EPI-001 is a selective peroxisome proliferator-activated receptor-gamma modulator with inhibitory effects on androgen receptor expression and activity in prostate cancer.

The androgen receptor (AR) is a driver of prostate cancer (PCa) cell growth and disease progression. Therapies for advanced PCa exploit AR dependence by blocking the production or action of androgens, but these interventions inevitably fail via multiple mechanisms including mutation or deletion of the AR ligand binding domain (LBD). Thus, the development of new inhibitors which act through non-LBD interfaces is an unmet clinical need. EPI-001 is a bisphenol A-derived compound shown to bind covalently and inhibit the AR NH2-terminal domain (NTD). Here, we demonstrate that EPI-001 has general thiol alkylating activity, resulting in multilevel inhibitory effects on AR in PCa cell lines and tissues. At least one secondary mechanism of action associated with AR inhibition was found to be selective modulation of peroxisome proliferator activated receptor-gamma (PPARγ). These multi-level effects of EPI-001 resulted in inhibition of transcriptional activation units (TAUs) 1 and 5 of the AR NTD, and reduced AR expression. EPI-001 inhibited growth of AR-positive and AR-negative PCa cell lines, with the highest sensitivity observed in LNCaP cells. Overall, this study provides new mechanistic insights to the chemical biology of EPI-001, and raises key issues regarding the use of covalent inhibitors of the intrinsically unstructured AR NTD.


Synthesis of EPI-001
General Chemical reagents were typically purchased from Sigma-Aldrich and used without additional purification unless noted. Bulk solvents were from Fisher Scientific. N,N-dimethylformamide (DMF) was rendered anhydrous by passing through the resin column of a solvent purification system (MBraun). Reactions were performed under an atmosphere of dry N 2 unless noted. Silica gel chromatography was preformed on a Teledyne-Isco Combiflash Rf-200 instrument utilizing Redisep R f Gold High Performance silica gel columns (Teledyne-Isco). Analytical HPLC analysis was performed on an Agilent 1200 series instrument equipped with a diode array detector and a Zorbax SB-C18 column (4.6 × 150 mm, 3.5 μm, Agilent Technologies). The method started with a 10% CH 3 CN (with 0.1% trifluoroacetic acid (TFA)) in H 2 O (0.1% TFA), the 10% CH 3 CN (with 0.1% TFA) was increased to 15% over 2 minutes, increased to 20% over 3 more minutes and then increased to 95% CH 3 CN (with 0.1% TFA) over 25 minutes. Nuclear magnetic resonance (NMR) spectroscopy employed a Bruker Ascend instrument operating at 500 MHz (for 1 H) and 125 MHz (for 13 C) at ambient temperature. Chemical shifts are reported in parts per million and normalized to internal solvent peaks or tetramethylsilane. Mass spectrometry was recorded in positive-ion mode on an Agilent MSD SL Ion Trap. (4-hydroxyphenyl)(4-(oxiran-2-ylmethoxy)phenyl) methanone (1). This compound was prepared by modification to the previously reported procedure [1]. To a stirred solution of NaH (60% dispersion in oil, 1.453 g, 36.331 mmol) in anhydrous DMF (150 mL) at 0°C was added bisphenol A (4.000 g, 16.514 mmol). The reaction was stirred for 30 min, then tetrabutylammonium iodide (1.220 g, 3.303 mmol) and epichlorohydrin (3.24 mL, 41.3 mmol) was added and the reaction was allowed to warm up to room temperature (rt) over 12 h. The reaction was quenched with water and extracted with ethyl acetate (3 × 50 mL). The organic layer was washed with water (2 × 30 mL), dried over Na 2 SO 4 , and concentrated in vacuo. The crude mixture was purified by silica gel chromatography (gradient CH 2 Cl 2 to 10% EtOAc in CH 2 Cl 2 ) to afford 1 (0.580 g, 12% yield) as a clear foam. 1  (4-(2,3-dihydroxypropoxy)phenyl)(4-(oxiran-2ylmethoxy)phenyl)methanone (2). This compound was prepared as previously described [1]. To a stirred solution of 1 (0.493 g, 1.735 mmol) in anhydrous DMF (15 mL) at rt was added K 2 CO 3 (0.480 g, 3.469 mmol) and glycidol (0.35 mL, 5.20 mmol). The reaction was stirred at 60°C for 12 h. The reaction was cooled to rt, quenched with water and extracted with ethyl acetate (3 × 15 mL). The reaction mixture was washed with water (2 × 10 mL), dried over Na 2 SO 4 , and concentrated in vacuo. The crude mixture was purified by silica gel chromatography (gradient 10% EtOAc in CH 2 Cl 2 to 50% EtOAc in CH 2 Cl 2 ) to afford 2 (0.258 g, 41% yield) as a clear foam. 1  EPI-001 [1][2][3][4] This compound was prepared by modification to the previously reported procedure. [1] To a stirred solution of 2 (0.164 g, 0.458 mmol) in CH 3 CN (10 mL) was added CeCl 3 × 7H 2 O (0.427, 1.146 mmol) and mixture was heated to reflux for 12 h. The reaction was cooled to rt, excess CH 2 Cl 2 was added to the white paste and

SUPPLEMENTARY METHODS
www.impactjournals.com/oncotarget/ the cerium salts were removed by filtration. The filtrate was concentrated in vacuo. The resulting crude material was purified by silica gel chromatography (gradient 50% EtOAc in CH 2 Cl 2 to 100% EtOAc) to afford EPI-001 (0.117 g, 64% yield) as a clear foam. 1  Two days after confluence, cells were subjected to twoday incubation in the adipocyte differentiation cocktail containing 10% fetal bovine serum (JRH Biosciences, Inc., Lenexa, KS), 115 mg/ml methylisobutylxanthine (Sigma Aldrich, Saint Louis, MO), 1mg/ml insulin (Sigma Aldrich, Saint Louis, MO), and 390 ng/ml dexamethasone (Sigma Aldrich, Saint Louis, MO). During the following 6 days, cells were maintained in DMEM containing 10% fetal bovine serum, 100 IU/ml penicillin/streptomycin and 1mg/ml insulin until fully differentiated with robust accumulation of lipid droplets. In the treated groups, Rosiglitazone at the concentration of 1 μM or EPI at various concentrations was added to the cultures during the entire 8-day treatment period. On day 8, cells were stained with Oil-red O or harvested for RNA extraction.

Plasmids
Gal4 tethering of AR TAU1 (amino acids 101-360) and TAU5 (amino acids 361-490) was achieved by PCR amplification of TAU1 or TAU5 from p5HBhAR-A using primers listed in Supplementary Table 1. Primers were designed to introduce in-frame recognition sites for EcoRI and BamHI, which were used to ligate the insert with EcoRI/BamHI-digested pM.

Cell transfection
LNCaP cells were transfected with siRNA (300 pmol) or reporter/transactivator plasmids (12 ug total DNA) via single-pulse electroporation at 305V for 10 ms using an ECM 830 Square-Wave cuvette electroporator (Harvard Apparatus) with 4.0 mm gap-width cuvettes, followed by seeding in RPMI + 10% FBS. C4-2 were transfected with Superfect reagent (Qiagen) according to manufacturer specifications. Transfections were performed in RPMI + 10% CSS, and cells were incubated at 37°C for 24 hours post transfection prior to further treatment. 293T were transfected with Lipofectamine 2000 (Life Technologies) according to manufacturer specifications. Cells were transfected overnight in serum-free DMEM, then fed with fresh DMEM + 10% FBS the following morning. Treatment of transfected cells with androgen and/or drug was performed for 8 hours or overnight in serum-free medium as indicated. Transfected cells were then harvested in assay-specific lysis buffers.

Western blot
Adherent cells were treated for 24 hours in serumfree RPMI containing drugs (1 nM DHT and/or EPI-001 at indicated doses). Cells were lysed in Laemmli buffer and equal masses of crude lysates were separated in denaturing polyacrylamide gels followed by transfer to nitrocellulose membranes (Bio-Rad). Membranes were blocked with 2.5% non-fat dry milk (w/v) in Tris-buffered saline with 1% Tween-20 (v/v) and probed with primary antibodies listed in Supplementary Table 2, followed by washing and incubation with horseradish peroxidase-conjugated secondary antibody (Santa Cruz). Chemiluminescence detection was performed using Super Signal West Pico reagent (Thermo) or WesternBright ECL (BioExpress) on HyBlot CL autoradiography film (Denville Scientific). Band intensity was assessed using NIH ImageJ software.

Proteasome inhibition
LNCaP and C4-2 were seeded in RPMI + 10% CSS. Cells were then serum-starved overnight and co-treated with 10 μM MG-132 (Sigma) and/or 50 μM EPI-001 and harvested at the indicated time points for western blot.

mRNA stability assay
LNCaP were seeded in RPMI + 10% CSS for 48 hours, then serum-starved overnight and co-treated with 10 μg/mL Actinomycin D (Sigma) and 50 μM EPI-001 in serum-free RPMI as indicated. Cells were harvested in guanidinium thiocyanate buffer at the indicated time points and RNA was extracted and analyzed via qRT-PCR.

Nascent RNA labeling and isolation
Nascent transcripts were labeled with biotin and subjected to streptavidin pull-down using the Click-iT Nascent RNA Capture Kit (Life Technologies) according to manufacturer specifications. Briefly, LNCaP cells were serum-starved overnight, then treated with 50 μM EPI-001 or vehicle control. After 7 hours incubation, cells were pulsed with 5-ethynyl Uridine (5EU) for an additional hour in the presence of EPI-001 or vehicle to label nascent transcripts, then harvested in Trizol (Life Technologies). Total RNA was then subjected to a Click chemistry reaction which attached a biotin molecule to 5EU-labeled nascent transcripts. RNA was re-precipitated, then bound to streptavidin-conjugated magnetic beads and washed 10X to remove unlabeled transcripts, leaving only biotin-5EU-labeled nascent RNA attached to the beads. First-strand cDNA synthesis was performed directly on RNA:bead conjugates using the SuperScript VILO cDNA synthesis kit (Life Technologies) according to manufacturer specifications, then subjected to qRT-PCR as described below.

Quantitative RT-PCR
LNCaP, C4-2, and PC-3 cells were seeded in RPMI 1640 with 10% CSS for 48 h to allow androgen signaling to nadir prior to drug treatment. Cells were co-treated with 50μM EPI-001 and/or 1 nM mibolerone or appropriate vehicle controls as indicated. RNA was extracted using an acid guanidinium thiocyanate-phenol-chloroform method as described [5] and reverse transcribed with a Transcriptor First-Strand cDNA synthesis kit (Roche) according to manufacturer specifications. Quantitative PCR for AR pre-mRNA and mature mRNA, PSA, hK2, TXNIP, CIDEC, LPL, FABP4 (aP2), PDK4, and either TBP, GAPDH, or Actin as housekeeping controls was performed using PerfeCTa SYBR Green Fastmix (Quanta Bioscience) according to manufacturer specifications. Primer sequences used in qPCR assays are listed in Supplementary Table 3. Fluorescence intensity was evaluated after every PCR cycle using a Bio-Rad My iQ thermal cycler set for default 2-step amplification (40 cycles). Gene expression was normalized to housekeeping controls (TBP, GAPDH, or Actin) using the formula 2 -∆∆Ct , where ∆∆Ct = threshold cycle of amplification difference between the gene of interest and the housekeeping control.

Prostate cancer explants
Patient tissues were obtained from the University of Texas Southwestern Medical Center tissue core under UTSW IRB STU 112013-056 and explant studies were performed as previously described [6,7]. Briefly, cancerous prostates were excised from patients with highrisk (GG8-10) high volume (> 2 positive cores) prostate cancers via robotic laparoscopic prostatectomy. Pathologyvalidated cancer tissue was dissected into 1 mm 3 cubes, and cultured on Surgifoam oral gelatin sponges (Ethicon) for 24 hours in RPMI + 10% FBS supplemented with insulin and hydrocortisone at 10 mg/L each. Explants were then washed 3 × 1 hr in RPMI + 10% CSS supplemented +/-1 nM Mibolerone alone or in combination with Troglitazone (10 or 50 μM) or EPI-001 (50, 100, or 200 μM) as indicated. Tissue explants were then cultured in RPMI + 10% CSS supplemented with 1 nM Mibolerone alone or in combination with Troglitazone or EPI-001 for 48 hrs at 37 o C. Explants were then removed from the sponges and lysed for western blot or qRT-PCR analysis using a tissue grinder in the appropriate lysis buffer.

Oil red O staining
Oil-red O staining was performed on 3T3-L1 cell cultures as previously described [8]. Briefly, cells were fixed in Baker's Formalin for 30 min at room temperature, followed by staining in a 60% (w/v) solution of Oil-red O (Sigma-Aldrich, Milwaukee, WI) in isopropyl alcohol for 10 min.
pH stability studies of EPI-001 and thiol reactivity assays General 2-Mercaptoethanol, cysteamine, and rose bengal were purchased from Sigma-Aldrich, and reduced glutathione and tris(2-carboxyethyl)phosphine hydrochloride salt (TCEP•HCl) were purchased from Alfa Aesar. All chemicals were used without additional purification unless noted.
A solution of thiol (10 equiv.) and TCEP•HCl (100 μL of a 0.5 M DMSO stock soln.) in 1x aqueous PBS (5 mL) was adjusted to the desired pH (2.4, 7.4, or 9.4) using either aqueous HCl (6 M) or aqueous NaOH (6 M) as determined by a pH meter (Thermo Scientific Orion 3 Star). Reduced l-glutathione, 2-mercaptoethanol, and cysteamine [9], were used as thiols in this assay. To a solution of the appropriate individual thiol (170 μL) was added either EPI-001 or compound 2 (1 equiv.; both compounds were solubilized as 50 mM DMSO stock solutions). The total composition of DMSO did not exceed 7.5% in any experiment and the ratio of thiol to EPI-001/compound 2 was 10:1. Note: This order of addition is key to achieving the appropriate pH environment for the reaction as the addition of reduced l-glutathione and TCEP acidify neutral solutions and cysteamine basifies neutral solutions. Aliquots of reactions were analyzed immediately (t ~ 30 min) following initial mixing by reverse phase HPLC and LC-MS. Reactions were gently shaken at 37°C for 12 h, then analyzed again using HPLC and LC-MS. Reactions were conducted in 2 mL clear screw cap glass vials (Agilent Technologies). Note: Thirty minutes was the earliest time point that could be collected after adding EPI-001, mixing, and injecting an aliquot onto the HPLC. The HPLC analytical method (Zorbax SB-C18 4.6 × 150 mm, 3.5 μm column, Agilent Technologies; flow rate = 1.0 mL/min) involved isocratic 10% CH 3 CN in 0.1% (v/v) aqueous CF 3 CO 2 H (0 to 2 min), followed by linear gradients of 10-85% CH 3 CN (2-24 min) and 85%-95% CH 3 CN (24-26 min). LC-MS analyses were performed on an Agilent 1100 series instrument equipped with an Agilent MSD SL Ion Trap mass spectrometer (positive-ion mode) and a Zorbax SB-C18 column (0.5 × 150 mm, 5 μm, Agilent Technologies). The analysis method (15 μL/min flow rate) involved isocratic 10% MeCN (containing 0.1% TFA) in ddH 2 O (containing 0.1% HCO 2 H; 0 to 2 mins) followed by a linear gradient of 10% to 90% MeCN (containing 0.1% TFA) in ddH 2 O (containing 0.1% HCO 2 H; 2 to 24 mins), and isocratic 90% MeCN (containing 0.1% TFA) in ddH 2 O (containing 0.1% HCO 2 H; 24-26 mins). The column was heated to 40°C. Wavelength monitored = 254 nm for all experiments unless otherwise noted. LC-MS analysis was performed on crude reaction mixtures. To quantify the amount of parent compound remaining, the area under the curve (AUC) of the parent compound was divided by the AUC of an internal standard. Rose bengal (7.5 μM) was used as the internal standard and was added immediately before HPLC analysis Experiments were performed in triplicate and values shown are the mean ± standard deviation (calculated in Microsoft Excel). Calibration curves to normalize for injection variances during HPLC analysis were generated for each compound (Supplementary Figure S20B). For both compounds, R 2 > 0.99.