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EPI-002 accelerates ligand dissociation from androgen receptor by disrupting N-terminus to C-terminus… Ding, Rick 2013

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EPI-002 Accelerates Ligand Dissociation from Androgen Receptor by Disrupting N-terminus to C-terminus Interaction   by   Rick Ding BSc., Simon Fraser University, 2005   A THESIS SUBMITTED IN PARTIAL FULFUILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF   MASTER OF SCIENCE   in   The Faculty of Graduate and Postdoctoral Studies (Pathology and Laboratory Medicine)   THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)   October 2013   ? Rick Ding, 2013   ii  Abstract Constitutively active splice variants of androgen receptor (AR) lacking the ligand-binding domain (LBD) are linked to the development and progression of castration-resistant prostate cancer (CRPC). Recent studies suggest a constitutively active splice variant, ARv567es, is capable of interacting with full-length AR, stabilizing and enhancing its ligand-dependent activities despite castrate levels of circulating androgen. EPI-001, an AR antagonist targeting the N-terminus domain (NTD) prevents N-terminus to C-terminus (N/C) interaction of AR, which is essential for AR antiparallel dimer formation. The ligand-dependent N/C interactions slow the dissociation of ligand from the LBD. Here we examine the effect of EPI-002, the most potent stereoisomer of EPI-001, on ARv567es complexed with full-length AR and test the hypothesis that EPI-002 will cause ligand to dissociate more quickly because it blocks N/C interaction. The aim of this study is two-fold as we first examined the effects of ARv567es on the dissociation rate of the full-length receptor. Then, we examined the effect of EPI-002 on the ligand dissociation rate of full-length AR with and without the presence of ARv567es. We have demonstrated that EPI-002 did not affect binding affinity of wild-type full-length AR nor the time for it to reach binding equilibrium. EPI-002 accelerated the ligand dissociation rate of wild-type full-length AR possibly by disrupting N/C interaction. Co-expression of ectopic ARv567es and wild-type full-length AR at 50:50 ratios did not alter the ligand dissociation rate of wild-type full-length AR but attenuated the effect of EPI-002. However, EPI-002 had minimal effect on the ligand dissociation rate of endogenous AR in LNCaP prostate cancer cells, consistent with the lack of effect when AR has a mutation in the LBD (T877A) that enhances the iii  N/C interaction and slows the ligand dissociation rate compared to the wild-type AR. Together these data begin to reveal 1) the unique mechanisms of splice variant ARv567es on the dissociation rate of full-length AR; and 2) the effect of an AR NTD inhibitor on the dissociation rate of full-length AR with and without the presence of splice variants.                   iv  Preface  This thesis is the unpublished and independent work by the author, and is prepared under the supervision of Dr. Marianne Sadar at the BC Cancer Agency Genome Sciences Centre. This thesis is an investigation into the molecular mechanism of small molecule drug EPI-002 on human androgen receptor. EPI-002 was prepared by members of Dr. Raymond Andersen?s laboratory in the departments of Chemistry and Earth & Ocean Sciences at the University of British Columbia.             v  Table of Contents  Abstract ......................................................................................................................................................... ii Preface ......................................................................................................................................................... iv Table of Contents .......................................................................................................................................... v List of Figures ............................................................................................................................................. viii Acknowledgements ....................................................................................................................................... x Chapter 1: Introduction .......................................................................................................................... 1 1.1 Prostate cancer ............................................................................................................................. 1 1.1.1 Epidemiology and diagnosis of prostate cancer ................................................................... 1 1.1.2 Progression to castration-resistant prostate cancer ............................................................ 2 1.1.3 Role of androgen receptor in castration-resistant prostate cancer ..................................... 3 1.2 Androgen receptor ........................................................................................................................ 5 1.2.1 Androgen receptor ................................................................................................................ 5 1.2.2 Amino-terminus domain ....................................................................................................... 8 1.2.3 DNA-binding domain ............................................................................................................. 9 1.2.4 Ligand-binding domain ......................................................................................................... 9 1.3 AR N-terminus to C-terminus interaction ................................................................................... 12 1.3.1 AR N-terminus to C-terminus interaction ........................................................................... 12 1.3.2 Factors impacting AR N/C interaction ................................................................................. 14   1.3.2.1   AR ligand specificity ............................................................................................................. 14   1.3.2.2   AR mutation in AIS ............................................................................................................... 15   1.3.2.3   AR mutation in prostate cancer .......................................................................................... 15   1.3.2.4   AR coregulators ................................................................................................................... 16   1.3.2.5   NTD polymorphism .............................................................................................................. 18   1.3.2.6   Small molecule drugs .......................................................................................................... 18 1.4 AR splice variants ........................................................................................................................ 19 1.4.1 AR splice variants ................................................................................................................ 19 1.4.2 ARv567es ................................................................................................................................. 20 1.4.3 AR-V7 ................................................................................................................................... 21 1.5 EPI compound ............................................................................................................................. 23 vi  1.5.1 EPI compound as a novel AR NTD inhibitor ........................................................................ 23 1.6 Androgen receptor-ligand kinetics ............................................................................................. 26 1.6.1 Androgen receptor-ligand kinetics ..................................................................................... 26 1.6.2 Ligand binding affinity ......................................................................................................... 28 1.6.3 Ligand dissociation .............................................................................................................. 29 1.6.4 Ligand dissociation and AR N/C interaction ........................................................................ 31 1.7 Hypothesis and specific aims ...................................................................................................... 32 Chapter 2: Materials and Methods ....................................................................................................... 34 2.1 Cell culture .............................................................................................................................. 34 2.2 Expression plasmids and transfection .................................................................................... 34 2.3 Ligand binding assay ............................................................................................................... 35 2.4 Ligand dissociation assay ........................................................................................................ 35 2.5 N/C interaction ........................................................................................................................ 36 2.6 Western blot ........................................................................................................................... 37 Chapter 3: Results ................................................................................................................................. 38 3.1 Background binding of 3H-R1881 in Cos-1 cells was negligible .............................................. 38 3.2 EPI-002 did not affect the binding affinity (Kd) of wt ARfl ....................................................... 39 3.3 EPI-002 did not alter the time for wt ARfl and 3H-R1881 to reach binding equilibrium ......... 40 3.4 Splice Variant ARv567es had no effect on the dissociation rate of 3H-R1881 from wt ARfl ....... 40 3.5 EPI-002 accelerated the dissociation rate of 3H-R1881 from wt ARfl ...................................... 41 3.6 EPI-002 blocked N/C interaction Cos-1 cells ........................................................................... 42 3.7 Variant ARv567es attenuated the effects of EPI-002 on the dissociation rate of 3H-R1881 from wt ARfl ...................................................................................................................................... 42 3.8 Effect of EPI-002 on the ligand dissociation rate of endogenous full-length AR in LNCaP cells .   ................................................................................................................................................. 43 3.9 Effect of EPI-002 on LNCaP-Cu3 cells expressing endogenous full-length AR and cumate-induced FLAG-ARv567es ............................................................................................................. 45 3.10 EPI-002 accelerated the rate of ligand dissociation of LNCaP95 cells expressing endogenous full-length AR and splice variant AR-V7 .................................................................................. 46 Chapter 4: Discussion ............................................................................................................................ 62 4.1 EPI-002 and AR ligand binding ................................................................................................ 62 4.2 EPI-002 and dissociation of ligand from wild-type full-length AR .......................................... 63 4.3 Splice variant ARv567es and dissociation of ligand from wild-type full-length AR .................... 64 vii  4.4 LNCaP prostate cancer cells and AR T877A mutant................................................................ 66 4.5 EPI-002 and LNCaP95 prostate cancer cells ............................................................................ 68 Chapter 5: Summary and Future Directions ......................................................................................... 71 References .................................................................................................................................................. 74                viii  List of Figures  Chapter 1 Figure 1.1 Structural organization of the human AR gene and protein??????????????.   6  Figure 1.2 Genomic pathway of AR signaling?????????????????????????????  7  Figure 1.3 Crystal structures of wild-type AR ligand-binding domain bound with DHT???.?? 10 Figure 1.4 Structure of AR FXXLF peptide bound to the AR LBD??????????????????. 11 Figure 1.5 The mRNA organization of full-length AR, and splice variants ARv567es and AR-V7?.. 22 Figure 1.6 Structure of EPI-001 and stereoisomer EPI-002 (2R, 20S)???????????????.. 24 Figure 1.7 Proposed model of covalent binding reaction of EPI-001 to AR AF1?????????.. 24 Figure 1.8 Law of mass action: simplified rate reaction and equations??????????????. 27 Figure 1.9 Analysis of saturation binding data using Scatchard plot and non-linear regression. 29  Figure 1.10 Analysis of ligand dissociation data using non-linear and linear regression????.. 30 Chapter 3 Figure 3.1 Specific binding of 3H-R1881 to full-length wild-type AR in Cos-1 cells???????? 47 Figure 3.2 EPI-002 did not affect the binding affinity (Kd) of wild-type full-length AR in Cos-1 cells????????????????????????????????????????????????. 48 Figure 3.3 EPI-002 did not alter the time for wild-type full-length AR to reach binding equilibrium???????????????????????????????????????????????? 49 Figure 3.4 ARv567es had no effect on the dissociation rate of 3H-R1881 from wild-type full-length AR in Cos-1 cells????????????????????????????????????????????? 50 Figure 3.5 EPI-002 accelerated the dissociation of 3H-R1881 from wild-type full-length AR in Cos-1 cells????????????????????????????????????????????????. 51 Figure 3.6 EPI-002 blocked AR N/C interaction induced by androgen??????????????.. 52 Figure 3.7 ARv567es attenuated the effect of EPI-002 on the dissociation of 3H-R1881 from wild-type full-length AR in Cos-1 cells???????????????????????????????????. 53 ix  Figure 3.8 Effect of EPI-002 on the protein levels of wild-type full-length AR in Cos-1 cells??  54 Figure 3.9 Effect of EPI-002 on the dissociation of 3H-R1881 from endogenous AR in LNCaP cells???????????????????????????????????????????????????? 55  Figure 3.10 EPI-002 had no effect on the dissociation of 3H-R1881 from AR T877A in Cos-1 cells???????????????????????????????????????????????????? 56 Figure 3.11 Levels of endogenous full-length AR and cumate-induced FLAG-ARv567es in LNCaP-Cu3 cells?????????????????????????????????????????????????. 58 Figure 3.12 Effect of EP-002 on the dissociation of 3H-R1881 from endogenous full-length AR and cumate-induced FLAG-ARv567es in LNCaP-Cu3 cells??????????????????????? 59 Figure 3.13 EPI-002 accelerated the dissociation of 3H-R1881 from endogenous AR in LNCaP95 cells???????????????????????????????????????????????????? 61 Chapter 4 Figure 4.1 Predicted binding model of FXXLF motifs in the AF2 region of wild-type (A) and  T877A AR (B) LBDs???????????????????????????????????????????.. 67              x  Acknowledgements I would like to thank my supervisor, Dr. Marianne Sadar, for giving me the opportunity to pursue graduate studies in her lab. Marianne has always dedicated time to discuss the progress of my project, and has never hesitated to review my abstracts, posters, presentations, reports and thesis. Marianne, I am grateful for your continued support and guidance, thank you for being a compassionate and optimistic mentor.  I have been very fortunate to be surrounded by a wonderful team of brilliant and intelligent scientific researchers throughout my graduate studies in the Sadar lab. I want to thank our lab manager, Rina Mawji, for her patience, guidance, and equipping me with the technical skills required to complete my project. Thanks to Drs. Adriana Ba?uelos, Amy Tien, Jaekyung Myung, and Simon Merhu for the insightful discussions. Thanks to my fellow graduate students Kevin Yang and Erica Osbourne for their support, friendship, and great sense of humor. I want to thank the staff and students at the BC Cancer Genome Sciences Centre who have made this experience both rewarding and enjoyable.  I want to thank my supervisory committee Drs. Marcel Bally, Sandra Dunn, and Raymond Andersen for their invaluable advice and input on my project. I am grateful for the funding provided by the National Institutes of Health/National Cancer Institute USA (NIH/NCI) Grant # 5R01CA105304, as well as the Department of Defense, US Army Medical Research and Materiel Command Prostate Cancer Research Program (W81XWH-11-1-0551). I am also thankful for the scholarship provided by UBC Faculty of Medicine Graduate Award and the travel award provided by John Bosdet Memorial Fund. xi  Finally, this work would not have been possible without the enduring love and support of my family and friends. I would like to express my deepest gratitude to my mother, JH Liu, for her encouragement, unconditional love and support.           1  Chapter 1: Introduction 1.1 Prostate cancer 1.1.1 Epidemiology and diagnosis of prostate cancer Prostate cancer is the third most common cause of cancer-related death in Canadian men, with 1 in 7 men expected to be diagnosed with the disease in their lifetime [1]. The risk factors of prostate cancer include age, race, family history, diet and lifestyle [2]. The incidence of prostate cancer is generally higher with increasing age. Prostate cancer is most frequently diagnosed in Canadian men aged 60?69 years but most prostate cancer death will occur in men aged 80 years and older [1]. In spite of its high incidence, the progression of prostate cancer is relatively slow. In fact, the mortality rate for prostate cancer has been declining since the mid 1990?s [1]. This decline in mortality has been attributed to early detection and improved treatments [3]. In Canada, screening for prostate cancer for early detection involves PSA (prostate specific antigen) testing in conjunction with a DRE (digital rectal exam) [4]. PSA is a serine protease produced by both benign and malignant prostate epithelial cells and is the most commonly used serum marker used to monitor disease development and tumour progression [5]. The PSA test is not currently recommended in Canada as a population-based screening test [1]. Early detection by population-wide PSA screening can result in over-diagnosis of clinically indolent cancers, over-treatment, and treatment-related morbidity [6]. Therefore, the decision to use PSA for the early detection of prostate cancer should be individualized [4]. According to the 2011 Canadian Urological Association (CUA) guideline, PSA testing should be offered to all men 50 years of age with a life expectancy of at least 10 years [7]. Men with increased risk of 2  prostate cancer such as those of African descent or with a family history of prostate cancer should consider testing at age 40 [7]. Once screening determines that a man has prostate cancer, a transrectal ultrasound-guided prostate biopsy is given to obtain the histological diagnosis regarding the staging and grade of the cancer [4].  1.1.2 Progression to castration-resistant prostate cancer  Active surveillance is a conservative treatment plan widely practised in Canada for men with low risk prostate cancer to avoid over-treatment [7]. During active surveillance, low risk prostate cancer is monitored without treatment until the disease is worsened and treatment is necessary. Localized, low grade prostate cancer can be effectively managed with primary therapies such as radical prostatectomy or radiation therapy [8]. In one study, a short course of androgen ablation therapy (discussed later) adjuvant to radiation therapy has been associated with significant improvement in overall survival for localized prostate cancer [9]. While the majority of men with prostate cancer do not require treatment or are cured with primary therapy, approximately 20 to 40% of these patients will experience tumor recurrence [10].  Recurrent prostate cancer after primary treatment is androgen-sensitive and androgen ablation therapy through surgical or chemical castration has been the standard treatment for more than half a century [11]. Suppression of serum testosterone in these patients can be achieved by bilateral orchiectomy, antiandrogens, and luteinizing-hormone releasing hormone (LHRH) analogues [12]. Androgen ablation therapy reduces serum testosterone to castrate levels (<0.5ng/mL) [12]. However, androgen ablation therapy only achieves transient therapeutic effect for a median time of approximately 18-36 months. In the absence of testicular androgen, 3  the tumours eventually return in a more aggressive form, and the disease becomes castration-resistant prostate cancer (CRPC). CRPC are advanced tumours that are now resistant to androgen ablation therapy but may not be 100% free from androgen since very low levels of androgens are still detectable in the serum and tissues of patients with CRPC [11]. The growth of castration-resistant tumours can be fueled by residual circulating androgens produced by the adrenal glands [13].  Ongoing steroidogenesis within the tumours and the maintenance of intratumoural androgens may also contribute to CRPC growth [14]. The standard first-line treatment for patients with symptomatic metastatic CRPC is docetaxel-based chemotherapy [15]. Recently, abiraterone acetate and enzalutamide have been approved by the US Food and Drug Administration (FDA) to treat those patients who have failed docetaxel treatment [15]. Despite the advances in the development of CRPC therapies, most patients succumb to the disease within 2-3 years [10] 1.1.3 Role of androgen receptor in castration-resistant prostate cancer Almost all cases of prostate cancer are adenocarcinomas that express androgen receptor (AR), whereas small cell neuroendocrine carcinomas do not express AR and account for no more than 1% of all tumour cells in prostate cancer [16]. In some cases, an increasing number of neuroendocrine tumour cells are observed after hormone therapy for adenocarcinoma, suggesting that the neuroendocrine tumour cells can promote AR-independent growth in CRPC [16].  AR is a ligand-dependent transcription factor and the action of androgen through AR has been studied extensively in prostate cancer [17]. AR is expressed throughout the progression of 4  prostate cancer. AR levels are amplified in CRPC patients compared to those patients with untreated primary prostate cancer [18]. The levels of androgen in CRPC tissues can be sufficient to transactivate AR and elevate the expression of androgen-regulated genes such as PSA. These evidences suggest CRPC is not AR independent. [13].  There are several mechanisms through which AR can sustain transcriptional activity in castration resistant tumours [19]. For example, the resurgence of AR transcriptional activity can be contributed by amplification of the AR gene, overexpression of AR protein, and gain-of-function somatic mutation in the AR ligand binding domain. Since AR requires interaction with a number of coregulatory proteins to mediate tissue-specific responses to androgen, perturbed interaction with normal AR co-activators and co-repressors can lead to persistent AR transactivation [19]. Intratumoural androgen synthesis has also been implicated to activate AR during the depletion of testicular androgen [14]. In addition, AR can be activated in CRPC through androgen-independent pathways by: 1) cytokines such as interleukin-6 (IL-6) and interleukin-8 (IL-8); 2) growth factors such as insulin-like growth factor (IGF-I) and epidermal growth factor (EGF); and 3) signaling with intracellular kinases such as mitogen-activated protein kinase (MAPK), cAMP-dependent protein kinase A (PKA), and protein kinase B also known as Akt [11]. Recently, constitutively active, truncated splice variants of AR lacking the c-terminal ligand binding domain have been discovered in CRPC by several groups [20-22]. The role of AR splice variants in CRPC will be discussed later in section 1.4. 5  1.2 Androgen receptor 1.2.1 Androgen receptor Androgen receptor (AR) is a member of the steroid and nuclear receptor superfamily [23]. AR mediates androgen actions by functioning as a ligand-dependent transcriptional factor. AR is expressed in various tissues including the prostate, seminal vesicles, epididymis, testis, skeletal muscle, skin and central nervous system with highest levels observed in the androgen-dependent tissues that include the prostate, adrenal gland, and epididymis [24]. The physiological functions of AR include male sexual differentiation during development and the maintenance of male sexual characteristics [24].  The AR gene is more than 90kb long and located on the X-chromosome at Xq11-12 [25, 26].  The AR mRNA consists of 8 exons that code for a 919 amino acid protein with a molecular weight of approximately 110kDa (Figure 1.1) [27-29]. The number of amino acids encoded by the AR gene may vary due to polymorphisms in the length of polyglutamine and polyglycine tracts in amino-terminal domain of  the receptor [24, 30]. AR protein contains multiple functional domains including an amino (N)-terminal transactivation domain (NTD), a DNA binding domain (DBD), a small hinge region, and a carboxyl (C)-terminal ligand binding domain (LBD) [31].  In addition, AR contains two transactivation functions: activation function 1 (AF1) in the NTD is ligand-independent and is responsible for most of AR transcriptional activities whereas the LBD activation function 2 (AF2) has limited transactivation function upon ligand binding [24]. 23FXXLF27 and 433WXXLF437 peptide motifs in the NTD interact with AF2 and contribute to AR 6  transcriptional activation (discussed in the section 1.2.2 and 1.2.4). A nuclear localization signal (NLS) spans the region between the DBD and the hinge region to allow AR nuclear translocation for transcriptional activation [24].  Figure 1.1 Structural organization of the human AR mRNA and protein. Schematic representation of the human androgen receptor mRNA containing 8 exons and the human AR protein containing NH2-terminal domain (NTD), DNA binding domain (DBD), hinge region (Hinge), and ligand binding domain (LBD). AR protein includes the follow features: activation function 1 (AF1), activation function 2 (AF2), FXXLF and WXXLF motif, and nuclear localization signal (NLS). Figure 1 from ? Gao, W et al. (2005). Chemistry and structural biology of androgen receptor. Chemical Reviews. 105(9): Page 3352-70 [27]. By permission from American Chemical Society Publications. In Figure 1.2, circulating testosterone (T) enters the target cell and is converted to the more potent androgen, dihydrotestosterone (DHT), by 5?-reductase in the cytosol [30]. AR protein associates with heat-shock proteins (HSP) in the cytosol in the absence of ligand [32]. The binding of androgen to AR ligand binding pocket initiates dissociation of AR from HSP. Then AR undergoes a series of conformational changes including phosphorylation, dimerization and translocation into the nucleus [27]. AR translocation into the nucleus is mediated by the recognition and binding of AR NLS to nuclear import factor importin-? [33]. The nuclear AR 7  binds to androgen response element (ARE) in the promoter or enhancer region of the AR target genes, followed by the recruitment of coregulatory proteins as well as the general transcriptional machinery to complete the transactivation of the AR target genes [27]. Androgen regulated genes such as PSA and TMPRSS2 are expressed as the result of this genomic pathway of ligand-dependent AR signaling [34, 35].  Figure 1.2 Genomic pathway of AR signaling.  Testosterone (T) enters the cell and is converted to dihydrotestosterone (DHT) by 5?-reductase. Upon binding to DHT, androgen receptor dissociates from heat shock proteins (HSP), translocates to the nucleus, binds to its target genes and regulates their expression. Adapted from Figure 2 from ? Lonergan, PE et al. (2011). Androgen receptor signaling in prostate cancer development and progression. Journal of Carcinogenesis. 10: Page 20 [29]. Under the terms of Creative Commons Attribution License.    8  1.2.2 Amino-terminus domain AR NTD has limited sequence homology compared to the NTD of other members of the steroid receptor family [36]. AR NTD is characterized by two peptide motifs that each forms an amphipathic ?-helix (23FXXLF27 and 433WXXLF437). Upon the binding of androgen, the FXXLF motif  preferentially binds to AF2 surface in the LBD to induce N-terminal and C-terminal (N/C) interaction that partly contributes to AR transcriptional activity [37, 38].  Another feature of the AR NTD is the presence of several polymorphic glutamine and glycine repeats [28, 39]. The glutamine repeat is on average the largest which can vary between 9-37 residues in normal human population [40]. Expansion of polyglutamine repeat beyond 40 residues is associated with the X-linked spinal bulbar muscular atrophy commonly known as Kennedy?s disease [41]. Deletion studies have shown that NTD is essential for ligand-dependent and ligand-independent transcriptional activity of AR [42]. The NTD AF1 region encompasses two transcriptional activation units (TAU); TAU1 and TAU5 are responsible for most of the constitutive transcriptional activity of NTD [29, 43]. AR NTD can communicate with cell?s transcriptional machinery through binding and interaction of AF1 region with a number of AR coregulatory proteins such as RAP74, SRC-1, CBP, and ART27 [36, 44, 45]. The crystal structure of AR NTD is not yet available due its highly disordered, flexible structure [45]. The structure of AR NTD is described as intrinsically disordered [36]. The AR AF1 has a collapsed disordered structure with only 13% helical secondary structure [45, 46]. The disordered structure of AR NTD can fold into a more ordered structure upon protein-protein 9  interactions, creating a docking platform for interaction with coregulatory proteins [46-48]. For example, the binding of transcription factor TFIIF induces the AF1 to adopt a more folded helical conformation [48]. In addition, the presence and the length of the polyglutamine repeat have been shown to modulate the structural plasticity of the AR NTD [49]. 1.2.3 DNA-binding domain The structure of AR DBD is highly conserved and is characterized by two zinc finger motifs that recognize and bind to selective AREs in the promoter or enhancer region of AR-regulated genes [27]. The consensus sequence for AR binding consists of two hexameric half-sites (5?-AGAACA-3?) arranged as an inverted repeat with a 3-bp spacer [50]. AR binds to DNA as a homodimer and this DNA-dependent dimerization of AR is mediated by the DBD [51]. 1.2.4 Ligand-binding domain The AR LBD facilitates the binding of ligands such as T and DHT. The crystal structures of AR LBD complexed with a variety of ligands as well as coregulator peptide motifs have been solved [52]. The AR LBD has a sandwich-like structure made of 12 ?-helices and 2 short ?-turns arranged in three layers with the ligand binding pocket buried in the interior of the protein domain (Figure 1.3) [53]. The ligand binding pocket is formed by helix 5, the N-terminal region of helix 3 and the C-terminal of helices 10 and 11. The binding of agonist repositions helix 12 to serve as the ?lid? to cover ligand binding pocket and to stabilize the ligand. The second ?-turn at the very end of the C-terminal region of the LBD works as a ?lock? to further stabilize the ligand. Agonist binding induces a conformational change in the LBD to expose the AF2 surface in the LBD [27]. AR AF2 forms a hydrophobic groove composed of helices 3, 4 and 12 and interacts with 10  coregulators containing nuclear receptor box LXXLL motif during coregulator recruitment for transcriptional activation [54]. AR AF2 can also interact with FXXLF or WXXLF motifs in the AR NTD to mediate N/C interaction [38]. In Figure 1.4, the hydrophobic groove of AF2 binds to the leucine and phenylalanine residues in the FXXLF peptide motif through hydrophobic interaction.  A charged clamp formed by lysine 720 and glutamate 897 surrounding the AF2 groove makes contact with the backbone of the FXXLF peptide to further stabilize the binding [55, 56]. Crystal structure of DHT-bound AR LBD showed a deep hydrophobic groove is formed in the AF2 to accommodate bulky side chain of phenylalanine. As the result, AR AF2 groove prefers the binding of the FXXLF motif in the NTD to mediate N/C interaction over the LXXLL motif in coregulators [55].     Figure 1.3 Crystal structures of wild-type AR ligand-binding domain bound with DHT (1I37.pdb). (A) front view; (B) ligand view. Space filled atoms are (black) carbon and (red) oxygen. The activation function 2 region (helices 3, 4, and 12) is highlighted in green. Figure 3 from ? Gao, W et al. (2005). Chemistry and structural biology of androgen receptor. Chemical Reviews. 105(9): Page 3352-70 [27]. By permission from American Chemical Society Publications. 11   Figure 1.4 Structure of AR FXXLF peptide bound to the AR LBD. Left: Helices 3, 4, 5 and 12 of AR LBD (green ribbon). The charged residues K720 (red) and E897 (blue) are indicated at the opposite ends of the groove. FXXLF peptide backbone is shown as the alpha coil in orange. Side chains of the FXXLF amino acid residues at positions +1, +4, and +5 are shown as sticks. Right: Close-up view of the AR AF2 binding groove with FXXLF peptide. Figure 4C & D from ? van de Wijngaart, DJ et al. (2012). Androgen receptor coregulators: Recruitment via the coactivator binding groove. Molecular and Cellular Endocrinology. 352(1-2): Page 57-69 [52]. By permission from Elsevier.              12  1.3 AR N-terminus to C-terminus interaction 1.3.1 AR N-terminus to C-terminus interaction The androgen-dependent AR NH2- and carboxyl-terminal (N/C) interaction is induced upon the high affinity binding of testosterone (T), dihydrotestosterone (DHT) or synthetic androgen methyltrienolone (R1881) to AR. AR N/C interaction is mediated by the FXXLF motif (23FQNLF27) in the NH2-terminal domain (NTD) and the activation function 2 (AF2) hydrophobic binding surface in the carboxyl-terminal ligand binding domain (LBD). Ligand induced N/C interaction contributes to AR transcriptional activation partly by slowing the dissociation rate of bound ligand and stabilizing AR against degradation without affecting the ligand binding affinity [37, 57-60]. AR N/C is required for normal androgen signaling because mutations in the AR AF2 characterized by disruption of N/C interaction and faster ligand dissociation rate have been found in patients with androgen insensitivity [59, 61]. Ligand-dependent N/C interaction has also been reported in other steroid receptors such as progesterone receptor [62], estrogen receptor-? [63] and mineralocorticoid receptor [64] . Ligand binding triggers a conformational change in the AR LBD where the LBD helix 12 is repositioned to seal the ligand binding pocket stabilizing the bound ligand. As the result, AF2, a hydrophobic groove is generated on the LBD surface to interact with AR NTD FXXLF motif and LXXLL motif present in AR coactivators [65]. Unlike most members of the steroid receptor family, AR AF2 preferentially interacts with the AR NTD FXXLF motif over the LXXLL motif in the coactivators [56]. AR AF2 can also interact with the NTD WXXLF motif (433WHTLF435) to mediate N/C interaction but with a much lower affinity than the FXXLF motif [37]. These evidences 13  suggest the AR AF2 hydrophobic groove prefers phenylalanine (F) at the +1 and +5 positions within the peptide sequence. A recent study has also identified a secondary interaction region in the AR LBD helix 12 structurally adjacent to the AF2 binding surface which can facilitate FXXLF or LXXLL motif binding [66]. The AR NTD FXXLF motif forms an amphipathic ?-helical structure with +1 and +5 phenylalanine residues that bind strongly to the deep hydrophobic groove on the surface of the AF2 [56, 67]. The +4 leucine (L) residue binds to a shallow hydrophobic depression that is partly exposed to solvent, whereas the two charged residues at +2 and +3 positions, glutamine (Q) and asparagines (N), are exposed to solvent and do not interact with the LBD surface [56]. Furthermore, complementary electrostatic interactions between the charged clusters surrounding the AF2 floor and the backbone of NTD FXXLF peptide facilitate the orientation and stabilization of FXXLF motif binding of NTD to AF2 [68]. Immediately after ligand binding in the cytoplasm, AR N/C interaction adopts an intramolecular conformation [69]. After translocation into the nucleus, two AR molecules come together to form an antiparallel homodimer through intermolecular N/C interaction [69]. A recent study suggests that the interaction between DNA-binding domains within AR homodimers in the nucleus drive the transition from monomeric intramolecular N/C interaction to dimeric intermolecular N/C interaction [70]. N/C interaction only occurs when AR is mobile and is lost when AR is DNA-bound, making AR a platform for docking and recruitment of coregulators [69, 71]. The spatial and temporal regulation of coactivator recruitment by AR N/C interaction is imperative for androgen-regulated gene expression. 14  1.3.2 Factors impacting AR N/C interaction A number of factors have been reported to regulate the AR N/C interaction. As described previously, the binding of agonists such as natural or synthetic androgens to AR induce N/C interaction, whereas the binding of antagonists such as antiandrogen can block the AR N/C interaction. AR mutations in the LBD that are associated with androgen insensitive syndrome (AIS) or prostate cancer can interfere or enhance AR N/C interaction, respectively. Various AR coregulators can interact with AR FXXLF motif or AR AF2 surface to either inhibit or mediate AR N/C interaction. Variations in polymorphic human AR polyglutamine repeat region have been shown to affect AR N/C interaction. In addition, small molecule drugs that are developed for the treatment of prostate cancer can specifically target the AR LBD or NTD to inhibit AR N/C interaction such as hydroxyflutamide, bicalutamide, and EPI-001. 1.3.2.1  AR ligand specificity   The ligand induced AR N/C interaction is specific to high affinity AR agonists such as T, DHT, mibolerone, and methyltrienolone (R1881). On the other hand, AR antagonist hydroxyflutamide and non-androgenic steroids including estradiol and progesterone can bind AR with moderate to high affinity but cannot stabilize AR nor induce AR N/C interaction [72]. Dihydrotestosterone is the 5?-reduced metabolite of T, and a more potent androgen in vivo. Both DHT and T bind to AR with similar equilibrium binding affinities to mediate AR transcriptional activities [73]. However, DHT is about 10 times more potent than T in mediating AR transcription [74]. T is a less potent androgen due to weaker induction of FXXLF motif binding to AR AF2 [75]. Both T and DHT dissociate with similar rate from the AR LBD, but T dissociates 3 times faster than DHT 15  from full-length AR because weaker AR N/C interaction results in a faster ligand dissociation rate [57]. T-bound AR exhibits subtle conformational instability at the AF2 surface which leads to weaker FXXLF motif binding and faster dissociation of T from AR [75].  1.3.2.2  AR mutation in AIS  AR AF2 germline mutations that cause partial and complete androgen insensitive syndrome (AIS) by disrupting AR N/C interaction have been extensively characterized [59, 61, 76, 77]. Those mutations that inhibit AR N/C interaction and binding of coactivator LXXLL motif to AR AF2 without altering equilibrium binding affinity of androgen are instrumental in identifying the region of AR LBD involved in mediating AR N/C interaction [59, 77]. Interruption of AR N/C interaction in these AR AF2 mutants results in a faster dissociation rate of bound androgen, which is consistent with the findings that AR N/C interaction slows ligand dissociation rate and is required for normal AR function [57].  1.3.2.3  AR mutation in prostate cancer  The increased genomic instability associated with prostate cancer progression leads to a higher frequency of AR mutations [78]. Somatic mutations in the AR LBD are the most common and they have been identified in human prostate cancer cell lines, xenografts, and prostate cancer specimens [79-81]. Notably, the human prostate cancer cell line, LNCaP, contains a point mutation (AR T877A) in the ligand binding pocket of the AR, which broadens its ligand specificity [80, 81]. The T877A mutant enhances N/C interaction and slowes dissociation rate of bound androgen without significantly affecting the equilibrium binding affinity of androgen [82, 16  83]. Molecular modeling has determined that T877A increases N/C interaction by increasing flexibility of amino acid residues in the AF2 and expanding the solvent accessible surface, which results in improved induced fit of NTD FXXLF peptide into AR T877A AF2 [83].  On the other hand, a mutation within the FXXLF motif has not been reported, while a mutation within the lower affinity WXXLF motif (W435L) has been detected. W435L is a gain-of-function mutation resulting from adaptation to antiandrogen treatment in prostate cancer. W435L mutation is positioned within the WXXLF motif of the AR NTD. WXXLF motif has a significant but more minor role compared to the FXXLF motif in mediating N/C interaction since WXXLF motif is weaker in its affinity for the AF2 [37, 38]. W435L mutation enhances AR N/C interaction by 50% compared to wild-type AR in PC3 cells [84].  1.3.2.4  AR coregulators  Various AR coregulatory proteins have been identified to modulate AR N/C interaction. For example, cyclinD1 is a potent corepressor of the AR. Cyclin D1 binds the FXXLF in the AR NTD and abrogates AR N/C interaction and subsequently its transcriptional activity [85]. Glycogen synthase kinase 3? (GSK3?) phosphorylates the amino terminus domain of AR and interrupts AR N/C interaction [86]. Tumour suppressor protein p53 inhibits human AR by disrupting N/C interaction [87]. FoxO1 mediates PTEN suppression of AR N/C interactions and coactivator recruitment [88]. Silencing mediator for retinoid and thyroid hormone receptor (SMRT) and nuclear receptor corepressor (N-CoR) inhibit AR N/C interaction by targeting AR LBD [89-91]. On the other hand, proto-oncogene c-Jun has been reported to enhance AR activity by promoting 17  N/C interaction [92]. SET9 is a histone methyltransferase that directly methylates AR on lysine 632. SET9 enhances transcriptional activity of AR by facilitating N/C interaction [93]. In addition, AR coactivators can act as bridging factors to mediate AR N/C interaction. Glucocorticoid receptor interacting protein 1 (GRIP-1) can indirectly facilitate AR N/C interaction by bridging the AR NTD and the LDB [94]. GRIP1 contains a LXXLL motif that interacts with AR AF2 in the LBD [95], as well as a carboxyl-terminal region that interacts with AR AF1 in the NTD [96]. GRIP1 enhances AR N/C interaction by stabilizing AR inter-domain communication [94]. Melanoma antigen-A11 (MAGE-A11) is a primate specific AR coregulatory protein that binds to the AR FXXLF motif and competes with AR AF2 in N/C interaction [97]. MAGE-A11 enhances AR transcriptional activity through interaction with transcriptional regulator p300 [98]. This presents a mechanistic dilemma because AR FXXLF binds to AR AF2 to facilitate N/C interaction while it binds to MAGE-A11 to mediate transcriptional activity. A recent study proposed a model which MAGE-A11 can form a molecular bridge linking two transcriptionally active AR dimers to form a multimeric complex. The two FXXLF motifs present in the AR dimer appear to serve distinctive functions: one FXXLF motif of the AR antiparallel dimer engages in androgen dependent AR N/C interaction, whereas the other AR FXXLF motif binds to MAGE-A11 and stabilizes the multimeric complex [99].     18  1.3.2.5  NTD polymorphism  The NTD of AR contains a polymorphic glutamine (polyQ) tract that is located close to the FXXLF motif [28]. PolyQ tract varies in length between 9-37 residues in normal human population [40]. Variations in polyQ tract length within and outside of normal range are associated with development of diseases. Glutamine repeat expansions greater than 40 residues cause spinal bulbar muscular atrophy or Kennedy?s disease [41]. Several studies have showed the length of the polyQ tract is inversely correlated with the risk of developing prostate cancer [100-102]. Interestingly, variation in glutamine repeat length does not affect androgen binding affinity [103]. 91-99% of AR alleles across different racial-ethnic groups encode polyQ tract in the range of 16-29 repeats, which a critical size for maintaining AR N/C function [104]. The functional significance of polyQ tract in contributing to AR N/C interaction remains unclear. While one group has reported that shorter polyQ tract length is associated with increased AR N/C interaction [83], another has showed that interruption of polyQ tract by two non-consecutive leucine residues inhibits N/C interaction [104].  1.3.2.6  Small molecule drugs  Antiandrogens such as hydroxyflutamide and bicalutamide competitively inhibit ligand induced N/C interaction [72]. High concentration of hydroxyflutamide at 10?M can acquire agonist activity with wild-type AR through stabilization of AR in the absence of androgen [105]. Furthermore, hydroxyflutamide can weakly induce N/C interaction in AR with T877A mutation, correlating with the agonist activity of hydroxyflutamide with the LNCaP endogenous AR [58]. These evidences may account for resistance to antiandrogen in prostate cancer patients. EPI-19  001 is a small molecule antagonist targeting the AR NTD that has been shown to block AR N/C interaction and binding of AR to coactivators CBP and RAP74 [106]. EPI-001 and its stereoisomers covalently bind to the AR AF-1 region and cause conformational change in the AR NTD [106, 107]. The change in AR NTD conformation may impair the binding of FXXLF motif to the AF2 surface in the LBD to mediate N/C interaction.  1.4 AR splice variants 1.4.1 AR splice variants Various independent clinical correlation studies support the potential role of constitutively active AR splice variants in the progression of CRPC [20, 21, 108]. ARv567es and AR-V7 are the two most clinically relevant AR splice variant species found in CRPC [20, 22, 108]. Expression of ARv567es and AR-V7 is enriched in CRPC bone metastasis specimens compared to those isolated from bone lesions of hormone-na?ve patients [108]. The presence of AR-V7 is also observed in normal prostate tissues indicating it is unlikely to be the initiation factor in prostate cancer [109]. However, the normal functions of AR splice variants remain unclear. Functionally, these constitutively active, ligand-independent AR splice variants have been demonstrated to support CRPC cell growth in vitro and in vivo [20-22].Treatment-induced AR variants activate a distinct expression signature enriched for cell-cycle genes without requiring the presence of full-length AR, indicating the essential role of variants in androgen-independent growth and proliferation in CRPC [110]. Increased expression of AR splice variants is believed to be a mechanism of resistance to current hormone therapy and development of CRPC. The levels of AR splice variants are 20  elevated in CRPC cells compared to androgen-dependent prostate cancer cells suggesting the increased variant expression is the result of selective pressure from hormone therapy [109].  Depletion of androgen induces AR gene transcription rate and splicing factor recruitment to AR pre-mRNA contributing to enhanced AR-V7 level in prostate cancer cells [111]. In addition, Dehm and coworkers have demonstrated that rearrangement of genomic DNA template could be a cause of disrupted AR splicing patterns [112]. 1.4.2 ARv567es The ARv567es transcript was discovered in LuCaP 86.2 and 136 xenografts derived from metastases obtained from patients with CRPC after prolonged exposure to androgen deprivation therapy [22]. ARv567es mRNA is generated by splicing of exon 4 to exon 8 to exclude exons 5-7, and a frameshift to generate a new stop codon. As the result, ARv567es protein contains the NTD, DBD, hinge region, and a unique 10-amino acid sequence (Figure 1.5). Currently, there is no antibody specific for the ARv567es protein. However, ARv567es can be recognized as an approximately 80kDa species with antibody specific to the AR NTD [22]. ARv567es is constitutively active and is localized in nucleus regardless of the presence of the ligand. ARv567es is the only AR variant that has been shown to physically interact with full-length AR when co-transfected into the AR negative prostate cancer cell line [22]. ARv567es stabilizes full-length AR and enhances nuclear localization of full-length AR in the absence of androgens. In human xenografts, transfection of ARv567es cDNA in human prostate 21  cancer cells yields castration resistant tumours. In 46 CRPC metastases that are positive for AR, as much as 20% of these metastases expressed only ARv567es [22] 1.4.3 AR-V7  AR-V7 (also known as AR3) is one of the best characterized AR splice variants to date [109]. AR-V7 transcript is derived from contiguous splicing of AR exons 1, 2, 3 and cryptic exon 3 (Figure 1.5). AR-V7 protein has NTD and DBD while lacking hinge region and LBD [20, 21]. AR-V7 has a unique 16-amino acid sequence in the C-terminus which allowed the development of AR-V7 specific antibody [20, 21]. Since AR-V7 does not have a hinge region containing the canonical AR NLS, the exact mechanism of AR-V7 nuclear translocation is still unclear. A recent study demonstrated AR splice variants can mediate nuclear translocation independent of canonical NLS through their NTD, DBD,  and the unique C-terminus amino acid sequences [113]. AR-V7 is primarily nuclear in prostate cancer cells under androgen depleted condition and is constitutively active [20, 21]. AR-V7 has been shown not to physically interact with full-length AR in 22Rv1 cells [20]. Interestingly, Watson and coworkers have shown the growth promoting effects of AR-V7 are mediated through full-length AR [114], while Hu and coworkers have demonstrated full-length is not required for induction of cell-cycle genes by AR-V7 [110]. More importantly, elevated AR-V7 expression in hormone na?ve prostate cancer is associated with poor clinical outcome following radical prostatectomy [21]. 22   Figure 1.5 The mRNA organization of full-length AR, and splice variants ARv567es and AR-V7. Top: exons 1-8 of full-length AR. Middle: skipping of exons 5-7 to give rise to ARv567es. Bottom: alternative splicing of exons 1-3 and CE3, cryptic exon 3 to give rise to AR-V7. Adapted from Figure 4B from ? Tindall, SM et al. (2011). Alternatively spliced androgen receptor variants. Endocrine-related Cancer. 18(5): Page R183-96 [109].                23  1.5 EPI compound 1.5.1 EPI compound as a novel AR NTD inhibitor The importance of the AR NTD in the progression of prostate cancer was demonstrated by decoy molecules of the AR NTD that competitively bind the interacting proteins required for activation of the endogenous full-length AR [115]. Deletion studies have demonstrated the necessity of NTD for AR transcriptional activity with or without the presence of androgen [42]. These findings suggest the NTD is a valid target for the development of AR antagonists to treat prostate cancer. Since the AR NTD is intrinsically disordered and lacks crystal structure, it would be impossible to identify inhibitors using structure-based drug design. Cell-based screening assays were used to identify EPI-001 (BADGE.H2O.HCl) from a library of marine sponge extracts for inhibition of AR NTD transcriptional activities in LNCaP prostate cancer cells [116]. EPI-001 is a metabolic byproduct of bisphenol A and is isolated from marine sponge Geodia lindgreni [106]. EPI-001 is mixture of 4 stereoisomers with 2 chiral carbons at the 2 and 20 positions (Figure 1.6). According to FDA requirement, each stereoisomer has to be evaluated separately when developing chiral drugs because of the potential differences in biological activity [107]. There is no stereospecificity in binding of EPI-001 stereoisomer to AR. All stereoisomers of EPI-001 showed similar activity with EPI-002 (2R, 20S) being the most potent [107].   24   Figure 1.6 Structure of EPI-001 and stereoisomer EPI-002 (2R, 20S).  Adapted from Figure 1A from ? Myung, JK et al. (2013). An androgen receptor N-terminus domain antagonist for treating prostate cancer. The Journal of clinical investigation. 123(7): Page 2948-60 [107]. By permission from American Society for Clinical Investigation.  The exact binding site of EPI compound on AR is still under investigation. It has been shown that EPI-001 interacts with AF1 region in the NTD and does not compete with androgen binding [106]. Fluorescence emission spectroscopy shows that EPI-001 alters folding of AF1 of AR but not glucocorticoid receptor (GR), indicating that the binding of EPI-001 is specific to AR [106]. Some secondary structure of AF1 is required for EPI-001 to bind. A proposed binding mechanism suggests EPI analogs irreversibly bind to AF1 region in the AR NTD and the chlorohydrin group of EPI is required to facilitate this covalent binding reaction (Figure 1.7) [107].   Figure 1.7 Proposed model of covalent binding reaction of EPI-001 to AR AF1. Binding of EPI compound to AR AF1 starts with a fast reversible reaction that places the secondary alcohol of the chlorohydrin group next to a basic site in the AF1. The base removes the proton from the secondary alcohol to form an intermediate epoxide in a slow rate limiting step. Then the reactive epoxide reacts irreversibly with a nucleophilic site on an AF1 amino residue to form a covalent bond. Figure 7 from ? Myung, JK et al. (2013). An androgen receptor N-terminus domain antagonist for treating prostate cancer. The Journal of clinical investigation. 123(7): Page 2948-60 [107]. By permission from American Society for Clinical Investigation. 25   In vitro characterization has shown that EPI-001 blocks transactivation of AR NTD and inhibits AR activity in the presence or absence of androgen; does not impact the transcriptional activities of related human steroid receptors such as progesterone receptor (PR) and glucocorticoid receptor (GR); prevents binding of AR interaction with AREs on the DNA and inhibits the expression of androgen responsive genes such as PSA and TMPRSS2;  inhibits protein-protein interaction with CBP, RAP74 and AR N/C interactions; and attenuates androgen-induced proliferation of prostate cancer cells [106]. Unlike antiandrogens, EPI-001 does not induce nuclear translocation of AR in the absence of androgen [107].  Studies using animal models have demonstrated that EPI-001 inhibits androgen-dependent as well as CRPC tumour growth in mice. EPI-001 is specific to AR and has no effect on androgen insensitive PC3 xenograft that do not express functional AR. Animals treated with EPI compound show no signs of toxicity as indicated by no loss of body weight, no changes in behavior, and no pathological changes in histology of internal organs [106, 107]. Furthermore, EPI-001 has 86% oral bioavailability and a half-life of 3.3 hours [107].    Expression of constitutively active, C-terminally truncated splice variant of AR devoid of LBD has emerged as a potential mechanism for resistance to androgen deprivation therapy in prostate cancer [22]. 20% of CRPC metastases solely express AR splice variants [22]. Treatments targeting AR LBD or lowering the levels of circulating androgen would have no effect on AR splice variants in CRPC. Since EPI compounds target the AR NTD, they are the first reported inhibitors of constitutively active splice variants. EPI has shown inhibition of transcriptional 26  activities of the variant in vitro. In addition, EPI-002 inhibits the growth of CRPC xenograft expressing AR splice variants [107]. 1.6 Androgen receptor-ligand kinetics 1.6.1 Androgen receptor-ligand kinetics  The steroid-binding and dissociation properties of human and rat AR have been studied since the 1970?s [26, 73, 117, 118]. The AR ligand binding domain shares approximately 50% sequence identity with the human progesterone receptor, glucocorticoid receptor, and mineralocorticoid receptor ligand binding domains [27]. Wilson and colleagues have extensively characterized the binding affinity and the rate of ligand dissociation of AR in whole cells using a tritium (3H) labeled synthetic androgen methyltrienolone (R1881) [59, 75, 82, 119]. 3H-R1881 is chosen for several reasons. Tritium label has a radioactive decay half-life 12.43 years and is very stable compared to other radioisotopes [120]. Synthetic androgen R1881 has better metabolic stability and higher water solubility than naturally occurring androgens such as DHT and T [119, 121, 122].  Since high concentration of unlabeled ligand is required to prevent the rebinding of dissociated radiolabeled ligand, R1881 is the preferred ligand for this assay.  On the other hand, measuring AR-ligand kinetics using purified recombinant AR protein in a buffered solution has several drawbacks. Recombinant full-length AR protein can be susceptible to aggregation in buffer [123]. The absence of intracellular chaperone can lead to incorrect protein folding and aggregation [124]. AR ligand dissociation rate is dependent on N/C interdomain interaction [59]. Commercially available ligand binding assay employs recombinant AR LBD alone (PolarScreenTM competitor assay kit, Invitrogen), and should not be used for 27  measuring ligand dissociation rate due to the lack of N/C interaction. In addition, the absence of normal interactions with AR coregulators in the intracellular environment may impact AR ligand binding affinity and dissociation rate [123]. Analysis of radioligand binding and dissociation experiments are based on the simple model of  law of mass action (Figure 1.8) where the following assumptions are made: 1) the receptor and ligand have reached binding equilibrium; 2) the binding is reversible and specific; and 3) there is no cooperative binding meaning the binding of a ligand to one binding site does not alter the affinity of another binding site; 4) A very small fraction (<5%) of the ligand is bound so that the free concentration of ligand is not significantly altered [120].   Figure 1.8 Law of mass action: simplified rate reaction and equations. Free concentrations of: [R], Receptor; [L], Ligand; [RL], Receptor-ligand complex. kon, association rate constant; koff, dissociation rate constant; Kd, Equilibrium dissociation constant. Equilibrium is reached when the rates at which ligand-receptor complexes are associated (kon) and dissociated (koff) are equal. Binding affinity of receptor for ligand is measured by Kd. Under the law of mass action, the relationship between the equilibrium dissociation constant (Kd ) and dissociation rate constant (koff) is simplified with the assumption that the receptor (R) is the only protein species available. In reality, the relationship between these rate constants 28  can be altered due to the interaction of receptor with various protein partners in a complex intracellular environment. 1.6.2 Ligand binding affinity  In addition to binding to AR, 3H-R1881 can bind to non-specific sites. Non-specific binding is detected by measuring 3H-R1881 in the presence of 100-fold molar excess of unlabeled R1881. Under these conditions, almost every AR is occupied with unlabeled R1881 so that 3H-R1881 can only bind to non-receptor sites. Specific binding of 3H-R1881 to AR can be assessed by subtracting nonspecific binding of 3H-R1881 from total binding of 3H-R1881 [120]. The maximum number of 3H-R1881 bound (Bmax) and equilibrium dissociation constant (Kd) can be obtained from saturation experiment where specific binding at equilibrium is measured by incubating cells that express AR with a concentration range of 3H-R1881. Kd is the concentration of 3H-R1881 that occupies half of the AR at equilibrium. Kd is typically expressed as a concentration in nanomolar. Kd value represents the affinity of AR for the R1881 at equilibrium. A smaller Kd means high affinity and a larger Kd means low affinity [120] . Scatchard plots are used to graphically visualize the equilibrium binding data (Figure 1.9 left). On a typical Scatchard plot, the X-axis represents specific binding or the amount of bound 3H-R1881. The Y-axis is a ratio of bound 3H-R1881 to free 3H-R1881. Equilibrium binding affinity Kd is the negative reciprocal of the slope and Bmax is the X-intercept [120]. Scatchard plot should only be used to display equilibrium binding data because linear transformation distorts experimental error of the binding data. Scatchard transformation also 29  violates the linear relationship between the X value (bound 3H-R1881) and the Y value (bound 3H-R1881/ free3H-R1881) because X is used to calculate Y. Thus Scatchard plot produces a less accurate estimate of Kd and Bmax [120]. More accurate Kd and Bmax values are determined from non-linear regression fit of the equilibrium binding data (one site specific binding, GraphPad Prism). Figure 1.9 right shows a hyperbolic non-linear saturation curve with the concentration range of 3H-R1881 initially added (i.e. free concentrations of 3H-R1881) on the X-axis and specific binding on the Y-axis.    Figure 1.9 Analysis of saturation binding data using Scatchard plot and non-linear regression. Binding affinity of receptor for R1881 is measured by equilibrium dissociation constant, Kd. Maximum number of R1881 bound, Bmax.  Left: Scatchard plot. The concentration of bound 3H-1881 is plotted against the ratio of bound 3H-1881 to free 3H-1881 to obtain a linear slope. Right: non-linear regression fit of saturation binding data. Specific binding is plotted against free concentration of 3H-1881 to obtain a hyperbolic curve.   1.6.3 Ligand dissociation  Ligand dissociation assay measures the off rate of 3H-R1881 dissociating from intracellular AR. Cells expressing AR are incubated with 3H-R1881 to reach receptor-ligand binding equilibrium. The dissociation reaction is a pseudo first order reaction and is initiated by the addition of a 30  large excess of unlabeled R1881. The unlabeled R1881 will bind to all unoccupied AR and block rebinding of dissociated 3H-R1881. The amount of bound 3H-R1881 over time is used to measure the rate of dissociation from AR. The off-rate can be expressed as dissociation rate constant (Koff) in inverse minutes or dissociation half time (T1/2) in minutes. T1/2 is the time required to reduce specific binding of 3H-R1881 by 50% [119, 120].  In Figure 1.10, the dissociation data can be displayed by plotting the logarithm of specific binding vs. time to give a linear graph where the slope is representative of the off rate. The linearity of this plot also confirms the AR-R1881 (receptor-ligand) system follows the law of mass action with a single affinity state. However, dissociation data analyzed using the nonlinear regression fit of specific binding (one phase exponential decay, GraphPad Prism) gives the most accurate off rate because there is no distortion of the raw data values by linear transformation [120].  Figure 1.10 Analysis of ligand dissociation data using non-linear and linear regression. Dissociation half-time (T1/2) is the time required to reduce specific binding by 50%. Left: non-linear plot of bound 3H-1881 over the time course of dissociation. Total, total binding of 3H-1881; NS, non-specific binding of 3H-1881. Right: linear regression of log(specific binding) over the time course of dissociation. 31  1.6.4 Ligand dissociation and AR N/C interaction  Wilson and colleagues have established that AR N/C interaction slows the dissociation of bound androgen resulting in prolonged ligand activation of AR [37, 58, 59]. Wilson and colleagues have demonstrated the direct relationship between N/C interaction and ligand dissociation using natural androgens such as T and DHT [75]. DHT has 3X slower dissociation rate than T as the result of enhanced N/C interaction when AR is DHT bound compared to T bound [75].                32  1.7 Hypothesis and specific aims Progression to CRPC is at least partially associated with continued activation of AR through increased intratumoural accumulation or synthesis of androgen [14]. Binding of androgen to AR ligand binding pocket leads to antiparallel dimerization of AR through N/C interaction, which results in slowed dissociation of bound androgen and prolong activation of AR [57, 58]. Expression of AR splice variants in CRPC tumours is believed to be another mechanism that enables prostate cancer progression in the presence of low level of androgen [22]. Most CRPC tumours express both full-length AR and varying levels of truncated AR splice variants [22]. Although several groups have shown the functions of AR variants are independent of the full-length AR [20, 22, 110], one other has shown the gain of function of AR variant is dependent on full-length AR [114]. Co-immunoprecipitation experiments have shown that ARv567es interacts with full-length AR, not only in prostate cancer cells overexpressed with the variant but also in human xenograft LuCaP 136 that expresses both full-length and variant ARs [22]. Furthermore, ARv567es causes nuclear translocation of full-length AR in the absence of androgen, resulting in enhanced AR transcriptional activity as well as cell proliferation in response to low levels of androgen [22]. These findings lead to the conclusion that ARv567es can physically and functionally interact with full-length AR by forming a heterodimer [22]. Taken together these findings, we tested two hypotheses in this thesis: Hypothesis A: Heterodimerization between full-length AR and ARv567es may alter the ligand dissociation rate from full-length AR. If the heterodimerization results in a slower dissociation rate, this would support enhanced response to low levels of intratumoural androgens. To 33  understand the mechanism of ARv567es, here we examined the effect of ARv567es on the dissociation rate of ligand from full-length AR. Hypothesis B: Ligand dissociates more quickly with reduced AR N/C interaction as deletion of the AR NTD quickens the dissociation rate of bound ligand [57]. AR NTD inhibitor EPI-001 blocks N/C interaction [106], and its most potent stereoisomer EPI-002 inhibits in vitro and in vivo activities of ARv567es [107]. However, the effect of EPI analogue on the ligand dissociation rate of AR has not been explored. Here we examined if EPI-002 can alter ligand dissociation rate of full-length AR with and without the presence of ARv567es. The following specific aims were addressed. Aim 1. To examine the effect of EPI-002 on the ligand dissociation rate of full-length AR complexed with ARv567es transiently transfected in Cos-1 cells (AR negative cell line). Aim 2. To examine the effect of EPI-002 on the ligand dissociation rate of endogenous full-length AR in LNCaP prostate cancer cells. Aim 3. To examine the effect of EPI-002 on the ligand dissociation rate of full-length AR in LNCaP-Cu3 cells (prostate cancer cell lines with endogenous full-length AR and cumate-inducible expression of AR variants).  Aim 4. To examine the effect of EPI-002 on the ligand dissociation rate of full-length AR in LNCaP95, an androgen-independent prostate cancer cell line expressing endogenous full-length AR and AR-V7.  34  Chapter 2: Materials and Methods 2.1 Cell culture   Monkey kidney Cos-1 cells are derived from a clone of SV-40 transformed CV-1 cells [125]. Cos-1 cells were maintained in phenol red free Roswell Park Memorial Institute (RPMI) medium 1640 (Gibco, Life Technologies) supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 ?g/mL streptomycin. LNCaP cells were provided by Dr. L.W.K. Chung (Cedar-Sinai Medical Center, CA, USA). LNCaP cells are androgen-sensitive human prostate adenocarcinoma cells derived from the left supraclavicular lymph node metastasis from a 50-year-old Caucasian male in 1977 [126]. LNCaP cells were maintained in phenol red free RPMI 1640 with 5% FBS, 100 units/mL penicillin, and 100 ?g/mL streptomycin. LNCaP-Cu3 cells were provided by Dr. Stephen Plymate (University of Washington School of Medicine, WA, USA). LNCaP-Cu3 cells were derived from parental LNCaP cells that were stably transduced with lentivectors containing the cumate switch inducible system (System Biosciences). LNCaP-Cu3 cells were maintained in phenol red free RPMI 1640 with 10% FBS and 1 ?g/mL puromycin.  LNCaP95 cells were provided by Dr. Stephen Plymate. LNCaP95 cells were maintained in phenol red free) RPMI 1640 with 10% charcoal stripped serum (CSS), 100 units/mL penicillin, and 100 ?g/mL streptomycin. 2.2 Expression plasmids and transfection  pSVAR0 plasmid was a gift from Dr. Guido Jenster (Erasmus University Medical Center, Rotterdam, Netherlands) and has been described [28]. AR T877A plasmid was a gift from Dr. Mark Trifiro (Lady Davis Institute for Medical Research, Montreal, QC, Canada). ARv567es and 35  pCDNA3 constructs were gifts from Dr. Stephen Plymate and have been described [22]. Gal4-ARLBD and VP16-ARTAD constructs were gifts of Dr. Karen Knudsen (Thomas Jefferson University, Philadelphia, PA, USA). 0.5-2 ?g of expression plasmids were transfected into Cos-1 cells with Fugene6 (Promega) according to manufacturer?s protocol. 2.3 Ligand binding assay  Apparent equilibrium binding affinity was determined in whole cell binding assay as previously described [82]. Cos-1 cells (7.5x104 cells/well in 12-well plates) were plated for 24 hours, resulting in approximately 50-70% confluency. This was followed by transfection with AR expression plasmids (0.5 ?g/well) using Fugene6 (Promega) in serum free RPMI 1640 for 24 hours. Cells were treated with DMSO or EPI-002 (25?M) for 1 or 20 hours and then incubated for 2 hours with 0.1, 0.2, 0.4, 0.8, 1.5, and 3nM of 3H-R1881 (PerkinElmer) in the absence and presence of 100-fold molar excess unlabeled R1881 (NEN Life Science Products) to determine non-specific binding. Free 3H-R1881 was determined after the 2-hour incubation by counting an aliquot of the medium. Cells were washed with phosphate-buffered saline (PBS). Cell lysates were harvested in 2% SDS, 10% glycerol, and 10mM Tris, pH6.8. Radioactivity was determined on scintillation counter (Beckman Coulter). Equilibrium binding constant (Kd) values were determined using GraphPad Prism (non-linear regression method). Scatchard plots were used to display the ligand binding data. 2.4 Ligand dissociation assay  Cos-1 cells (1.5x105 cells/well in 6-well plates) were plated for 24 hours in RPMI 1640 with 10% FBS to reach approximately 50-70% confluency. Then Cos-1 cells were transfected for 24 hours 36  with AR expression plasmids (1-2 ?g/well) using Fugene6 in serum free RPMI 1640 media. LNCaP cells (1.5x105 cells/well in 6-well plates) were plated for 24 hours in RPMI 1640 with 5% FBS followed by incubation in serum free RPMI 1640 for 24 hours. LNCaP-Cu3 cells (1.25x105 cells/well in 12-well plates) were plated for 24 hours in RPMI 1640 with 10% CSS followed by the incubation with 30 ?g/mL cumate (System BioSciences) for 48 hours to induce the expression AR variant. LNCaP95 cells (1.5x105 cells/well in 6-well plates) were plated for 48 hours in RPMI 1640 with 10% CSS.  Next, ligand dissociation experiments were performed as previously described [119]. Cos-1 cells transfected with AR expression plasmids, LNCaP cells, LNCaP-Cu3 cells, or LNCaP95 cells were treated with DMSO or EPI-002 (25?M or 35 ?M) for 1 or 20 hours before labeling with 5nM of 3H-R1881 for 2 hours at 37?C to reach binding equilibrium. Dissociation was initiated by the addition of 10,000-fold molar excess of unlabeled R1881. The dissociation time points were 0, 0.5, 1, 1.5, 2, and 2.5 hours. Non-specific binding (5nM 3H-R1881 plus 100-fold molar excess unlabeled R1881) was subtracted from total binding (5nM 3H-R1881) to obtain specific binding. Cells were washed in PBS, cell lysates were harvested in 2% SDS, 10% glycerol, and 10mM Tris, pH6.8. Radioactivity was determined on scintillation counter. Ligand dissociation rate (T1/2) was measured as the time required to reduce specific binding by 50%. 2.5 N/C interaction  Two-hybrid N/C interaction assays used Cos-1 cells (7.5x104 cells/well in 12-well plates) that were plated for 24 hours followed by transfection with Fugene6 with 0.5?g/well Gal4-TATA-Luc reporter vector with five copies of Gal4 upstream enhancer element, 0.5?g/well VP16-ARTAD 37  (1-565) that encodes the VP16 transactivation domain fused to amino residues 1-565 of AR NTD, and 0.5?g/well Gal4-ARLBD (628-919) encoding the wild-type AR LBD C-terminus amino acid residues 628-919 fused to Gal4 DNA-binding domain. Five hours after transfection, cells were incubated for 24 hours at 37?C in the absence and presence of 1nM R1881 with or without inhibitors: EPI-002 (25?M) or Bicalutamide (10?M). Cell lysates were harvested in passive lysis buffer (Promega) and luciferase activity was measured on Glowmax luminometer (Promega). Luciferase activities were normalized to protein concentrations of the sample. Protein concentrations were quantified using Bradford protein assay (Bio-Rad).  2.6 Western blot  Whole Cell lysates were collected in RIPA buffer containing 50mM Tris, 150mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS), 20mM sodium fluoride, 20nM sodium molybdate, 1nM sodium orthovanadate and cOmplete mini EDTA free (Roche) and phosSTOP (Roche) protease inhibitors. Protein was quantified using BCA protein assay reagent (Pierce). Proteins in cell lysate were separated on a 8.5% SDS-polyacrylamide, by gel electrophoresis (Bio-Rad) and transferred to Hybond C extra nitrocellulose membrane (Amersham Biosciences). Blocking was carried out in 5% milk in PBS-T (phosphate-buffered saline and 0.05% Tween-20). Membranes were washed in PBS-T. The following primary antibodies were used in our studies: anti-AR PG21 (Millipore), anti-AR N20 (Santa Cruz Biotechnologies), anti-AR C19 (Santa Cruz Biotechnologies), anti-Beta actin (Abcam), anti-VP16 (Santa Cruz Biotechnologies), or anti-FLAG (Sigma-Aldrich). Protein of interest was detected using enhanced chemiluminescence (GE Healthcare Life Sciences). 38  Chapter 3: Results 3.1 Background binding of 3H-R1881 in Cos-1 cells was negligible The choice of cell lines for ligand binding and dissociation assays is important. Cos-1 cells are SV40 transformed monkey kidney cells that do not express detectable levels of endogenous AR [127]. When Cos-1 cells are transfected with plasmid constructs containing both the SV40 promoter and AR cDNA, the SV40 large T antigens in Cos-1 cells amplify the plasmid copy numbers to produce high AR protein expression [119]. To determine levels of background binding in Cos-1 cells, these cells were transfected with empty vector (control) or an expression vector for wild-type full-length AR (wt ARfl) for 24 hours followed by incubation with 5nM of 3H-R1881 (?hot?)  for 2 hours to reach binding equilibrium . Non-specific binding condition was done in parallel by incubation with ?hot? + ?cold? (?cold? refers to non-radioactive R1881), 5nM of ?hot? 3H-R1881 plus 100-fold molar excess ?cold? unlabeled R1881. Non-specific binding was subtracted from total binding (?hot? 5nM 3H-R1881) to obtain specific binding. Cells were washed, cell lysates were harvested and radioactivity was determined. Background binding of 3H-R1881 in cells that did not express AR (empty vector) constituted <2% of the 3H-R1881 bound in cells that were transiently transfected with wt ARfl (Figure 3.1). Since the amount of 3H-R1881 binding was negligible in Cos-1 cells lacking AR (cells with transfected empty vector), we excluded repeating this negative control in ligand binding and dissociation assays in subsequent experiments using these cells.   39  3.2 EPI-002 did not affect the binding affinity (Kd) of wt ARfl Before we can assess the effect of EPI-002 on ligand dissociation rate of full-length AR in Cos-1 cells, it was essential to determine if EPI-002 had an effect on the affinity of full-length AR for R1881. To do this, the equilibrium dissociation constant (Kd) was measured according to published protocols [128]. Cos-1 cells were transfected with an expression plasmid of wt ARfl for 24 hours and then treated with either 25?M of EPI-002 or equivalent concentration of DMSO (0.05% v/v) for 1 or 20 hours prior to addition of 0.1, 0.2, 0.4, 0.8, 1.5, and 3nM of ?hot? 3H-R1881 in the absence and presence of 100-fold molar excess ?cold? unlabeled R1881 to determine non-specific binding. Free 3H-R1881 was determined after 2 hours of incubation by sampling an aliquot of the medium. Cells were washed, cell lysates were harvested, and radioactivity was determined. Kd values were determined using the non-linear regression method in GraphPad Prism (one site specific binding). Scatchard plot was used to graphically visualize the maximum binding Bmax (X-intercept) and the Kd (negative inverse of slope). Figure 3.2A and 3.2C showed representative Scatchard plots of 3H-R1881 binding to wt ARfl after 1 hour and 20 hours EPI-002 treatment, respectively. The slopes of DMSO and EPI-002 treated conditions were parallel on the Scatchard plots, indicating the approximated Kd values were similar (0.25?0.07 nM). The maximum binding (X-intercept on Scatchard plot) was reduced with EPI-002 but the maximum binding was independent of binding affinity constant. The average Kd values determined by the non-linear regression in Graphpad Prism were shown in Figure 3.2B and 3.2D. There were no differences between the DMSO and EPI-002 after 1 hour and 20 hours of exposure indicating EPI-002 did not affect the binding affinity (Kd) of the wt ARfl. Kd values of 0.45?0.04 nM were within previous reported values of 0.48?0.25 nM [128].  40  3.3 EPI-002 did not alter the time for wt ARfl and 3H-R1881 to reach binding equilibrium  Interaction between drug and cell membrane phospholipids can alter the physiological properties of cell membrane, affecting the permeability and uptake of biomolecules [129, 130]. The effect of EPI-002 on cell membrane is beyond the scope of this thesis. However, we had to ensure EPI-002 treatment in our experiments was not affecting the subsequent uptake and binding of ligand to AR. Therefore, EPI-002 was tested for effects on the time for wt ARfl and its ligand to reach binding equilibrium. To do this, Cos-1 cells were transfected for 24 hours with an expression plasmid for wt ARfl and then treated with either DMSO (0.05% v/v) or EPI-002 (25?M) for 1 hour. Cells were incubated with ?hot? 3H-R1881 in the absence and presence of 100-fold molar excess unlabeled ?cold? R1881 to assess non-specific binding. The amount of 3H-R1881 bound was assessed at the following time points: 0, 0.5, 1, 1.5, 2 and 2.5 hours. Cells were washed, cell lysates were prepared and radioactivity was determined. In Figure 3.3, EPI-002 did not delay the time for wt ARfl and 3H-R1881 to reach binding equilibrium when compared to the DMSO control. 3.4 Splice Variant ARv567es had no effect on the dissociation rate of 3H-R1881 from wt ARfl Most CRPC patients have numerous tumours, each with potentially varying levels of expression of full-length and truncated variant AR. To understand the effect of truncated splice variant ARv567es on the dissociation of ligand from full-length AR, we transfected Cos-1 cells with the following expression vectors: 1) wt ARfl; or 2) a 50:50 mixture of wt ARfl to ARv567es. Ligand 41  dissociation experiments were performed in Cos-1 cells. A representative ligand dissociation plot of wt ARfl alone compared to wt ARfl and ARv567es in a 50:50 mixture is shown in Figure 3.4A. The average ligand dissociation rate (T1/2) was 34?6 minutes for wt ARfl on three independent experiments (Figure 3.4B). A 50:50 ratio of wt ARfl to ARv567es revealed that the truncated variant ARv567es did not alter the ligand dissociation rate of wt ARfl (Figure 3.4C). Western blot analysis using lysates of cells treated in parallel confirmed ectopic expression of wt ARfl and the truncated ARv567es variant at the desired 50:50 ratio (Figure 3.4D). Furthermore, levels of AR proteins achieved by transfection of expression plasmids were within physiological levels when compared to the level of endogenous AR expression in LNCaP cells.  3.5 EPI-002 accelerated the dissociation rate of 3H-R1881 from wt ARfl EPI compound inhibits N/C interaction [106] in CV-1 cells and should therefore quicken the rate of ligand dissociation. To test this, we examined the effect of EPI-002 on the ligand dissociation rate of wild-type full-length AR alone. Cos-1 cells were transfected with wt ARfl for 24 hours followed by treatment for 1 hour with DMSO vehicle (0.05% v/v) or EPI-002 (25?M). A representative ligand dissociation plot of wt ARfl treated with DMSO and EPI-002 is shown in Figure 3.5A. The average ligand dissociation rate (T1/2) of wt ARfl was 35?11 minutes for DMSO treated samples in three independent experiments (Figure 3.5B). The change in T1/2 was determined by normalizing the average T1/2 of EPI-002 treated samples to the DMSO treated samples (Figure 3.5C). Consistent with the anticipated results, EPI-002 accelerated the dissociation rate from wt ARfl transfected in Cos-1 cells by approximately 30% in three independent experiments (p <0.05). 42  3.6 EPI-002 blocked N/C interaction Cos-1 cells In order to support these findings, we also examined the effect of EPI-002 on AR N/C interaction in Cos-1 cells using an androgen dependent mammalian two-hybrid assay. Cos-1 cells transfected with Gal4-ARLBD (C-terminus), VP16-ARTAD (N-terminus) and Gal4-TATA-luciferase reporters were pretreated with vehicle (0.14% v/v), EPI-002 (25?M) or Bicalutamide (10 ?M) for 1 hour before the addition of R1881 (1nM) for 24 hours. Cell lysates were collected and luciferase activity was determined. The positive control Bicalutamide completely inhibited the AR N/C interaction induced by synthetic androgen R1881 (Figure 3.6A). Consistent with our finding from the ligand dissociation data in Cos-1 cells (Figure 3.5), EPI-002 inhibited AR N/C interaction in Cos-1 cells by approximately 30% (p <0.01). These data suggest EPI-002 accelerated ligand dissociation rate of wild-type full-length AR possibly by disrupting N/C interaction. Western blot analysis verified the levels of Gal4-ARLBD and VP16-ARTAD protein expression in the mammalian two-hybrid experiment (Figure 3.6B).  3.7 Variant ARv567es attenuated the effects of EPI-002 on the dissociation rate of 3H-R1881 from wt ARfl  To determine the effect of EPI-002 on wt ARfl in the presence of ARv567es, we transfected expression plasmids to achieve approximately a 50:50 ratio of wt ARfl to ARv567es in Cos-1 cells. Western blot analysis of cell lysates confirms the expression of wt ARfl and ARv567es at the desired 50:50 ratio and the level of wt ARfl was comparable to the physiological level of endogenous AR in LNCaP cells (Figure 3.7A). Twenty-four hours after transfection of the expression plasmids, the cells were incubated for an additional 20 hours with EPI-002 (35?M) or 43  equivalent concentration of DMSO (0.07% v/v). A higher concentration and longer incubation period were used to optimize conditions which now had doubling of drug target (AR NTD) compared to previous conditions using only full-length AR. A representative ligand dissociation plot of wt ARfl and ARv567es treated with DMSO and EPI-002 is shown in Figure 3.7B. The average ligand dissociation rate (T1/2) of wt ARfl in the presence of ARv567es was 34?5 minutes for DMSO treated samples in four independent experiments (Figure 3.7C).The amount of 3H-R1881 bound at time 0 was noticeably reduced under EPI-002 treated condition compared to DMSO treated condition (Figure 3.7B). Since EPI-002 did not affect binding affinity of wt ARfl (Figure 3.2) nor the time for it to reach binding equilibrium (Figure 3.3), we investigated whether 20-hour incubation of EPI-002 affected the full-length AR protein levels. Western blot analysis showed 20-hour treatment of EPI-002 decreased the levels of ectopic wt ARfl levels in Cos-1 cells (Figure 3.8). EPI-002 had no effect on the dissociation rate of a 50:50 mixture of wt ARfl to ARv567es in Cos-1 cells (Figure 3.7D). These findings suggest expression of ARv567es reduced the impact EPI-002 on the dissociation rate of 3H-R1881 from wt ARfl.  3.8 Effect of EPI-002 on the ligand dissociation rate of endogenous full-length AR in LNCaP cells  Next, we investigated the effect of EPI-002 on ligand dissociation rate of endogenous full-length AR in LNCaP cells. LNCaP cells are androgen-sensitive human prostate adenocarcinoma cells that express endogenous full-length AR with a point mutation in the ligand binding domain (T877A) [80]. AR T877A stabilizes the bound ligand and slows ligand dissociation rate compared to wild-type AR [128]. LNCaP cells were treated with DMSO (0.05% v/v) or EPI-002 (25?M) for 1 44  hour. Although statistically significant (p <0.05), EPI-002 had minimal effect (5% average change relative to DMSO) on the ligand dissociation rate of endogenous full-length AR in LNCaP cells (Figure 3.9A and 3.9B). The ligand dissociation rate of LNCaP endogenous AR in was 67?5 minutes, consistent with the reported value of 83?7 minutes [128]. Western blot analysis revealed 1-hour treatment of EPI-002 did not affect the levels of full-length AR in LNCaP cells (Figure 3.9C).  In order to determine if the lack of effect of EPI-002 on the ligand dissociation rate of LNCaP endogenous AR was caused by the presence of T877A mutation, we verified the sequence of expression plasmid encoding AR T877A and transfected it in Cos-1 cells for 24 hours followed by treatment for 1 hour with DMSO vehicle (0.05% v/v) or EPI-002 (25?M). A representative ligand dissociation plot of AR T877A treated with DMSO and EPI-002 is shown in Figure 3.10A. The ligand dissociation rate (T1/2) of AR T877A transfected in Cos-1 cells was 74?11 minutes (Figure 3.10B), which was comparable to the ligand dissociation rate of LNCaP endogenous AR T877A in previous data [128]. Consistent with our finding in LNCaP cells, EPI-002 did not affect the ligand dissociation rate of AR T877A expressed in Cos-1 cells (Figure 3.10C). The levels of AR T877A protein achieved by transient transfection were within the levels of endogenous AR expression in LNCaP cells (Figure 3.10D). In our experiments, the ligand dissociation rate of AR T877A transfected in Cos-1 (74?11 minutes) was significantly slower than wt ARfl transfected in Cos-1 (34?6 minutes), whereas the reported values for AR T877A and wt ARfl were 228?12 minutes and 144?18 minutes, respectively [128]. These findings highlight the importance of expressing AR protein at physiological levels when assessing ligand dissociation rates.  45  3.9 Effect of EPI-002 on LNCaP-Cu3 cells expressing endogenous full-length AR and cumate-induced FLAG-ARv567es To further address the role of variant on the dissociation rate from full-length AR, LNCaP-Cu3 cells were employed. LNCaP-Cu3 cells are derived from parental LNCaP cells that were stably transduced with lentivectors containing the cumate switch inducible system [131]. LNCaP-Cu3 cells express endogenous AR and cumate-inducible FLAG tagged AR variant. First, we determined the levels of FLAG-ARv567es protein that were induced with cumate at 24 and 48 hours. After 48 hours of induction, the amount of FLAG-ARv567es protein was approximately 40% of the endogenous full-length AR in LNCaP-Cu3 cells (Figure 3.11A). The expression FLAG-ARv567es protein was also detected using anti-FLAG antibody (Figure 3.11B).  A representative ligand dissociation plot of LNCaP-Cu3 cells expressing full-length AR alone and full-length AR with FLAG-ARv567es treated with DMSO and EPI-002 is shown in Figure 3.12A. The average T1/2 values from three independent experiments are shown in Figure 3.12B. The average ligand dissociation rate of full-length AR in LNCaP-Cu3 cells (83?16 minutes) was comparable to the endogenous full-length AR in parental LNCaP cells (67?5 minutes) and in reported data (83?7 minutes) [128]. This finding suggests the addition of cumate did not affect the ligand dissociation rate of the full-length AR in LNCaP-Cu3 cells.  In Figure 3.12C, EPI-002 did not affect the ligand dissociation rate of full-length AR alone. In the presence of cumate-induced FLAG-ARv567es (Figure 3.12B and 3.12D), the effect of EPI-002 on full-length AR was minimal (7% average change relative to DMSO) but statistically significant (p < 0.05). The presence of FLAG-ARv567es did not affect the ligand dissociation rate of full-length 46  AR in LNCaP-Cu3 cells (Figure 3.12E). These results are consistent with our findings in Cos-1 cells with ectopic wt ARfl and ARv567es. Levels of full-length AR and cumate-induced FLAG-ARv567es protein in the ligand dissociation experiment are shown in Figure 3.12F. Densitometric analysis revealed the amount of FLAG-ARv567es protein was approximately 13% of full-length AR protein indicating the cumate induction was less than optimal for the ligand dissociation experiments. 3.10 EPI-002 accelerated the rate of ligand dissociation of LNCaP95 cells expressing endogenous full-length AR and splice variant AR-V7 The effect on EPI-002 on the ligand dissociation rate of LNCaP95 cells that express endogenous full-length AR and splice variant AR-V7 was next examined. LNCaP95 is an androgen independent prostate cancer cell line derived from the parental LNCaP cells that have been cultured since 1995 in charcoal stripped serum media depleted of hormones. Similar to ARv567es, AR-V7 lacks the ligand binding domain and is constitutively active. However, AR-V7 lacks the hinge region and there is no reported evidence that AR-V7 physically interacts with full-length AR.  Figure 3.13A shows a representative ligand dissociation plot of LNCaP95 cells treated with DMSO and EPI-002. The dissociation rate of 3H-R1881 from endogenous full-length AR in LNCaP95 was 107?15 minutes (Figure 3.13B). This value was slower than the ligand dissociation rate of endogenous full-length AR in parental LNCaP (Figure 3.9A). In Figure 3.13C, EPI-002 accelerated the ligand dissociation rate of endogenous full-length AR in LNCaP95 cells by approximately 13% (p < 0.05), whereas EPI-002 accelerated the ligand dissociation rate of endogenous full-length AR in parental LNCaP cells by approximately 5% (Figure 3.9A). Western 47  blot analysis showed the levels of AR-V7 protein were substantially lower than the levels of full-length AR protein in these cells (Figure 3.13D). Densitometric analysis revealed the amount of AR-V7 protein present was approximately 25% of the endogenous full-length AR protein in LNCaP95 cells.   Figure 3.1 Specific binding of 3H-R1881 to full-length wild-type AR in Cos-1 cells.  Cos-1 cells were transfected with 500ng of expression plasmid for full-length wild-type AR or empty vector. Twenty-four hours after transfection, cells were incubated for an additional 2 hours with 3H-R1881 (5nM). Bars represent mean of specific binding of 3H-R1881 ? standard deviation (SD) from three technical replicates. Two-sample student?s T-test for equal variance. Total protein amount in whole cell lysates was measured using BCA protein assay. Protein levels from cells transfected with wild-type full-length AR was 48.3 ?g comparable to cells transfected with empty vector at 43.5 ?g.      48    Figure 3.2 EPI-002 did not affect the binding affinity (Kd) of wild-type full-length AR in Cos-1 cells. (A) A representative Scatchard plot of 3H-R1881 binding to wild-type full-length AR in Cos-1 cells after 1-hour of EPI-002 treatment using 100-fold excess unlabeled R1881 at the initiation of the binding reaction to determine nonspecific binding. (B) The average Kd (nM) between cells treated for 1 hr with DMSO (control) or EPI-002. Kd values were determined using non-linear regional method in Graphpad Prism. Bars represent the mean ? SD of three technical replicates. (C) A representative Scatchard plot of 3H-R1881 binding to wild-type full-length AR in Cos-1 cells after 20-hour of EPI-002 treatment using 100-fold excess unlabeled R1881 at the initiation of the binding reaction to determine nonspecific binding. (D) The average Kd (nM) between cells treated for 20 hrs with DMSO (control) or EPI-002. Kd values were determined using non-linear regional method in Graphpad Prism. Bars represent the mean ? SD of three technical replicates. Two-sample student?s T-test for equal variance. Cells were labeled with 3H-R1881 (0.1, 0.2, 0.4, 0.8, 1.5, and 3nM) for 2 hrs at 37?C. The level of binding was measured by the total 3H-R1881 bound after subtraction of 3H-R1881 binding in the presence of 100-fold excess unlabeled R1881. Cells were washed, harvested in SDS sample buffer, and radioactivity determined by scintillation counting. B/F indicates the ratio of bound-to-free ligand. DMSO, Vehicle; 25 ?M EPI-002.     49   Figure 3.3 EPI-002 did not alter the time for wild-type full-length AR to reach binding equilibrium. A representative of 3H-R1881 binding curve in Cos-1 cells transfected with full-length wild-type AR for 24 hrs prior to treatment with DMSO vehicle or EPI-002 (25?M) for 1 hr before the addition of 3H-R1881 (5nM) and incubation at 37?C. The amount of 3H-R1881 bound was measured at 0, 30, 60, 90, 120 and 150min. Cells were washed, harvested in SDS sample buffer, and radioactivity determined by scintillation counting. Non-specific-binding was assessed using parallel cells treated with 100-fold excess unlabeled R1881 at the initiation of the binding reaction. Total binding represents specific plus non-specific binding of 3H-R1881. Data points represent mean ? stand error of mean (SEM) from three technical replicates.                    50    Figure 3.4 ARv567es had no effect on the dissociation rate of 3H-R1881 from wild-type full-length AR in Cos-1 cells. Cos-1 cells were transfected with expression plasmids for wt ARfl, ARv567es, or empty vector (pcDNA3) for 24 hrs before labeling with 3H-R1881 (5nM) for 2 hrs at 37?C. Dissociation was initiated by addition of 10,000-fold molar excess of unlabelled R1881. Cells were washed, harvested in SDS sample buffer, and radioactivity determined by scintillation counting. Non-specific-binding was assessed using parallel cells treated with 100-fold excess unlabeled R1881 at the initiation of the binding reaction. (A) A representative ligand dissociation curve. Data points represent specific 3H-R1881 bound (mean ? SEM from three technical replicates). (B) The average T1/2 from 3 independent experiments. Change relative to wt ARfl was calculated by normalizing the T1/2 of samples expressing wt ARfl and ARv567es to T1/2 of samples expressing wt ARfl and empty vector in each experiment. (C) Fold change was calculated by normalizing the mean T1/2 (min) of samples expressing wt ARfl and ARv567es to the samples expressing wt ARfl and empty vector. In doing this, Fold change for samples expressing wt ARfl and empty vector became one and SD zero. Error bars represent +/- SD for three independent experiments. One-sample Student?s T-test. NSS, Not Statistically Significant (p > 0.05). (D) Levels of wt ARfl and ARv567es proteins in transfected Cos-1 cells used for ligand dissociation assays. Protein (1.5?g) from whole cell lysates prepared 24 hrs after transfection were loaded to each lane. Both wt ARfl and ARv567es proteins were detected using anti-AR antibody to the common epitope within the first 21 amino acids of the AR N-terminus domain (PG21).  Untransfected, negative control; LNCaP whole cell lysate (1.5 ?g protein); beta-actin, loading control. Samples from 3 independent experiments are shown (n=3).    51    Figure 3.5 EPI-002 accelerated the dissociation of 3H-R1881 from wild-type full-length AR in Cos-1 cells. (A) A representative 3H-R1881 dissociation curve of Cos-1 cells transfected with full-length wild-type AR for 24 hrs prior to treatment with DMSO vehicle or EPI-002 (25?M) for 1 hr before the addition of 3H-R1881. Cells were labeled with 3H-R1881 (5nM) for 2 hrs at 37?C. Dissociation was initiated by addition of 10,000-fold molar excess of unlabelled R1881. Cells were washed, harvested in SDS sample buffer, and radioactivity determined by scintillation counting. Non-specific-binding was assessed using parallel cells treated with 100-fold excess unlabeled R1881 at the initiation of the binding reaction. Data points represent specific 3H-R1881 bound in transfected cells (mean ? SEM from three technical replicates). (B) The average T1/2 from 3 independent experiments. Change relative to DMSO was calculated by normalizing the T1/2 of EPI-002 treated samples to the DMSO treated samples in each experiment. (C) Fold change was calculated by normalizing the mean T1/2 (min) of EPI-002 treated samples to the DMSO vehicle treated samples. In doing this, DMSO treatment fold change became one and SD zero. Error bars represent +/- SD for three independent experiments. One-sample Student?s T-test (*p < 0.05).     52    Figure 3.6 EPI-002 blocked AR N/C interaction induced by androgen.  Cos-1 cells transfected with Gal4-ARLBD, VP16-ARTAD and Gal4-TATA-luciferase reporter were pretreated with EPI-002 (25?M) or Bicalutamide (10?M) for 1 hour before the addition of R1881 (1nM) for 24 hours. Cell lysates were collected and luciferase activity was determined.  (A) The data are representative of the androgen dependent mammalian two-hybrid AR N/C interaction assay. RLU, Relative Luciferase Units; R1881, synthetic androgen 1nM; EPI-002 25?M; BIC, Bicalutamide 10?M. Bars represent the mean ? SD from three technical replicates. Two-sample student?s T-test for equal variance. (B) Levels of Gal4-ARLBD and VP16-ARTAD proteins in transfected Cos-1 cells used for N/C assay. Protein (6?g) from whole cell lysates prepared 24 hrs after transfection were loaded to each lane. Untransfected, negative control; VP16 antibody; C19 antibody targets AR LBD; beta-actin, loading control.     53     Figure 3.7 ARv567es attenuated the effect of EPI-002 on the dissociation of 3H-R1881 from wild-type full-length AR in Cos-1 cells. (A) Levels of wt ARfl and ARv567es proteins expressed in transfected Cos-1 cells used in ligand dissociation experiments. Protein (1.5?g) from whole cell lysates prepared 24 hrs after transfection were loaded to each lane. Both wt ARfl and ARv567es proteins were detected using anti-AR antibody to the common epitope within the first 21 amino acids of the AR N-terminus domain (PG21).  Untransfected, negative control; LNCaP whole cell lysate (1.5 ?g protein); beta-actin, loading control. Samples from 4 independent experiments are shown (n=4). (B) A representative 3H-R1881 dissociation curve of Cos-1 cells transfected with full-length wild-type AR and ARv567es for 24 hrs prior to treatment with DMSO control or EPI-002 (35?M) for 20 hrs before the addition of 3H-R1881 (5nM). Cells were labeled with 3H-R1881 (5nM) for 2 hrs at 37?C. Dissociation was initiated by addition of 10,000-fold molar excess of unlabelled R1881. Cells were washed, harvested in SDS sample buffer, and radioactivity determined by scintillation counting. Non-specific-binding was assessed using parallel cells treated with 100-fold excess unlabeled R1881 at the initiation of the binding reaction. Data points represent specific 3H-R1881 bound in transfected cells (mean ? SEM from three technical replicates). (C) The average T1/2 from 4 independent experiments. Change relative to DMSO was calculated by normalizing the T1/2 of EPI-002 treated samples to the DMSO treated samples in each experiment. (D) Fold change was calculated by normalizing the mean T1/2 (min) of EPI-002 treated samples to the DMSO vehicle treated samples. In doing this, DMSO treatment fold change became one and SD zero. Error bars represent +/- SD for four independent experiments. One-sample Student?s T-test. NSS, Not Statistically Significant (p > 0.05).  54     Figure 3.8 Effect of EPI-002 on the protein levels of wild-type full-length AR in Cos-1 cells.  Cos-1 cells transfected with full-length wild-type AR for 24 hrs prior to treatment with DMSO control or EPI-002 (35?M) for 20 hrs. Protein (1.5?g) from whole cell lysates were loaded to each lane. AR proteins were detected using anti-AR antibody to the common epitope within the first 21 amino acids of the AR N-terminus domain. LNCaP whole cell lysate (1.5 ?g protein); beta-actin, loading control.                         55    Figure 3.9 Effect of EPI-002 on the dissociation of 3H-R1881 from endogenous AR in LNCaP cells. LNCaP cells were treated with DMSO vehicle or EPI-002 (25?M) for 1 hr before the addition of 3H-R1881 (5nM) for 2 hrs at 37?C. Dissociation was initiated by addition of 10,000-fold molar excess of unlabelled R1881. Cells were washed, harvested in SDS sample buffer, and radioactivity determined by scintillation counting. Non-specific binding was assessed using parallel cells treated with 100-fold excess unlabeled R1881 at the initiation of the binding reaction. (A) The average T1/2 from 4 independent experiments. Change relative to DMSO was calculated by normalizing the T1/2 of EPI-002 treated samples to the DMSO treated samples in each experiment. (B) Fold change was calculated by normalizing the mean T1/2 (min) of EPI-002 treated samples to the DMSO vehicle treated samples. In doing this, DMSO treatment fold change became one and SD zero. Error bars represent +/- SD for three independent experiments. One-sample Student?s T-test (*p < 0.05). (C) Levels of full-length AR proteins expressed in LNCaP cells used in ligand dissociation experiments as described above. Protein (1.5?g) from whole cell lysates were loaded to each lane. AR proteins were detected using anti-AR antibody to the common epitope within the first 21 amino acids of the AR N-terminus domain. LNCaP whole cell lysate (1.5 ?g protein); beta-actin, loading control.      56    Figure 3.10 EPI-002 had no effect on the dissociation of 3H-R1881 from AR T877A in Cos-1 cells. (A) A representative 3H-R1881 dissociation curve of Cos-1 cells transfected with AR T877A mutant for 24 hrs prior to treatment with DMSO control or EPI-002 (25?M) for 1 hr before the addition of 3H-R1881 (5nM). Cells were labeled with 3H-R1881 (5nM) for 2 hrs at 37?C. Dissociation was initiated by addition of 10,000-fold molar excess of unlabelled R1881. Cells were washed, harvested in SDS sample buffer, and radioactivity determined by scintillation counting. Non-specific-binding was assessed using parallel cells treated with 100-fold excess unlabeled R1881 at the initiation of the binding reaction. Data points represent specific 3H-R1881 bound in transfected cells (mean ? SEM from three technical replicates). (B) The average T1/2 from 3 independent experiments. Change relative to DMSO was 57  calculated by normalizing the T1/2 of EPI-002 treated samples to the DMSO treated samples in each experiment. (C) Fold change was calculated by normalizing the mean T1/2 (min) of EPI-002 treated samples to the DMSO vehicle treated samples. In doing this, DMSO treatment fold change became one and SD zero. Error bars represent +/- SD for three independent experiments. One-sample Student?s T-test. NSS, Not Statistically Significant (p > 0.05). (D) Levels of AR T877A proteins expressed in transfected Cos-1 cells used in ligand dissociation experiments as described above. Protein (1.5?g) from whole cell lysates prepared 24 hrs after transfection were loaded to each lane. AR T877A proteins were detected using anti-AR antibody to the common epitope within the first 21 amino acids of the AR N-terminus domain.  Untransfected, negative control; LNCaP whole cell lysate (1.5 ?g protein); beta-actin, loading control. Samples from 3 independent experiments are shown (N=3).                    58    Figure 3.11 Levels of endogenous full-length AR and cumate-induced FLAG-ARv567es in LNCaP-Cu3 cells. (A) LNCaP-Cu3 cells induced with ethanol control or cumate (30?g/mL) for 24 and 48 hours in charcoal stripped serum. Protein (6?g) from whole cell lysates were loaded to each lane. Both endogenous full-length AR and FLAG-ARv567es were detected using anti-AR antibody targeting NTD of AR. (B) FLAG-ARv567es protein level is also detected using anti-FLAG antibodies. LNCaP whole cell lysate (6?g protein); beta-actin, loading control.  Vector, Empty vector.        59    60   Figure 3.12 Effect of EPI-002 on the dissociation of 3H-R1881 from endogenous full-length AR and cumate-induced FLAG-ARv567es in LNCaP-Cu3 cells.  (A) A representative 3H-R1881 dissociation curve for endogenous AR in LNCaP-Cu3 cells induced with ethanol control or cumate (30?g/mL) for 48 hours in charcoal stripped serum followed by treatment with DMSO control or EPI-002 (25?M) for 1 hr before the addition of 3H-R1881 (5nM). Cells were labeled with 3H-R1881 (5nM) for 2 hrs at 37?C. Dissociation was initiated by addition of 10,000-fold molar excess of unlabelled R1881. Cells were washed, harvested in SDS sample buffer, and radioactivity determined by scintillation counting. Non-specific binding was assessed using parallel cells treated with 100-fold excess unlabeled R1881 at the initiation of the binding reaction. Data points represent specific 3H-R1881 bound in LNCaP-Cu3 cells (mean ? SEM from three technical replicates). (B) The average T1/2 from 3 independent experiments. Change relative to DMSO was calculated by normalizing the T1/2 of EPI-002 treated samples to the DMSO treated samples in each experiment. (C) & (D) Fold change was calculated by normalizing the mean T1/2 (min) of EPI-002 treated samples to the DMSO vehicle treated samples. In doing this, DMSO treatment fold change became one and SD zero. Error bars represent +/- SD for three independent experiments. One-sample Student?s T-test. *p < 0.05. NSS, Not Statistically Significant (p > 0.05). (E) Fold change was calculated by normalizing the mean T1/2 (min) of cells expressing ARfl and ARv567es to the cells expressing ARfl. In doing this, Fold change for cells expressing ARfl became one and SD zero. Error bars represent +/- SD for three independent experiments. One-sample Student?s T-test. NSS, Not Statistically Significant (p > 0.05). (F) Levels of AR proteins in LNCaPCu3 cells incubated as described above. Protein (6?g) from whole cell lysates were loaded to each lane. Both endogenous full-length AR and cumate induced FLAG-ARv567es were detected using anti-AR antibody targeting NTD of AR. FLAG-ARv567es protein level is also detected using anti-FLAG antibodies. beta-actin, loading control.    61    Figure 3.13 EPI-002 accelerated the dissociation of 3H-R1881 from endogenous AR in LNCaP95 cells.  (A) A representative 3H-R1881 dissociation curve for endogenous AR in LNCaP95 cells treated with DMSO control or EPI-002 (25?M) for 1 hr before the addition of 3H-R1881 (5nM). Cells were labeled with 3H-R1881 (5nM) for 2 hrs at 37?C. Dissociation was initiated by addition of 10,000-fold molar excess of unlabelled R1881. Cells were washed, harvested in SDS sample buffer, and radioactivity determined by scintillation counting. Non-specific binding was assessed using parallel cells treated with 100-fold excess unlabeled R1881 at the initiation of the binding reaction. Data points represent specific 3H-R1881 bound in LNCaP95 cells (mean ? SEM from three technical replicates). (B) The average T1/2 from 3 independent experiments. Change relative to DMSO was calculated by normalizing the T1/2 of EPI-002 treated samples to the DMSO treated samples in each experiment. (C) Fold change was calculated by normalizing the mean T1/2 (min) of EPI-002 treated samples to the DMSO vehicle treated samples. In doing this, DMSO treatment fold change became one and SD zero. Error bars represent +/- SD for three independent experiments. One-sample Student?s T-test. *p < 0.05. NSS, Not Statistically Significant (p > 0.05). (D) Levels of AR proteins in LNCaP95 cells incubated as described above. LNCaP whole cell lysate (1.5 ?g protein); beta-actin, loading control.    62  Chapter 4: Discussion 4.1 EPI-002 and AR ligand binding EPI-001 (BADGE.H2O.HCl) and the inactive analogue (BADGE.2H2O) are bioaccumulated derivatives of Bisphenol A Digylcidic Ether (BADGE) isolated from marine sponge Geodia lindgreni [106]. A study by Nagai and colleagues have suggested high concentration of inactive analogue 185-9-1 (BADGE.2H2O) and BADGE.2HCl are required to bind full-length AR by competing with specific testosterone binding [132]. However, this study has not looked at EPI-001 (BADGE.H2O.HCl) or the binding site on AR for these BADGE-related compounds. Sadar and colleagues have demonstrated that EPI-001 does not compete with the binding of synthetic androgen R1881 to the AR LBD [106], and the EPI-001 chlorohydrin group specifically and covalently binds to the AR NTD AF1 region [107]. Functionally, EPI-001 inhibits the transactivation of AR NTD in LNCaP cells but not BADGE.2H2O [106]. In androgen-dependent and castration-resistant prostate cancer tumour models in mice, EPI-001 inhibits tumour growth without apparent toxicity to the animals [106]. EPI-002 (2R, 20S) is the most potent stereoisomer of EPI-001 [107]. The AR LBD functions independent of other domains to bind ligand [42]. The equilibrium binding affinity of AR with deleted NTD for 3H-R1881 is similar to that of wild-type full-length AR, indicating that the absence of N/C interaction does not affect the binding of androgen [57]. Unlike antiandrogens such as hydroxyflutamide and bicalutamide that target the AR LBD, EPI-002 is an NTD inhibitor and it does not compete for the binding of androgen to the LBD [106, 107]. Here we have demonstrated that EPI-002 did not affect the binding affinity of wild-type 63  full-length AR and the measured Kd values for 3H-1881 were comparable to the reported values [128]. Potential interaction between drug and cell membrane can affect the rate of penetration of biomolecules into the cytoplasm [130]. Although the interaction between EPI-002 and cell membrane is beyond the scope of this thesis, we tested to ensure that EPI-002 did not affect the uptake and binding of ligand to AR in the cytoplasm. We have determined EPI-002 treatment did not delay the time for ligand to bind AR. It was important to establish these premises before we could determine the effect of EPI-002 on ligand dissociation rate. 4.2 EPI-002 and dissociation of ligand from wild-type full-length AR Deletion of the AR NTD quickens the dissociation of bound ligand [57], and AR N/C interaction results in slower dissociation of bound ligand [57]. Slower androgen dissociation resulting from NTD FXXLF motif binding to LBD AF2 suggests that structural changes are transmitted from AF2 to the ligand binding pocket [82]. High affinity FXXLF motif binding may generate a stabilizing effect on helix 12 that is passed on to amino acid residues located above the ligand binding pocket, resulting in slower ligand dissociation [82].  We have demonstrated that EPI-002 accelerated the ligand dissociation rate of wild-type full-length AR in Cos-1 cells by approximately 30%, which is consistent with the finding that EPI-002 blocked AR N/C interaction in Cos-1 cells by approximately 30%. These data support the hypothesis that EPI-002 accelerates the ligand dissociation rate of AR by disrupting N/C interaction. This is the first report that demonstrates the effect of an AR NTD inhibitor on ligand dissociation rate of AR. Conventional therapies that target the AR are dependent on the LBD, 64  and these therapies include the reduction of androgen by castration and the application of antiandrogens [10]. Antiandrogens are pharmacological AR ligands that block AR N/C interaction by competing for androgen binding to the AR LBD [72, 106]. Antiandrogens such as hydroxyflutamide and bicalutamide are nonsteroidal AR ligands that bind to AR with high affinity [27], and these antiandrogens have their own dissociation rates [72]. Since we were studying the dissociation rate of androgen from AR, antiandrogens cannot be used as the control compounds in our experiments.  Interestingly, we have shown the ligand dissociation rate of wt ARfl was 34?6 minutes (Figure 3.4B). This value is substantially less than the reported values ranging from 78?11 to 144?18 minutes for ectopic wild-type full-length AR in Cos-1 cells [66, 82, 128]. Possible reasons for these different values are likely due to the fact that ectopic expression of AR protein were within physiological levels in our experiments. Supraphysiological levels of expression of AR protein may not be ideal for evaluating ligand dissociation rates, especially when considering physiological levels of AR protein allows interaction with normal intracellular coregulators that can either positively or negatively impact AR N/C interaction and ligand dissociation rate. At the same time, the levels of AR protein should be realistic for the drug to target in order to accurately assess the effect of EPI-002 on ligand dissociation rate. 4.3 Splice variant ARv567es and dissociation of ligand from wild-type full-length AR Castration of the adult men with prostate cancer lowers the circulating testosterone from the normal level (3-10ng/mL) to castrate level (<0.5ng/mL) [12]. Levels of androgen are still measurable in the tissues of patients with CRPC [11]. CRPC growth can be fueled by residual 65  circulating androgens produced from the adrenal glands or by intratumoural androgens [13, 14]. Splice Variant ARv567es is expressed more abundantly in tumours with the lower levels of androgen [22]. The C-terminally truncated ARv567es does not bind ligand and has no N/C interaction, but it can interact with full-length AR. Heterodimerization between full-length AR and ARv567es enhances the response of full-length AR to low levels of ligand [22].  Our goal was to understand the mechanism of truncated variant ARv567es on the ligand dissociation rate of full-length AR. We have determined ARv567es had no effect on the dissociation rate of 3H-R1881 from wild-type full-length AR when both proteins are ectopically expressed in Cos-1 cells. This finding does not support the hypothesis that ARv567es may slow ligand dissociation from the full-length AR. However, variant ARv567es attenuated the effects of EPI-002 on the dissociation rate of 3H-R1881 from wild-type full-length AR. This data suggest expression of ARv567es reduced the impact EPI-002 on the dissociation rate of 3H-R1881 from wild-type full-length AR. It is possible that in accordance with our hypothesis, ARv567es exerted a stabilizing effect on the wild-type full-length AR through hetero-dimerization [22] and neutralized the accelerating effect of EPI-002 on ligand dissociation from the wild-type full-length AR. Another possibility is that twice the amount of AR NTD was present when ARv567es was co-expressed with the wild-type full-length AR (Figure 3.7A), rendering EPI-002 ineffective on the dissociation of ligand under these conditions. EPI-002 is a suicide inhibitor that covalently binds to AF1 region of the AR NTD in a dose-dependent manner [107]. Therefore, it is not surprising that the concentration of EPI-002 may be the limiting factor in the presence of doubled the amount of targets contributed by full-length AR and ARv567es. However, the same concentration of EPI-001 (25?M) can sufficiently block AR transcriptional activity in LNCaP cells 66  that express 50:50 levels of full-length AR (T877A) and ARv567es [107]. The modulation of AR transcriptional activity by AR N/C interaction is complex process that involves the binding and retention of androgen to AR as well as multiple interacting peptide motifs, activation functions, and coregulatory proteins [133]. In addition to slowing the rate of ligand dissociation and stabilizing AR [57], N/C interaction facilitates AR binding to androgen response element in the chromatin and recruits chromatin remodeling complexes to direct transcription [134], indicating N/C interaction has multiple functional roles in the regulation of AR transactivation. EPI-001 also blocks AR binding to androgen response element (ARE) as well as protein-protein interactions with CBP and RAP74 that are required for AR transcriptional activity [106], and feasibly the effect of EPI compound on AR ligand dissociation rate demonstrated here only contribute in part to the overall inhibitory effect on AR transcriptional activity. Nevertheless, this is the first demonstration of the effect of ARv567es on the ligand dissociation rate of full-length AR since the discovery of AR splice variant as an emerging mechanism of CRPC development. 4.4 LNCaP prostate cancer cells and AR T877A mutant AR T877A is a mutant with promiscuous ligand binding property, and is found in LNCaP prostate cell line as well as in prostate cancer tissue specimens of patients with advanced disease [81]. The T877 residue is located in the ligand pocket and the replacement of T877 by alanine increases the space around the bound ligand to accommodate other ligands such as progestins, estrogens, cortisols and antiandrogens [135]. The T877A mutation is also adjacent to the AF2 region in which subtle local conformational changes can alter N/C and coregulatory protein 67  interactions. Comparison of the crystal structures of wild-type and T877A AR LBD reveal an increase in the flexibility of the amino acids residues surrounding the FXXLF binding region as well as a larger solvent accessible surface in the T877A AF2 [83]. In Figure 4.1, enhanced N/C interaction is evident in AR T877A compared to wild-type AR where favorable hydrophobic packing of the +4 leucine residue of FXXLF motif is shown in the T877A AF2 [83]. Consistent with the finding that enhanced N/C interaction slows dissociation of bound androgen [58], slower R1881 dissociation rate was also observed in AR T877A when compared to wild-type AR without significantly altering the ligand binding affinity [82, 128].  Figure 4.1 Predicted binding model of FXXLF motifs in the AF2 region of wild-type (A) and T877A AR (B) LBDs.  The AR LBD surfaces are shown in yellow (wild-type) and white (T877A) with a stick models of the FXXLF peptides. Note the altered conformation of the +4 leucine (LEU) residues in the T877A AR LBD. The binding +1 and +5 phenylalanine (PHE) residues are similar in the wild-type and T877A AR LBD. Figure 7A &B from ? Southwell, J et al. (2008). An investigation into CAG repeat length variation and N/C terminal interactions in the T877A mutant androgen receptor found in prostate cancer. The Journal of steroid biochemistry and molecular biology. 111(1-2): Page 138-46 [83]. By permission from Elsevier.   68  We have determined that EPI-002 had statistically significant, but minimal effect on ligand dissociation rate of LNCaP endogenous AR, consistent with the lack of effect when AR has the T877A mutation in the LBD. Consistent with the reported data [82, 128], we have shown that AR T877A has longer ligand retention than wild-type AR as indicated by a significantly slower ligand dissociation rate. Since EPI-002 blocked the N/C interaction and quickened ligand dissociation rate of wild-type by only 30%, we believe the overpowering effect of T877A mutation on ligand retention rendered EPI-002 ineffective on ligand dissociation rate. Despite these findings, it is important to point out that EPI-001 can effectively inhibit the transcriptional activity of ectopic wild-type AR in the AR-negative PC3 cells [106] as well as endogenous AR (T877A) in LNCaP cells [107]. These evidences suggest the inhibitory effect of EPI compound on AR T877A transcriptional activity may be independent of effect of the EPI compound on ligand dissociation rate.  4.5 EPI-002 and LNCaP95 prostate cancer cells  Increased AR-V7 transcript and protein levels are detected in CRPC specimens [20, 21] and the expression of AR-V7 is associated with poor prognosis in CRPC patients [108]. Unlike the ARv567es, there are no reports of AR-V7 interaction with the full-length AR. LNCaP95 is an androgen-independent cell line that expresses endogenous full-length AR and AR-V7 protein [110]. Unpublished data from our lab have shown LNCaP95 contains a functional full-length AR that induces PSA gene expression in the presence of R1881. We have determined the dissociation rate of 3H-R1881 from endogenous full-length AR in LNCaP95 was 107?15 minutes (Figure 3.13B). This value was slower than the ligand dissociation rate of endogenous full-length 69  AR in parental LNCaP cells (67?5 minutes, Figure 3.9A). These findings suggest the rate of ligand dissociation may be cell specific and dependent on culture conditions. Reasons for these different values are unclear but are likely due to the one of the following differences between LNCaP95 and the parental LNCaP: 1) the presence of androgen in culture media; 2) the presence of AR-V7; 3) mutations in the endogenous full-length AR; and 4) expression of co-regulators impacting AR N/C interaction.  AR does not act independently to mediate tissue specific response to androgen, but requires interaction with a distinct profile of cell-specific as well as ubiquitous coregulatory proteins [136]. Ligand-bound AR in the nucleus can initiate protein-protein interactions with coregulators to mediate remodeling of chromatin structure at target promoter, recruitment of basal transcriptional machinery, and the transactivation of AR target genes [136]. Over 160 AR-interacting proteins have been identified to date and these coregulators can be either coactivators or corepressors that exert positive or negative effect on AR function [137]. For example, glucocorticoid receptor interacting protein 1 (GRIP1) is a member of the p160 coactivators that has been demonstrated to behave as a bridging factor to mediate AR N/C interaction [94], whereas nuclear receptor co-repressor (N-CoR) has been shown to impair ligand-induced N/C interaction [90]. Since enhanced N/C interaction results in slower ligand dissociation rate, differential coregulator recruitment may account for the different ligand dissociation rates observed between LNCaP and LNCaP95 cells.  To our surprise, EPI-002 accelerated the rate of ligand dissociation of LNCaP95 cells expressing endogenous full-length AR and splice variant AR-V7 by 13% (Figure 3.13C) but accelerated the 70  rate of ligand dissociation of endogenous full-length AR in parental LNCaP cells by 5%. Since different prostate cancer cell lines may contain different sets or different levels of co-regulatory proteins that can affect AR N/C interaction and ligand dissociation, we believe the effect of EPI-002 on the ligand dissociation rate of AR can also be cell-specific.                   71  Chapter 5: Summary and Future Directions The nomenclature castration-resistant prostate cancer (CRPC) indicates advanced tumours are resistant to castration but may not be completely free from androgen [11]. Residual androgen produced by the adrenal glands and intracrine steroidogenesis may permit CRPC tumours to circumvent low levels of circulating androgens [13, 14]. Increased intratumoural androgen and restoration of AR activity make a significant contribution to the development of CRPC [19]. Recently discovered constitutively active splice variants of AR lacking the LBD in CRPC metastases can also contribute to resistance to castration and development of CRPC [21, 22]. ARv567es is one such variant that can interact with full-length AR to stabilize and enhance its androgen-dependent activities [22]. AR N-terminus and C-terminus (N/C) interaction slows the dissociation of bound androgen from the ligand binding pocket [58]. Enhanced N/C interaction increases the transcriptional activity of AR in response to ligand [38]. Here we have examined the specific mechanism by which splice variant ARv567es and an AR NTD antagonist can affect the ligand dissociation rate of full-length AR.  Consistent with our hypothesis, EPI-002 accelerated the ligand dissociation rate of wild-type full-length AR possibly by disruption of N/C interaction. Furthermore, we have demonstrated that EPI-002 did not affect the binding affinity of wild-type full-length AR nor the time for it to reach binding equilibrium. Co-expression of ectopic ARv567es and wild-type full-length AR at a 50:50 ratio did not alter the ligand dissociation rate of wild-type full-length AR but attenuated the effect of EPI-002. However, EPI-002 had minimal effect on the ligand dissociation rate of LNCaP endogenous AR consistent with the lack of effect when AR has a mutation in the LBD 72  (T877A) that enhances the N/C interaction and slows the ligand dissociation rate. Together these data begin to reveal 1) the unique mechanisms of splice variant ARv567es on the dissociation rate of full-length AR; and 2) the effect of an AR NTD inhibitor on the dissociation rate of full-length AR with and without the presence of splice variants. Remarkably, prostate cancer cells LNCaP and LNCaP95 have demonstrated different ligand dissociation rates, and the effect of EPI-002 on these cells are also inconsistent and difficult to interpret. In order to have a better understanding of the fundamental differences between these cell lines that lead to the different ligand dissociation profiles, we may investigate the following: 1) mutation profiles of the endogenous AR; and 2) the expression levels of endogenous coactivators and co-repressors that are known to influence the N/C interaction. A number of factors have been reported to regulate AR N/C interaction and it is conceivable that the changes in N/C interaction can alter ligand dissociate rate. For example, polymorphism of the glutamine repeat (polyQ) is intrinsic to the AR NTD and the length of the glutamine tracts vary in the normal human population [40]. Shorter polyQ tract is associated with increased risk of prostate cancer development [101]. While variation of polyQ tract length does not affect ligand binding affinity [103], a critical size of 16-29 repeats is required to maintain AR N/C function [104]. PolyQ tract does not directly contribute to N/C interaction. However, variable spacing between the FXXLF and WXXLF motif in the NTD, as the result of different glutamine repeat length, may result in structural conformation change in the NTD impacting N/C interaction. Shorter polyQ tract length has been shown to enhance AR N/C interaction [83]. Based on this finding, we can postulate that the ligand dissociation rate of AR may also be 73  affected as the result of variation in the length of polyQ tract. A somatic AR gene mutation has been detected in human prostate tumour in which two non-consecutive leucine residues interrupted the polyQ tract and disrupted AR N/C interaction [104]. We believe such mutation may also affect the ligand dissociation rate. Therefore, it will be interesting to establish a relationship between of length of polyQ tract and the ligand dissociation rate of AR in future experiments, as well as the effect of EPI-002. Furthermore, it is feasible that long-term passage of LNCaP95 cells in culture media supplemented with charcoal stripped serum depleted of hormones could either alter the length of polyQ tract or generate mutations within polyQ tract, resulting in a slower ligand dissociation rate in LNCaP95 cells compared to the parental LNCaP cells. AR W435L is a novel, gain-of-function mutation identified from antiandrogen resistant CRPC specimens [84]. AR W435 is located within the NTD WXXLF motif that has a significant but more minor role than FXXLF in mediating N/C interaction [37]. AR W435L demonstrates enhanced N/C interaction compared to the wild-type AR [84], and it is reasonable to believe that this mutation may also display a slower ligand dissociation rate. Another clinically relevant AR mutation, AR F876L, has been recently identified to confer resistance to second generation antiandrogens enzalutamide and ARN-509 [138]. F876 is located in helix 11 of the AR LBD and is believed to contribute to ligand binding by influencing ligand-induced conformational change in helix 12 [138]. Since N/C interaction is mediated by helix 12, the F876L mutation might affect the ligand dissociation rate [138]. It will be interesting to examine the effect of EPI-002 on the ligand dissociation rate of these newly discovered NTD and LBD mutants.  74  References  1. Statistics, C.C.S.s.A.C.o.C., Canadian Cancer Statistics 2013. 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