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CARDIOVASCULAR

The Prototypical Inhibitor of Cholesterol Esterification, Sah 58-035 [3-[Decyldimethylsilyl]-N-[2-(4-methylphenyl)-1-phenylethyl]propanamide], Is an Agonist of Estrogen Receptors

Institut National de la Santé et de la Recherche Médicale U-563, Département Innovation Thérapeutique et Oncologie Moléculaire/Centre de Physiopathologie de Toulouse Purpan, Toulouse, France (P.d.M., N.B., G.F., S.S.-P., M.P.); Institut Claudius Regaud, Toulouse, France (G.F., S.S.-P., M.P.); Université Paul Sabatier, Toulouse, France (G.F., S.S.-P., M.P.); Affichem, Toulouse, France (P.d.M.); and Institut National de la Santé et de la Recherche Médicale U-540, Endocrinologie Moléculaire et Cellulaire des Cancers, Montpellier, France (P.B.)

Received March 10, 2006; accepted July 7, 2006.

	Abstract

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We have shown recently that estrogen receptor (ER) ligands share a diphenyl ethane pharmacophore with Sah 58-035 [3-[decyldimethylsilyl]-N-[2-(4-methylphenyl)-1-phenylethyl]-propanamide], a prototypical inhibitor of the acyl-cholesterolacyl-transferase (ACAT), which enabled us to establish that ER ligands were potent inhibitors of ACAT and blocked the formation of foam cells. In the present study, we have tested whether this structural similarity means that Sah 58-035 is an ER modulator. We report that Sah 58-035 bound to ER and ER with an IC₅₀ of 2.9 and 3.1 µM, respectively. Docking studies using molecular modeling of Sah 58-035 with the X-ray structure of the ER showed that Sah 58-035 fits well into the ligand binding site known for 4-hydroxy-tamoxifen. Despite having high three-dimensional structural similarities with the pure antiestrogen ICI 164,384 [(N-n-butyl-N-methyl-11-[3,17-di-hydroxyestra-1,3, 5(10)-trien-7-yl]-undecanamide], we showed that Sah 58-035 is an agonist of ER for transcription and cellular proliferation. These data showed that Sah 58-035 was an estrogen receptor agonist and that the size and the chemical nature of the side chain were critical for agonist versus antagonist activity on ER. This new molecular mechanism of action for Sah 58-035 has to be taken into account in understanding better its pharmacological activities. Moreover, these data give new structural insights into the understanding of agonist versus antagonist activities of ER ligands and also for the conception of new drugs with a dual ACAT inhibition and ER modulation potential and their evaluation in different pathologies where both targets are involved, such as atherosclerosis, Alzheimer's disease, and cancer.

Sah 58-035 is known as a prototypical and selective inhibitor of cholesterol esterification, catalyzed by ACAT (Ross et al., 1984). ACAT has been considered as a candidate target for the development of antiatherosclerosis drugs (Sliskovic et al., 2002) and recently as a potential target for the treatment of Alzheimer's disease (Hutter-Paier et al., 2004) and cancer (Tosi and Tugnoli, 2005).

ERs are ligand-dependent transcription factors that belong to the nuclear receptor superfamily. Through binding to the ER, estrogens are known to stimulate the development of estrogeno-dependent breast and endometrial cancers (Jensen and Jordan, 2003), to exert neurotrophic and neuroprotective effects in vitro (Nilsen and Diaz Brinton, 2003) and in vivo (Wise et al., 2005), and to protect against the development of cardiovascular diseases in different mammalian atherosclerosis models via a mechanism that involved the ER (Pare et al., 2002). In the present study, we report for the first time that Sah 58-035, which is widely used as a specific ACAT inhibitor, binds specifically to the ER and ER and selectively activates ER-dependent transcription at concentrations comparable with those required for pharmacological activities.

	Materials and Methods

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Chemicals. [³H]17-Estradiol and [³H]oleic acid were purchased from GE Healthcare (Little Chalfont, Buckinghamshire, UK). ICI 164,384 was provided by Zeneca Pharmaceuticals plc (Macclesfield, UK), Sah 58-035 was kindly provided by A. Suter from Novartis (Basel, Switzerland), and 447C88 was a generous gift of M. Issandou from GlaxoSmithKline (Les Ulis, France). TMP-153 was from BIOMOL Research Laboratories (Plymouth Meeting, PA). 1-2-(4-Benzyl-phenoxy)-ethyl]-N-pyrrolidine hydrochloride (PBPE) was synthesized in our laboratory as described previously (Poirot et al., 2000). Unlabeled R1881 was purchased form NEN Life Science Products (Paris, France). Hydroxyflutamide (2-hydroxy-2-methyl-N-(4-nitro-3-(trifluoromethyl)phenyl)propanamide was a gift from Theramex (Monaco). Other compounds and chemicals were from the Sigma-Aldrich (St. Louis, MO). All solvents were from VWR (Fontenay sous Bois, France).

Molecular Structure Analysis. Computational chemical calculations were performed on a Silicon Graphics Indigo workstation using Insight II version 2000 (Accelrys, San Diego, CA). Minimal energy conformations were calculated using the Discover module (2.9.7/95.0/3.0.0) with CVFF force field. van der Waals volumes and van der Waals volume intersections were determined using the Search-Compare module version 95.0 (Accelrys). We first compared the structure of Sah 58-035 with that of ICI 164,384. Superimposition was carried out between the energy minimized structure of Sah 58-035 and of ICI 164,384 in the conformation adopted in the crystallographic structure of the ER-ICI 164,384 (Pike et al., 2001) (Protein Data Bank 1HJ1 [PDB] ). Superimposition was conducted using the diphenylethane part of Sah 58-035 that was superimposed carbon to carbon onto the steroidal backbone of ICI 164,384. For the superimposition with steroids, carbon 3 of the phenol group of ICI 164,384 was adjusted to carbon 4 of the toluol group of Sah 58-035, and the benzylic carbon linked to carbon 6 of the phenol of ICI 164,384 was superimposed onto the benzylic carbon of the phenyl of the diphenylethane of Sah 58-035. We have compared the van der Waals volume of Sah 58-035 and that of ICI 164,384. The percentage of superimposition was calculated by measuring the ratio of the intersection of the van der Waals volume of ICI 164,384 with the van der Waals volume of Sah 58-035. The percentage of superimposition between the steroidal backbone of ICI 164,384 with the diphenylethane of Sah 58-035 was also measured. We then proceeded to the comparison of ICI 164,384 with compound 447C88 using the same procedure as with Sah 58-035. TMP-153 does not contain a diphenyl ethane substructure, so the superimposition was between each phenyl ring of TMP-153 and the phenolic ring of ICI 164,384.

Estrogen Receptor Binding Assay. ER binding experiments were conducted using ERs extracted from COS-7 cells transfected with expression vectors coding for the human ER and ER. Expression of the receptors was checked by Western blotting using ER- and ER-specific antibodies (Doisneau-Sixou et al., 2003). Transfected cells were grown to 80% confluence in estrogen-depleted medium. Cells were then scraped and washed twice with PBS. After centrifugation for 10 min at 1500 rpm, the cells were resuspended in TM buffer (20 mM Tris-HCl, pH 7.4, and 20 mM sodium molybdate) and were lysed by freeze-thawing in an equal volume of TM buffer. Cytosolic receptors were prepared by a 105,000g x 60-min centrifugation at 0°C, and the supernatant was stored at -80°C. This receptor suspension was diluted to 60% in TM buffer and then incubated with different concentrations of Sah 58-035, compound 447C88, TMP-153, tamoxifen, or E₂ for 18 h at 4°C in a total volume of 100 µl containing 50 µg of protein and 1 nM [³H]17-estradiol. Assays were terminated by loading 65 µl of the incubate onto a 1.2-ml Sephadex LH-20 column equilibrated with TM buffer. The eluate was collected and counted for radioactivity in Ready Safe scintillant (Beckman Coulter, Fullerton, CA).

Microsomal Antiestrogen Binding Site Binding Assay. Binding assays were performed exactly according to a previously published procedure (Kedjouar et al., 2004). In brief, MCF-7 microsomes (10 µg of protein) were incubated in a binding buffer (20 mM Tris-HCl, 2.5 mM EDTA, pH 7.4, and 2.5 mM thioglycerol) with 3 nM [³H]tamoxifen (specific activity, 84 Ci/mmol; GE Healthcare) with 10 concentrations of the test ligands from 10 to 10,000 nM for 18 h at 4°C. Assays included 1 µM E₂. Free radioligands were separated using Sephadex-LH20 gel filtration (1.5 ml). The eluate was collected and counted for radioactivity in Ready Safe scintillant (Beckman Coulter).

Molecular Modeling with Estrogen Receptors. Sah 58-035, generated as described above, was prepositioned in the 4-hydroxytamoxifen (OHT)-ER LBD crystal structure (Protein Data Bank 3ERT [PDB] ) (Shiau et al., 1998) using the Search-Compare module of Insight II (Accelrys), and the superimposition of OHT and Sah 58-035 was done as described previously (de Medina et al., 2004b). Once prepositioned, OHT was unmerged from the OHT-ER complex and deleted, whereas Sah 58-035 was merged to the receptor. The resulting complex was submitted to energy minimization using 250 steps of the steepest descent followed by a conjugated gradient until the root mean square gradient was less than 0.001 kcal/mol/Å. A distant-dependent dielectric term ( = r) and 20-Å nonbonded cut-off distance were chosen, whereas the hydrogen bond involved in the conformation of the helices was preserved by applying a generic distance constraint between the backbone oxygen atoms of residue i and the backbone nitrogen atoms of residue i + 4, excluding prolines. This was performed using the Discover calculation engine with the CVFF force field (Insight II version 2000.1; Accelrys). The minimized coordinates of the receptor were then used as the starting point for 100 ps at 300k using the Verlet algorithm whereas the constraint used during minimization was maintained. The resulting conformation was then further minimized using 250 steps of the steepest descent followed by a conjugated gradient until the root mean square gradient was less than 0.001 kcal/mol/Å. The same procedure was carried out for the complex ICI 164,384-ER by replacing the OHT with ICI 164,384.

The ER molecular model was generated using Biopolymer (Accelrys) by first substituting Leu-384 with Met-384 and Met-421 with Ile-421 in the Sah 58-035-ER molecular model and then conducting an initial minimization of these two residues while keeping the remaining atoms fixed. This complex was then submitted to molecular dynamic simulation and energy minimization as described above. All minimizations were done with CVFF force field.

Reporter Cell Lines and Luciferase Assay. Stably transfected cells were obtained as described previously (Joyeux et al., 1997). The MELN cells and the MRLN cells were established by transfecting MCF-7 cells with the ERE--globin-Luc-SV-Neo plasmid and a RARE3-tk-Luc-SV-Neo plasmid, respectively. MELN cells expressed luciferase in an estrogen-dependent manner, and MRLN cells expressed luciferase in a retinoid-dependent manner. Cells were routinely grown in RPMI 1640 growth medium and MELN in DMEM growth medium, supplemented with 5% fetal bovine serum (Invitrogen, Carlsbad, CA). Cells were incubated at 37°C in a humidified 5% CO₂ incubator. For experiments, cells were grown for 5 days in phenol red-free medium, containing 5% dextran-coated charcoal-treated FCS. The medium was changed after 2 days. At day 5, cells were treated or not with the test compounds dissolved in ethanol. For each condition, 15 x 10³ cells per well were seeded in 12-well plates and treated, as described above, for8hina final volume of 0.5 ml. At the end of the treatment, cells were washed with PBS and lysed in 150 µl of lysis buffer (Promega, Charbonnières, France). Luciferase activity was measured using the luciferase assay reagent (Promega), according to the manufacturer's instructions. Protein concentrations were measured using the Bradford technique to normalize the luciferase activity data. For each condition, the mean luciferase activity was calculated from the data of three independent wells.

Cotransfection Assays. COS-7 cells were from the American Type Culture Collection (Manassas, VA) and were grown in DMEM supplemented with 10% fetal bovine serum. Cells were transfected with 1 µg of the expression vector for the ER and ER (pSG5-ER or pSG5-ER) and 10 µg of the estrogen-dependent luciferase reporter construct (ERE--globin-luc-SV-Neo) by the DEA-dextran methodology. For androgen receptor (AR)-dependent regulation of transcription, COS-7 cells were cotransfected with 1 µg of pSG5-AR and the androgen-dependent luciferase reporter construct (MMTV-luc) as described above. In each case, cells were cotransfected with 30 ng of a -galactosidase expression construct (pCMV-lacZ) to measure the efficiency of transfection. -Galactosidase activity was measured by the luminescence derived from 10 µl of each sample incubated in 200 µl of 1 mg/ml O-nitrophenyl--D-galactopyranoside and used to correct transfection efficiency among the different treatment groups (luminescent -galactosidase detection kit; BD Biosciences Clontech, Palo Alto, CA). Cells were treated as described above for permanent transfection cell lines. Luciferase activity was measured as described above and normalized with -galactosidase activity. For each condition, the mean luciferase activity was calculated from the data of three independent wells.

Cell Proliferation Study. MCF-7 (ER+) and MDA-MB-231 (ER-) cells were obtained from the American Type Culture Collection and grown as described above. T47D (ER+) were provided by J. F. Savouret (Institut National de la Santé et de la Recherche Médicale U587, Paris, France). For experiments, cells were grown for 5 days in phenol red-free medium containing 5% dextran-coated charcoal-treated FCS. Cells were seeded into 96-well plates at 2000 cells/well. Treatment media (150 µl/well) were added on the following day and replaced at 48-h intervals until the end of the experiment. Cell density was measured using the tetrazolium reduction assay (Sigma-Aldrich) (Cory et al., 1991) after 0, 2, 4, and 6 days. The absorbance at 490 nm of the formazan was measured directly in the 96-well plates with a multiscan multisoft reader from Thermo Electron Corporation (Waltham, MA).

Progesterone Receptor Expression Analysis. For each condition, 9 x 10⁵ cells were seeded in 140-mm-diameter dishes and treated, as described above, in a final volume of 15 ml. Cells were incubated for 48 h with E₂, tamoxifen, or Sah 58-035. Quantification of the progesterone receptor (PR) was performed on the cytosolic fraction of cells exactly as described in a previous article (Doisneau-Sixou et al., 2003). In brief, after treatment, the culture medium was removed, and the cells were washed twice with PBS and scraped into 350 µl of homogenization buffer (10 mM Tris buffer, pH 7.4, containing 20 mM molybdic acid and 12 mM monothioglycerol). The cells were lysed by three cycles of freezing/thawing (-170°C/20°C) and then centrifuged at 100,000g for 60 min at 4°C. We used the Abbott progesterone receptor-EIA monoclonal kit, according to the manufacturer's instructions (Abbott, Rungis, France). Cytosolic protein concentrations were measured using the Bradford technique to normalize the progesterone receptor expression data. For each condition, the mean receptor concentration was calculated from the data of two independent dishes.

Measurement of the Effect of Compounds on Cholesterol Esterification in Tumor Cell Lines. MCF-7 cells and MDA-MB-231 cells were grown as described above. To investigate the effect of the ACAT inhibitors Sah 58-035, 447C88, and TMP-153 and other compounds such as E₂, tamoxifen, ICI 164,384, and PBPE on cholesteryl ester formation, cells were incubated for 8 h with [³H]oleate. In brief, the cells were preincubated for 15 min with 10 concentrations of compounds ranging from 10 nM to 10 µM and then incubated in DMEM containing 5 µCi/ml [³H]oleate and 1% FCS in the CO₂ incubator for 8 h. The [³H]oleate incorporation process was studied, and the cholesteryl oleates were quantified as described previously (de Medina et al., 2004b).

	Results

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Sah 58-035 Shares Structural Similarities with Estrogen Receptor Ligands. The secondary structure of ACAT inhibitors (Sah 58-035, 447C88, and TMP-153), ER ligands (E₂, ICI 164,384, and tamoxifen), and microsomal antiestrogen binding site (AEBS) ligand (PBPE) are shown in Fig. 1A. These compounds have targets that are common with tamoxifen (de Medina et al., 2004a). We have shown previously that there was an overlap of 82.6% between the van der Waals volumes of E₂ and the diphenylethane part of Sah 58-035 (de Medina et al., 2004b). Sah 58-035 shows a close similarity with steroidal antiestrogens that contain a long hydrophobic side chain, such as ICI 164,384, when drawn (Jordan et al., 2001) in a two-dimensional representation (Fig. 1A). Therefore, we have investigated structural similarities that may exist between the active structure of ICI 164,384 (Fig. 1B) cocrystallized with the ER (Pike et al., 2001) and a minimal energy conformation of Sah 58-035 in a three-dimensional representation (Fig. 1B). The van der Waals volumes of Sah 58-035 and ICI 164,384 were 425 and 469 Å, respectively (Fig. 1B). Superimposition of the compounds is shown in Fig. 1B and reveals that ICI 164,384 and Sah 58-035 shared a common volume of 297 Å, which represented 70% of the van der Waals volume of Sah 58-035. The hydrophobic side chain of both compounds gave a perfect superimposition, with the exception of the ultimate ethyl group of the side chain of ICI 164,384. This showed that the molecular volume defined by Sah 58-035 was included into the ligand-accessible volume in the ER and that the orientation of the hydrophobic side chain of Sah 58-035 corresponds to that of the 7/11 substituent on the steroid backbone known to induce a high-affinity interaction with the ER (Jordan, 2003a). Altogether, these data suggest a possible interaction of Sah 58-035 with the ER. Compound 447C88 is another potent ACAT inhibitor structurally related to Sah 58-035 (Matsuda, 1994) that contains a diphenyl ethane backbone and an aliphatic side chain grafted onto the ortho position of the phenyl 2 related to the phenylethane group. The superimposition of compound 447C88 with ICI 164,384 gave an overlap of 82.6%, when comparing the DPE moiety with the steroidal backbone of ICI 164,384, but of only 33% for the whole molecule. This is because of the lack of super-imposition between the side chains of both compounds. TMP-153 is another ACAT inhibitor with a poor similarity to Sah 58-035 that does not contain a DPE motif. The superposition of TMP-153 with ICI 164,384 gave a maximal overlap of 37%, confirming the poor similarity between both compounds. The poor similarity of PBPE with ICI 164,384 has been described previously (de Medina et al., 2004b).

View larger version (23K): [in this window] [in a new window]   Fig. 1. Chemical structures of ACAT inhibitors, Sah 58-035, 447C88, and TMP-153; estrogen receptor ligands, ICI 164,384, E2, and tamoxifen; and a non-antiestrogen tamoxifen derivative that binds selectively to the microsomal AEBS, PBPE. In these molecules, the chemical groups that may be superimposable with the diphenyl ethane part of Sah 58-035 are in boldface. A, Sah 58-035 is very similar to the pure estrogen antagonist ICI 164,384. Three-dimensional structures of the calculated minimal energy conformation of Sah 58-035 and the conformation of ICI 164,384 taken in the crystallographic structure of rat ER-ICI 164,384 are shown. Calculated minimal energy was carried out using the Discover module of Insight II (version 2000) as described under Materials and Methods. Overlay of Sah 58-035 and ICI 164,384 as well as van der Waals volume calculations and intersection measurements were done using the Search-Compare module. The van der Waals volume intersection is depicted as the gray grid and illustrates the structural similarities between Sah 58-035 and ICI 164,384. The van der Waals volume of Sah 58-035 and of ICI 164,384 are 425.03 and 468.77 Å3, respectively. Seventy percent of the van der Waals volume of Sah 58-035 is in common with that of ICI 164,384.

Sah 58-035 Binds to the Estrogen Receptors. We then decided to evaluate the binding of ACAT inhibitors to ERs. Competition binding experiments were performed on extracts from COS-7 cells transfected with plasmids expressing ER or ER or extracts from MCF-7 cells for the AEBS measurement. We show in Table 1 that Sah 58-035 bound to the ER and ER in a concentration-dependent manner, with an IC₅₀ of 2.9 and 3.1 µM, respectively, whereas compounds 447C88 and TMP-153 had no measurable affinity.

View this table: [in this window] [in a new window]   TABLE 1 Binding of Sah 58-035, compound 447C88, and TMP-153 on ER, ER, and AEBS Binding of radiolabeled E2 on the ER and ER, or tritiated tamoxifen on the AEBS were measured at different concentrations of E2, tamoxifen, PBPE, Sah 58-035, compound 447C88, and TMP-153 as described under Materials and Methods. Values are the mean ± S.E.M. from three independent experiments.

To verify the selectivity of binding of Sah 58-035 to the ER and because the DPE moiety is present on most AEBS ligands (de Medina et al., 2004a), we have investigated whether Sah 58-035 might be a ligand of the AEBS. In Table 1, we show that the ACAT inhibitors Sah 58-035, compound 447C88, and TMP-153 had no measurable interaction with the AEBS.

The binding affinity of Sah 58-035 for the ERs in the micromolar range is not surprising since Sah 58-035 does not bear a phenolic group, which is known to be important for affinity to the ER (Anstead et al., 1997). The similarity of the rest of the molecule with ICI 164,384 might be sufficient to compensate for the absence of the phenol and enable the compound to recognize the ER. Interestingly, the IC₅₀ obtained for Sah 58-035 corresponded to the concentrations used for in vitro tests on tumor (this study) or other cell lines (5-30 µM) (Rodriguez et al., 1999).

Molecular Modeling of the Complex Sah 58-035-ER. The ability of Sah 58-035 to interact with the ER despite the absence of a phenol group raises the question of which molecular features underlie an interaction with the ER. In the absence of a crystal structure of the Sah 58-035-ER complex, we have investigated this issue by molecular modeling. The docking of Sah 58-035 into the ER taken from the X-ray structure of the complex ER-4-hydroxy-tamoxifen (Shiau et al., 1998), and energy minimization gave a complex in which the Sah 58-035 fitted well into the LBD. In Fig. 2A, we show the chemical interactions between Sah 58-035 and the ER. Interestingly, the methyl of the toluol group of Sah 58-035, which is isosteric to the phenol group of E₂ or OHT, defined a cluster of van der Waals interactions with the side chains of Glu-353, Arg-394, and Leu-349. The phenyl part of the toluol group produced a T-shape interaction with the phenyl side chain of Phe-404 and van der Waals contacts with the methyl groups of Leu-391 and Leu-384. The ethyl spacer of the DPE backbone of Sah 58-035 established van der Waals contacts with Leu-346 and Leu-384. The phenyl group of Sah 58-035 interacted with Met-343, Met-421, Ile-424, His-524, and Leu-525. These data showed that the DPE backbone of Sah 58-035 can occupy the same cavity as E₂ or DES (Brzozowski et al., 1997; Shiau et al., 1998). The side chain of Sah 58-035 protruded into the 11 cavity of the LBD of the ER and produced multiple van der Waals interactions with hydrophobic amino acids such as Ala-350, Leu-525, and Trp-383. No interaction could be observed between Sah 58-035 and Asp-351, which is involved in the antiestrogenic activity of SERM such as raloxifene (Levenson and Jordan, 1998). Van der Waals interactions can be seen between Leu-525 and Trp-583, with methyl groups carried by the silicon present on the side chain of Sah 58-035. The upper part of the side chain interacted with Val-533, Leu-536, Leu-539, Leu-540, and Met-543. These latter amino acids belong to helix H12, thus showing in this model an interaction between the upper part of the side chain of Sah 58-035 and helix H12. Analysis of the crystallographic data suggested that, when the LBD is occupied by the pure estrogen antagonist ICI 164,384, the extremity of the hydrophobic side chain of ICI 164,384 prevents the interaction of helix H12 with helices H3 and H5 (NR box) of the LBD (Pike et al., 2001). This is reinforced by our modeling studies in which we observed that when OHT is replaced by the ICI 164,384, the steroid binding domain is dramatically destructured, and the key interactions of the steroid backbone of ICI 164,384 with Glu-353, Arg-394, Phe-404, and His-524 are lost, whereas the helix H12 remains in interaction with helices H3 and H5. This suggests that the ligand accessible volume in the ligand binding pocket is not sufficient to make possible the interaction of ICI 164,384 with Glu-353, Arg-394, Phe-404, and His-524 when helix H12 interacts with helices H3 and H5. In Fig. 2B, we can see that the helix H12 did not move compared with the crystal of the ER-OHT complex, although the orientation of the side chains of leucine amino acids has changed. For example, the side chains of Leu-539 and Leu-536 have moved from 3 to 5.6 Å and are more accessible to the solvent. This may have consequences to the production of coregulator interaction surfaces. It is obvious that this does not rule out that the helix H12 could interact differently with helix H3 and H5 but makes it possible the formation of the NR box compatible with a SERM or an agonist activity. When we applied the same procedure after replacing OHT with compound 447C88, we observed a loss of the different points of interaction observed with Sah 58-035 or OHT, in particular the absence of an interaction between the phenyl ring and the Phe-404 of the ER. The side chain of 447C88 cannot fit into the 11 pocket of the ER (data not shown).

View larger version (32K): [in this window] [in a new window]   Fig. 2. A, cross-sectional view of the Sah 58-035 interactions with the ER ligand binding domain. Blue, Sah 58-035. Amino acid side chains that interact with Sah 58-035 are represented in the figure. Green, carbon atoms; white, hydrogen; red, oxygen; and yellow, sulfur. B, ribbon representation of the molecular model of Sah 58-035 liganded with the ER. Sah 58-035 is drawn in stick form and colored in blue. Helical elements are numbered (H3, H5, H7, H11, and H12) and colored in green.

View larger version (26K): [in this window] [in a new window]   Fig. 3. A, dose-response curve of Sah 58-035 on luciferase activity on MCF-7 cells stably transfected with ERE--globin-tk-Luc (MELN cells) as described under Materials and Methods. Results are represented as percentage of ER-dependent luciferase activity obtained with 10 nM E2. B, evaluation of the estrogenic activity of Sah 58-035, compound 447C88, and TMP-153 on MELN cells. Cells were incubated with 10 nM E2, with or without 100 nM or 10 µM Sah 58-035, 10 µM compound 447C88, and TMP-153 or with 10 nM E2 and 10 µM Sah 58-035 in combination with 1 µM antiestrogen ICI 164,384 or 1 µM tamoxifen, and assayed for luciferase activity. C, ER and ER transcriptional activation were compared in transient transfection experiments in COS-7 cells. Cells were transfected with the ER expression plasmids pSG5-ER or pSG5-ER and ERE--globin-tk-Luc. Cells were incubated with 10 nM E2, with or without 10 µM Sah 58-035 and 10 µM Sah 58-035 in the presence of 1 µM ICI 164,384, and assayed for luciferase activity. D, retinoid receptor (RAR) transcriptional activity measured in MCF-7 cells stably transfected with RARE3-tk-Luc+SV-Neo (MRLN) as described under Materials and Methods. Cells were incubated with 100 nM 9-cis-retinoic acid (cRA) and 10 µM Sah 58-035 alone or in combination and assayed for luciferase activity. Errors bars, mean values ± S.E.M. from three independent experiments. E, AR transcriptional activity was measured in transient transfection experiments in COS-7 cells. Cells were transfected with the AR expression plasmid pSG5-AR and the MMTV-Luc. Cells were incubated with 1 nM R1881 in the presence or in the absence of 1 µM hydroxyflutamide or Sah 58-035 and assayed for luciferase activity. *, p < 0.0005 compared with control.

Sah 58-035 Functions as an Agonist for Estrogen Receptor-Mediated Transcription. The ability of Sah 58-035 to bind to ERs raised the possibility that this compound might act as an ER agonist or antagonist since the presence of a long side chain was associated with either antagonist or agonist activity in the steroidal series (Wakeling and Bowler, 1988) or in the stilbene series (Zablocki et al., 1987). Our molecular modeling studies suggested that Sah 58-035 might not act as a pure antagonist. Therefore, we decided to evaluate experimentally the agonist/antagonist properties of Sah 58-035. We used MCF-7 cells stably transfected with a plasmid encoding an estrogen-responsive promoter fused to the luciferase gene that were called MELN (Doisneau-Sixou et al., 2003). In Fig. 3A, we show that Sah 58-035 stimulates expression of luciferase in MELN cells in a concentration-dependent manner with an EC₅₀ of 4.6 ± 0.5 µM(p < 0.0005) and reached a plateau at 10 µM, representing 74% of the maximal response observed with E₂. This agonist activity was observed in a range of concentrations consistent with the binding affinity of Sah 58-035 to the ER, and antiestrogens inhibited this stimulation. In Fig. 3B, we show that Sah 58-035, compound 477C88, and TMP-153 did not inhibit the stimulation of the transcription produced by E₂ and were therefore not ER antagonists. In contrast, pure or partial antagonists, such as ICI 164,384 and tamoxifen, inhibited the stimulation of the transcription by E₂. This suggests that conformational modifications and in particular the coregulator interactions surface produced by Sah 58-035 are different from those obtained with tamoxifen and more comparable with those produced by estrogens but still different because Sah 58-035 produced only a 74% maximal transcriptional activation. Only the ACAT inhibitor Sah 58-035, but not compounds 477C88 and TMP-153, stimulated the transcription of the luciferase reporter gene. Transient expression experiments in COS-7 cells (Fig. 3C) confirm that Sah 58-035 is an agonist on ER-dependent expression of luciferase on both ER and ER subtypes.

View larger version (13K): [in this window] [in a new window]   Fig. 4. Effect of the ACAT inhibitor Sah 58-035 on PR expression in MCF-7 cells. Cells were treated for 48 h with solvent vehicle, 10 nM E2, or 1 and 10 µM Sah 58-035. Analyses were performed as described under Materials and Methods. Results are expressed as the ratio of the amount of PR protein measured with treated cells versus the amount of PR found in solvent vehicle-treated cells. Errors bars, mean values ± S.E.M. from three independent experiments. *, p < 0.005 compared with control.

View larger version (16K): [in this window] [in a new window]   Fig. 5. Effect of Sah 58-035 on estrogen-regulated growth. A, MCF-7 cells were treated with 10 nM E2 and 5 µM Sah 58-035, with or without 100 nM ICI 164,384 (ICI) or with the solvent vehicle. Cell density was determined at the indicated time intervals. Each point represents the mean ± S.E.M. as described under Materials and Methods; p < 0.005 compared with control. B, comparison of the 6-day treatments of estrogen-sensitive cells, T47D cells, and estrogen-unresponsive cells MDA-MB-231, with E2, and Sah 58-035 with or without the pure antiestrogen ICI 164,384. Results are shown as a mean ± S.E.M. of triplicates from three independent experiments. *, p < 0.005, ** p < 0.001.

Sah 58-035 Stimulates the Proliferation of Estrogen-Dependent Breast Cancer Cells. MCF-7 and T47D cells, estrogen-positive human breast cancer cell lines, and MDA-MB-231 cells, an ER-negative breast cancer cell line, were used to compare the effect of the ACAT inhibitor Sah 58-035 on cell growth. As shown in Fig. 5A, Sah 58-035 (5 µM) induced a significant stimulation of proliferation the MCF-7 cells but to a lower extent than E₂ (10 nM) over a 6-day period. The stimulation of the proliferation observed with Sah 58-035 and E₂ was blocked by the estrogen receptor antagonist ICI 164,384. E₂ and Sah 58-035 stimulated the proliferation of T47D, another ER-expressing cell line, whereas they did not stimulate the proliferation of MDA-MB-231 cells that do not express ERs (Fig. 5B). Altogether, these data showed that Sah 58-035 acts as an agonist of the ER at pharmacological concentrations.

Sah 58-035 Inhibits the Esterification of Cholesterol in Mammary Tumor Cells. To evaluate a potential role of ACAT inhibition on the ER agonist activity stimulated by Sah 58-035, we evaluated the effect of ACAT inhibitors (Sah 58-035, compound 447C88, and TMP-153), estrogen receptor ligands (E₂, ICI 164,384, and tamoxifen) and AEBS ligands (tamoxifen and PBPE) on the esterification of cholesterol in ER-positive and -negative breast cancer cell lines. We then measured whether cholesterol esterification could occur and be inhibited in MCF-7 and MDA-MB-231 cells. First, we showed that cholesterol esterification occurred in these tumor cells and represented less than 5% of the total sterols. In Table 2, we report that ACAT inhibitors were efficient in inhibiting cholesterol esterification with respective IC₅₀ values of 8.5 (MCF-7) and 8.3 (MDA-MB-231) µM for Sah 58-035, 7.3 (MCF-7) and 6.7 (MDA-MB-231) µM for compound 447C88, and 2.1 (MCF-7) and 3.1 (MDA-MB-231) µM for TMP-153. As previously shown with intact macrophages (de Medina et al., 2004b), tamoxifen and the pure antiestrogen ICI 164,384 are inhibitors of cholesterol esterification in both cell lines, with IC₅₀ values of 7.8 (MCF-7), 8.1 (MDA-MB-231), 9.8 (MCF-7), and 11.1 (MDA-MB-231) µM, respectively, showing that these compounds are potent inhibitors of cholesterol esterification in human tumor cell lines. E₂ and PBPE, which is a nonantiestrogen diphenylmethane derivative of tamoxifen, displayed no measurable inhibition of cholesterol esterification with intact cells. In the breast cancer cell lines used, Sah 58-035 but also TMP-153 and 447C88 were inhibitors of cholesterol esterification, whereas only Sah 58-035 exerted estrogenic activity. These data rule out a role of ACAT inhibition in the estrogenic activity of Sah 58-035.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
We have shown previously that Sah 58-035 shared three-dimensional structural homologies with ER ligands. These data led us to demonstrate that antiestrogens were potent inhibitors of ACAT (de Medina et al., 2004b). Here, we report for the first time that Sah 58-035 behaves like an estrogen. This compound competed with radiolabeled E₂ for binding to the ERs, and it induced an ER-dependent transcriptional activation that was inhibited by antiestrogens. Moreover, Sah 58-035 was selective for a promoter containing an estrogen receptor element because it had no effect on promoter containing a retinoid receptor or androgen receptor-responsive element. Sah 58-035 also stimulated the proliferation of ER-positive breast cancer cell lines MCF-7 and T47D, whereas it had no effect on ER-negative MDA-MB-231 cells.

The selectivity of the ACAT inhibitor Sah 58-035 has never been questioned before. We show that Sah 58-035 displays estrogenic activities at concentrations used for pharmacological studies. Sah 58-035 has been reported to inhibit cholesterol accumulation in human macrophages (Rodriguez et al., 1999), and this activity seems to be important for preventing foam cell formation, which is an early step in the formation of atheromatous plaques (de Medina et al., 2004b). However, because estrogen receptor modulators have been reported to display atheroprotective properties in mammals, the estrogenic activity of Sah 58-035 may explain some of its biological activities, notably those cited above. Indeed, E₂ has been reported to stimulate the expression of the ATP binding cassette and apolipoprotein A-I, both of which are involved in cholesterol efflux by high-density lipoprotein formation (Kramer and Wray, 2002). Moreover, in most studies, the duration of the treatment with Sah 58-035 was sufficient to induce ER-dependent transcriptional activity. In addition, ERs have been detected in human macrophages, used for studies with Sah 58-035 (Rodriguez et al., 1999). Therefore, the estrogenic activity of Sah 58-035 must be taken into account to understand better its biological activity such as the inhibition of foam cell formation.

We have shown that Sah 58-035 shared 70% structural homology with the active conformation of ICI 164,384. We note a perfect superimposition between both compounds along the diphenylethane and the steroidal backbone and side chains, with the exception of the last ethyl of the antiestrogen side chain. In this study, we have shown by molecular modeling that the positioning of Sah 58-035 into the LBD is compatible with the structure of the ER-OHT determined by X-ray analysis. This suggests that when the ER is occupied by Sah 58-035, the helix H12 can be positioned between the helices H3 and H5. On the contrary, ICI 164,384 does not fit into this model. These data confirm that in the crystallographic structure of ICI 164,384/ER, the last ethyl of the ICI 164,384 hydrophobic side chain (Fig. 6A) prevents the positioning of the helix H12 between helices H3 and H5. Importantly the positioning of helix H12 between H3 and H5 associated with the intact carboxylate of Asp 351 might constitute a binding site for a coactivator and give rise to estrogenic activity in breast cancer cells (Jordan et al., 2001). Despite strong structural similarities with pure antiestrogen, the Sah 58-035/ER complex fulfills these characteristics. Moreover, Sah 58-035 does not interact with the carboxylate of Asp 351. The nature of the side chain of ER ligands has been shown to be crucial for pure ER antagonist activity (Wakeling and Bowler, 1988). As shown in Fig. 6A, the two common properties of pure ER antagonists are: the presence of a 20- to 22-Å-long side chain (ICI 164,384, ICI 182,780, EM-319, ZK-164,015); the presence of a polar group such as an amide (ICI 164,384, EM-319), a sulfinyl (ICI 182,780), or a sulfonyl (ZK-164,015) group localized at 10 to 12 Å from the steroidal(-like) backbone that may be directed toward the bulk solvent. This polar group may drive the orientation of the side chain within the LBD. Modifications to the size of this side chain have been made and showed that some minor structural modifications can have dramatic repercussions on activity. Figure 6B shows the secondary structure of partial ER agonists. The presence of a smaller side chain can produce ER agonists such as 2,3-bis-(4-hydroxyphenyl)-pentyl-3-(N,N-dimethylaminopropylsulfide) (Zablocki et al., 1987). The side chain of the Sah 58-035 is 19 Å long and does not contain a polar group, which is consistent with our observation that Sah 58-035 is not an antagonist of ERs. The absence of the N-methyl,N'-n-butyl amide substituent induced a strong diminution of affinity for the ER and gave a compound that displays partial agonist activities, suggesting that the length and the absence of a negative charge could both be important parameters. The fact that ICI 160,325 is also a partial agonist with high affinity for ER, whereas it differs only from the pure antagonist ICI 164,384 by the absence of the methyl group on the amidic nitrogen, is very interesting. Two main hypotheses can explain this effect: the amide bond can be hydrolyzed to give the above-mentioned carboxylic compound, which is a low-affinity and partial agonist for ER; and the amide group lead to an enol-amide tautomerism that can give E and Z isomers that may have different properties. If one considers, as pointed out by Pike et al. (2001), that the amide group present on the side chain of ICI 164,384 is positioned on a solvent-accessible part of the LBD, the positioning of the ultimate butyl group of the side chain may be different in both isomers and thus may have different properties in term agonist-antagonist activity. Thus, it would be of interest to test in these series the impact of the introduction of groups of various sizes and physicochemical properties and see the consequences in terms of agonist/antagonist action.

View larger version (15K): [in this window] [in a new window]   Fig. 6. A, chemical structures of pure antiestrogens, ICI 164,384, ICI 182,780, EM-319 (Jordan, 2003b), and ZK-164,015 (Biberger and von Angerer, 1996). The chemical groups involved in pure antagonist activity are highlighted in gray. B, agonists and partial agonists 2,3-bis (4-hydroxyphenyl)pentyl 3-(N,N-dimethylaminopropylsulfide) (Zablocki et al., 1987), Sah 58-035, 11-(3,17-hydroxy-estra-1,3,5(10)-triene-7-yl)-undecanoic acid (Wakeling and Bowler, 1988), and ICI 160,325 (Wakeling and Bowler, 1988).

Altogether, this study shows for the first time that the prototypical ACAT inhibitor Sah 58-035 is a modulator of the estrogen receptor at pharmacological concentrations, and this new mechanism of action must be taken into account to better understand the effects of this compound. Moreover, this study gives new structural insights not only for a better understanding of agonist versus pure antagonist activity on ERs but also for the conception of selective ligands for ERs or ACAT or both of these targets.

Among the different ACAT inhibitors that we have tested, Sah 58-035 was the only one that displayed estrogenic properties, showing that this property was restricted to some structural feature. The use of a DPE motif and the presence of a substituent grafted onto a benzylic carbon of the DPE might constitute an interesting way to develop dual activities of ACAT inhibition and estrogen receptor modulation and to analyze the efficiency of these compounds compared with the association of compounds selective for each target separately.

These results as well as our preceding report showing that tamoxifen was an inhibitor of ACAT as well as a modulator of the ER may raise the question of what activity may be mainly responsible to the atheroprotective effect. Because both estrogen receptors and ACAT are potential targets for atherosclerosis, the answer then will come from in vivo studies in which the effect of selective ACAT inhibitors will be evaluated and compared with dual ER modulators and ACAT inhibitors. Dual modulators of ERs and inhibitors of ACAT may represent new compounds of high medical interest because they will target two important proteins involved in atherosclerosis, Alzheimer's disease, and cancer.

	Acknowledgements

 
We thank P. Chambon, H. Richard-Foy, and F. Bayard for the gift of constructs and expression vectors used in this study.

	Footnotes

 
S.S.-P. is in charge of research for the Centre National de la Recherche Scientifique. This work was supported by the Institut National de la Santé et de la Recherche Médicale, by the French Ministry of Education and Research, and by grants from the Institut Claudius Regaud.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.104349.

ABBREVIATIONS: ACAT, acyl-cholesterol-acyl-transferase; Sah 58-035, 3-[decyldimethylsilyl]-N-[2-(4-methylphenyl)-1-phenylethyl]propanamide; ER, estrogen receptor; DPE, diphenylethane; DES, diethylstilbestrol; E₂, 17-estradiol; LBD, ligand binding domain; ICI 164,384, (N-n-butyl-N-methyl-11-[3,17-di-hydroxyestra-1,3,5(10)-trien-7-yl]-undecanamide; 447C88, (N-heptyl-N'-(2,4 difluoro-4,6-(2(-4-(2,2 dimethylpropyl)phenyl)ethyl)phenyl) urea); TMP-153, N-[4-(2-chlorophenyl)-6,7-dimethyl-3-quinolyl]-N'-(2,4-difluorophenyl)urea; PBPE, 1-2-(4-benzylphenoxy)-ethyl]-N-pyrrolidine hydrochloride; R1881, 17-methyltrienolone; PBS, phosphate-buffered saline; AEBS, antiestrogen binding site; OHT, 4-hydroxy-tamoxifen; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; AR, androgen receptor; PR, progesterone receptor; tk, thymidine kinase; SERM, selective estrogen receptor modulator; ICI 182,780, fulvestran; ICI 160,325, N-n-butyl-11-[3,17-di-hydroxyestra-1,3,5(10)-trien-7-yl]-undecanamide; EM-319, N-n-butyl-N-methyl-11-[3,17-di-hydroxy-16-chloro-estra-1,3,5(10)-trien-7-yl)-undecanamide; ZK-164,015, 5-hydroxy-2-(4-hydroxyphenyl)-3-methyl-1-[10-(pentylsulfonyl)decyl]indole; CVFF, consistent valence force field.

Address correspondence to: Dr. Marc Poirot, Institut National de la Santé et de la Recherche Médicale U 563, Unit on Metabolism, Oncogenesis, and Cell Differentiation, ITOM/CPTP, Institut Claudius Regaud, 20-24 rue du Pont Saint Pierre, 31052 Toulouse cedex, France. E-mail: Marc_Poirot{at}hotmail.com

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