for yeast cells were as described previously [19]. 2.2. Yeast estrogen and androgen bioassays with fluorescence measurement The estrogenic, anti-estrogenic, androgenic, and antiandrogenic properties of the compounds were tested as described previously [19]. In short, cultures of the yeast estrogen and andro-
For transactivation studies the stably transfected CHO cells were used according to the procedures described by Schoonen et al. [22]. These CHO cells were cultured in DMEM/HAM F12 medium supplemented with 5% defined bovine calf serum from Hyclone (UT, USA) at 37 ◦ C in Roux flasks (175 cm3 ) and flushed with 5% CO2 in air until pH 7.2–7.4 was reached. Complete medium was refreshed every 2–3 days. These CHO cells express luciferase upon exposure to estrogens. 3. Results The structures of six 11-substituents of E2 are presented in Fig. 1 and the responses of these SERMs when tested in the yeast estrogen bioassay, expressing human estrogen receptor ␣ (hER␣) and yeast enhanced green fluorescence protein (yEGFP) in response to estrogens, are shown in Fig. 2. All six 11-substituted compounds show an agonistic response and five are active as real partial anti-estrogens. E2 with the 11-hydroxypentyl substituent gave only an agonistic effect (90% of E2 being 100%), while a decrease from agonistic activity towards 22% was found in the sequence of 11-3-cyclopropylidenepropyl (65%), 11pentenyl (65%), 11-pentyl (53%), 11-butyl (37%), and 11-n-yl carbon chain (22%). The 11-hydroxypentyl compound is with respect to potency the weakest estrogen receptor agonist, demonstrated by its EC50 of 153 nM, followed directly by the 11-butyl compound. Than with a 10-fold higher potency the 11-pentyl and 11-pentenyl estrogens were found, while the 11-carbon chain and 11-3-cyclopropylidenepropyl were with EC50 values
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Fig. 2. Responses of 11-substituents of estradiol in the yeast estrogen bioassay. (a–f) The responses of six 11-substituents of E2 alone () and by co-administration of 1 nM E2 () or 3 nM E2 (). In addition (a) the response of E2 (). Fluorescence signals are the mean of a triplicate with SD.
of, respectively 7 and 8 nM the most potent agonists. As already described above E2 with the 11-hydroxypentyl substituent did not show antagonism, even at the level of 3000 nM, while the 11-butyl E2 was the next weakest anti-estrogen. Than the ranking of the four other compounds for anti-estrogenic activity was from 75 to 17 nM as follows: 11-pentenyl, 11-pentyl, 11-3cyclopropylidenepropyl, and 11-n-yl carbon chain. Comparing the dose–response curves for these substituents demonstrates that an increasing chain length on the 11 side chain, from
butyl to n-yl, increases the antagonistic potency and more or less also the agonistic potency of the compound in yeast. Table 1 shows a summary of these yeast results as well as of the transactivation data as obtained with a CHO cell line. In the CHO cells the 11-butyl (70%) and 11-3-cyclopropylidenepropyl (64%) substituted compounds were the strongest partial agonists, followed by an increasing weaker effect from 55% to 14% from 11-pentenyl, 11-pentyl, 11-hydroxypentyl, and 11-nyl carbon chain. With respect to antagonistic activities in CHO
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Table 1 The estrogenic and anti-estrogenic properties of E2 with various 11-substituents in yeast and CHO assays. Compounds
Yeast hER␣ a
E2 +11-Butyl +11-Pentyl +11-n-yl +11-Pentenyl +11-HO-pentyl +11-3-Cyclopropylidenepropyl
CHO hER␣ a
b
b
Agonist (nM)
Antagonist (nM)
Ratio (ago/ant)
Efficacy (%)
Agonist (pM)
Antagonist (pM)
Ratio (ago/ant)
Efficacy (%)
1 63 12 7 11 153 8
>1000 600 60 17 75 >3000 50
0.001 0.11 0.20 0.41 0.15 0.05 0.16
100 37 53 22 65 90 65
50 200 227 53 93 36 42
>1000 2100 2830 3230 2700 1270 5400
0.001 0.095 0.080 0.016 0.034 0.028 0.008
100 70 46 14 55 15 64
a The EC50 and IC50 values for agonistic and antagonistic human estrogen receptor ␣ activities, respectively of E2 and E2 with various 11-substituents in yeast and CHO assays. b The ratio for agonism and antagonism as well as the overall agonist efficacy is given for these compounds compared to the maximal response caused by E2 (E2 = 100%).
Fig. 3. Anti-androgenic responses in the yeast androgen bioassay of 7 compounds that show strong binding to the hAR according to a QSAR model. The AR antagonist activities of EE, DHEA, 5␣-androstane-3-ol-17-one (5␣-3-ol-17-one), 5-androstane-3-ol-17-one (5-3-ol-17-one), 5␣-androstane-3,17␣-diol (5␣3,17␣-diol), dexamethasone (Dex) and 16-hydroxystanozolol (16-OH-Stan) are examined by co-administration of 70 nM T (). In addition, the agonistic response of T () is shown. Fluorescence signals are the mean of a triplicate with SD.
cells these compounds were active in the range of 1.27 to 5.4 nM. Fig. 3 shows the anti-androgenic responses of 7 compounds that were not active as AR agonists in the yeast androgen bioassay [20]. The data in Fig. 3 show that 6 of these 7 com-
pounds, namely 5␣-androstane-3-ol-17-one, 5-androstane3-ol-17-one, 5␣-androstane-3,17␣-diol, 17␣-ethinylestradiol (EE), dehydroepiandrosterone (DHEA), and 16-hydroxystanozolol (16-OHStan) showed a clear antagonistic response in the yeast androgen bioassay. Both 5␣-androstane-3-ol-17-one and 16hydroxystanozolol also showed a weak agonistic response (data not shown). Only dexamethasone (Dex) was almost inactive in yeast and only showed a small anti-androgenic response at very high concentrations (mM). The strength and specificity of the yeast androgen bioassay was further demonstrated by the analysis of the steroidal AR agonists desoxymethyltestosterone (DMT), fluoxymesterone, and oxandrolone, the partial steroidal AR antagonist CPA, and the non-steroidal AR antagonists bicalutamide, flutamide, hydroxyflutamide (HF), and nilutamide. In Fig. 4a it is shown that the steroidal AR agonists DMT, fluoxymesterone, and oxandrolone indeed acted as AR agonists in the yeast androgen bioassay, being, respectively about half as potent as T, 100-fold, and 4-fold less potent than T. These three steroidal agonists were not able to inhibit a response caused by T (data not shown). On the other hand, Fig. 4b shows that the partial steroidal AR antagonist CPA acted as an antagonist, almost completely inhibiting (80%) the response caused by 70 nM T. CPA also showed an agonistic response of 5% (data not shown) and is thus also classified in yeast as a partial AR antagonist, which is in line with responses as obtained in the human U2OS-based AR receptor and hAR-MMTV-LUC CHO cell lines [23,24]. Moreover, epi-oxandrolone was not active as a pure AR agonist (data not shown), but acted as a pure AR antagonist in the yeast androgen bioassay (see Fig. 4b), once more demonstrating the importance of
Fig. 4. Responses of known AR agonists and antagonists in the yeast androgen bioassay. (a) The androgenic responses of the known steroidal AR agonists desoxymethyltestosterone (DMT), fluoxymesterone, and oxandrolone. This agonistic response is shown as % of the maximal response by T. (b) The anti-androgenic responses of several compounds tested by co-administration of 70 nM T. Fluorescence signals are the mean of a triplicate with SD.
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Fig. 5. Chemical structures of SARMs. The given structures of the arylpropionamide-derived SARMs are APA1, APA2, and APA3 and of the 2-quinoline-based SARMs are Q1, Q4, and Q2. In addition the structure of the major metabolite M11 of APA3 is given.
the hydroxyl group in the 17 position for agonistic activity (see oxandrolone in Fig. 4a) [20,25]. With respect to the non-steroidal AR antagonists flutamide, hydroxyflutamide (HF), and nilutamide, Fig. 4b shows that they acted as pure AR antagonists in the yeast androgen bioassay and completely inhibited the response induced with 70 nM T. These compounds showed no agonistic activities. Bicalutamide, however, was completely inactive in yeast and unable to show its antiandrogenic properties. Fig. 5 shows the structures of the three arylpropionamidebased compounds and the three quinoline-based compounds, together with the structures of the non-steroidal AR antagonists bicalutamide, flutamide, nilutamide, and M11. The M11 compound is the proposed metabolite of the arylpropionamide-based SARMs and bicalutamide. Fig. 6 shows the responses of the active arylpropionamide-based and quinoline-based SARMs as obtained in the yeast androgen bioassay. All three arylpropionamide-based compounds, APA1, APA3 and APA4 are inactive as an agonist in the yeast androgen bioassay (data of the latter not shown), but Fig. 6 shows that APA1 and APA3 have clear antagonistic properties at high concentrations. From the three quinoline-based SARMs tested, Q1, Q2 and Q4, Q1 acts as a weak agonist and Q2 as a weak antagonist, while Q4 turns out to be inactive in the yeast androgen bioassay (data of the latter not shown). As expected, when tested in the yeast estrogen assay all these 6 SARMs turned out to be inactive as an agonist and antagonist (data not shown).
4. Discussion In general agonistic and additive effects of compounds can best be observed in combination with a half-maximal stimulating dose of E2 or T. Antagonistic effects, on the other hand, can best be observed with a near maximal stimulating dose of E2 or T. However, the results in the present study show that it is crucial to include also a combination with the half-maximal stimulating dose of T in order to elicit the very weak antagonistic properties of the SARMs APA1, APA3, and Q2. All six 11-substituents of E2 showed clear estrogenic or antiestrogenic responses in the yeast estrogen bioassay and even for some of the compounds combined estrogenic and anti-estrogenic SERM properties can be identified in these yeast cells. Comparison of the yeast transactivation data with those of the CHO cells learned that the ranking of the compounds with respect to agonistic potency was slightly different. The largest difference is observed with the 11-hydroxypentyl substituent, while the 11n-yl and 11-3-cyclopropylidenepropyl substituted compounds are the most potent estrogen receptor agonists in both assay types and show similar efficacies. The relative low agonistic and antagonistic potencies of the 11hydroxypentyl substituent in the yeast estrogen bioassay point into the direction that very polar compounds might have difficulties to diffuse through the yeast cell wall or membrane or are more actively transported from the cytoplasm towards the exter-
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Fig. 6. Responses of two arylpropionamide-based and two quinoline-based SARMs in the yeast androgen bioassay. SARMs were tested alone () and by co-administration of 70 nM T () or 1 M T (). In addition (c) the response of T (). Fluorescence signals are the mean of a triplicate with SD.
nal environment. However, other compounds with at least three hydroxyl groups, like genistein, estriol (E3) and ␣-zearalanol, and that are even more polar than 11-hydroxypentyl-E2, were all very potent in the yeast estrogen bioassay and showed relative estrogenic potencies that were exactly in line with those determined in Ishikawa, U2-OS, and MCF-7 mammalian cells [18,19,23,26,27]. Even the in vivo Allen Doisy test showed similar relative activities for genistein, estriol and estradiol [23]. With respect to the antagonistic activity, the ranking of the compounds was different with both assay types and the ratio of the EC50 and IC50 values between agonistic and antagonistic activities in the CHO cells therefore showed a larger difference than those determined in yeast cells. There are several possible explanations for observed differences between different assay types, as differences in co-repressor or co-activator recruitment, metabolism, and even crosstalk can cause large discrepancies [28]. However, in general receptor systems based on different cell types show similar results and once the differences in responses as obtained with different in vitro cell systems are understood, it will help to elicit a compound’s mode of action and predict its real in vivo activity [23,28]. The responses of the known steroidal AR agonists DMT, fluoxymesterone, and oxandrolone, the known partial steroidal AR antagonist CPA and the known pure non-steroidal AR antagonists flutamide, hydroxyflutamide, and nilutamide in the yeast androgen bioassay were all assessed as predicted. Showing that this bioassay can specifically detect these drugs. Moreover, cyproterone was not active as an AR agonist or antagonist in the yeast (data not shown), which correlates well with the general idea that CPA is the active metabolite of cyproterone and the finding that the 17␣-
acetate group of CPA induces movement of the Leu-701 side chain of the hAR, resulting in a disconnection of the loop between helices 11 and 12 [29]. This probably explains the antagonistic properties of CPA and the inactivity of cyproterone on the hAR. Only bicalutamide was completely inactive in yeast and unable to show its known anti-androgenic properties. We have no straightforward explanation why bicalutamide is inactive in the yeast androgen bioassay. Although the permeability of yeast cells is sometimes an issue, the molecular weight of bicalutamide is with 430.37 g/mol not a likely obstacle. As it has been demonstrated that the isolated yeast cell wall is permeable to solutes with an average Einstein–Stokes hydrodynamic radius less than 0.8 nm and an average molecular weight of 620 g/mol [30]. The wall of living yeast cells can be even more permeable, allowing much larger molecules into the cell [31]. The plasma membrane is a lipid bi-layer that might provide a relatively impermeable barrier to hydrophilic compounds, but this lipid bi-layer is also part of mammalian cell membranes [32]. So far, effects in yeast cells that point into the direction that small (<600 g/mol) relatively polar or a-polar compounds might have difficulties to diffuse through the yeast cell wall or membrane in comparison to mammalian cell systems, have never been observed for these kind of hormonal compounds. Moreover, bicalutamide’s structure analogues APA1 and APA3 showed a clear anti-androgenic effect in the yeast androgen bioassay. Six out of seven compounds, namely 5␣-androstane-3-ol-17one, 5-androstane-3-ol-17-one, 5␣-androstane-3,17␣-diol, 17␣-ethinylestradiol (EE), dehydroepiandrosterone (DHEA), and 16-hydroxystanozolol (16-OHStan), which were previously shown to be without agonistic potential in the yeast androgen
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bioassay, although QSAR modelling indicated a strong binding to the hAR [20], are now shown to possess clear anti-androgenic properties in the yeast androgen bioassay. The synthetic steroid EE is known to posses anti-androgenic properties and the results for the other 5 AR antagonists in yeast might thus point into directions of possible in vivo anti-androgenic activities of these compounds too, as we have demonstrated before that the agonistic effect of TAM in the yeast estrogen assay was not an artefact, but correlated well with the agonistic effect of this compound on endometrial cells [19]. The latter is clinically important as the agonistic effect of TAM is known to be responsible for the increased chance of developing endometrial cancer [11]. The compounds 5␣-androstane-3-ol-17-one and 16hydroxystanozolol also showed a weak androgenic response and thus can be classified as partial AR antagonists. Moreover, both DHEA and 5␣-androstane-3,17␣-diol were also inactive as AR agonists in the U2OS-AR cell line and 5␣-androstane-3-ol-17-one was only slightly active as an AR agonist in this bone cell line, and thus confirm our observations [33]. Dexamethasone (Dex) was hardly active in the yeast androgen bioassay and only showed a weak antagonistic response at huge concentrations (mM). As it is not likely that Dex is a strong AR agonist or antagonist [20,23,33], the strong binding of Dex to the hAR as predicted by the QSAR model might need some improvement by fine-tuning of the free energy calculations. Two out of the three arylpropionamide-based compounds showed clear antagonistic properties in the yeast androgen bioassay at relatively high concentrations (APA1 and APA3) and from the three quinoline-based compounds, Q1 acts as a weak AR agonist and Q2 acts as a weak AR antagonist. These outcomes are hard to interpret, not only because these compounds were not tested before, but especially since the mechanism of action by which SARMs mediate tissue selective anabolic, androgenic, and anti-androgenic effects via the AR is not yet resolved [16,34,35]. Although, the three arylpropionamide-based compounds APA1, APA2, and APA3 are mainly described as androgens, they are structurally related towards the pure anti-androgen bicalutamide. Also the three quinoline-based compounds Q1, Q2, and Q4 are mainly described as androgens, but their structure analogue nilutamide acts as a pure androgen receptor antagonist. Nilutamide was also tested as a pure AR antagonist in this study. It is not unlikely that these SARMs, just as the case with the well known SERMs TAM, RAL, and chlomiphene citrate, will show different characteristics, agonistic or antagonistic, with different cell based systems [19,28]. Formation of active metabolites often represents an important mechanism for observed differences between different in vitro bioassays and between in vitro bioassays and the real in vivo situation [28]. The pharmacological activity of the nonsteroidal AR antagonist flutamide for instance is mainly mediated by its more active metabolite hydroxyflutamide [13,36] and structurally resembles the hydroxylated metabolite M11 of both bicalutamide and the arylpropionamide-based SARMs [37]. This metabolic fate of bicalutamide and the most widely described arylpropionamide-derived SARMs were only recently studied by employing microsomal and S9 human liver enzymes in vitro and in vivo with rat [37,38]. As both flutamide and hydroxyflutamide are potent AR antagonist in yeast and are structurally almost equal to this M11 metabolite of bicalutamide, it can be hypothesised that the in vivo anti-androgenic effect of bicalutamide is circumstanced by its related M11 metabolite. More efforts are needed to get hold of milligram amounts of these M11 metabolites and subsequently to determine whether these metabolites are active compounds in vitro and in vivo, including yeast, and whether they could be responsible for the androgenic, anabolic and anti-androgenic effects of bicalutamide and its structural analogues.
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