Castration-recurrent prostate cancer (CRPC) is suspected to depend on androgen receptor (AR). The AF-1 region in the amino-terminal domain (NTD) of AR contains most, if not all, of the transcriptional activity. Here we identify EPI-001, a small molecule that blocked transactivation of the NTD and was specific for inhibition of AR without attenuating transcriptional activities of related steroid receptors. EPI-001 interacted with the AF-1 region, inhibited protein-protein interactions with AR, and reduced AR interaction with androgen-response elements on target genes. Importantly, EPI-001 blocked androgen-induced proliferation and caused cytoreduction of CRPC in xenografts dependent on AR for growth and survival without causing toxicity.

Hormone therapies for advanced prostate cancer target the androgen receptor (AR) ligand-binding domain (LBD), but these ultimately fail and the disease progresses to lethal castration-resistant prostate cancer (CRPC). The mechanisms that drive CRPC are incompletely understood, but may involve constitutively active AR splice variants that lack the LBD. The AR N-terminal domain (NTD) is essential for AR activity, but targeting this domain with small-molecule inhibitors is complicated by its intrinsic disorder. Here we investigated EPI-001, a small-molecule antagonist of AR NTD that inhibits protein-protein interactions necessary for AR transcriptional activity. We found that EPI analogs covalently bound the NTD to block transcriptional activity of AR and its splice variants and reduced the growth of CRPC xenografts. These findings suggest that the development of small-molecule inhibitors that bind covalently to intrinsically disordered proteins is a promising strategy for development of specific and effective anticancer agents.

The androgen receptor (AR) is a member of the nuclear receptor superfamily. Sequences within the large amino-terminal domain of the receptor have been shown to be important for transactivation and proteinprotein interactions; however, little is known about the structure and folding of this region. In the present study we show that a 344-amino acid polypeptide representing the main determinants for transactivation has the propensity to form ␣-helical structure and that mutations which disrupt putative helical regions alter conformation. Folding of the AR was observed in the presence of the helix-stabilizing solvent trifluoroethanol and the natural osmolyte trimethylamine N-oxide (TMAO). TMAO resulted in the movement of two tryptophan residues to a less solvent-exposed environment and the formation of secondary/tertiary structure resistant to protease cleavage. Critically, binding to the RAP74 subunit of the general transcription factor TFIIF resulted in extensive protease resistance, consistent with induced folding of the receptor transactivation domain. These data indicate that this region of the AR is structurally flexible and folds into a stable conformation upon interactions with a component of the general transcription machinery.The actions of the male sex hormones testosterone and dihydrotestosterone are mediated by the intracellular androgen receptor (AR) 1 (reviewed in Refs. 1 and 2). The AR belongs to a large family of nuclear receptors, whose members mediate the actions of steroid and thyroid hormones, retinoic acid, vitamin D 3 , and fatty acid derivatives. The majority of these receptor proteins share a common domain organization (see Fig. 1A). The ligand binding domain (LBD) in the carboxyl terminus binds ligand and participates in dimerization and ligand-dependent transactivation. A hinge region connects the LBD to the DNA binding domain (DBD), a region responsible for recognition of and binding to DNA target sequences and receptor dimerization. The amino-terminal domain (NTD) of these proteins is the most variable in length and amino acid sequence but is often critical for transactivation. In the case of the AR, the major transactivation domain has been mapped to the amino-terminal domain (3-8). Sequences within this domain have been shown to mediate protein-protein interactions with the carboxyl-terminal LBD (9 -15), the general transcription factors TFIIF (16) and TFIIH (17), members of the p160 family of nuclear receptor coactivator proteins (18 -21), cyclin E (22), and an AR-associated protein 160 (23). This region of the receptor also contains homopolymer stretches of the amino acids glutamine (Gln), glycine (Gly), and proline (Pro). The largest stretch of glutamines is of particular interest because expansion of this sequence from on-average 22 to greater than 40 residues results in the neuromuscular degenerative condition spinal bulbar muscular atrophy or Kennedy's disease (reviewed in Refs. 24 and 25). In addition, polymorphisms in this polyglutamine stretch have been associated with prosta...

The androgen receptor (AR) is a member of the nuclear hormone receptor family of transcription factors that plays a critical role in regulating expression of genes involved in prostate development and transformation. Upon hormone binding, the AR associates with numerous co-regulator proteins that regulate the activation status of target genes via flux to the post-translational modification status of histones and the receptor. Here we show that the AR interacts with and is directly methylated by the histone methyltransferase enzyme SET9. Methylation of the AR on lysine 632 is necessary for enhancing transcriptional activity of the receptor by facilitating both inter-domain communication between the N- and C-termini and recruitment to androgen-target genes. We also show that SET9 is pro-proliferative and anti-apoptotic in prostate cancer cells and demonstrates up-regulated nuclear expression in prostate cancer tissue. In all, our date indicate a new mechanism of AR regulation that may be therapeutically exploitable for prostate cancer treatment.

Poly-amino acid repeats, especially long stretches of glutamine (Q), are common features of transcription factors and cell-signalling proteins and are prone to expansion, resulting in neurodegenerative diseases. The amino-terminal domain of the androgen receptor (AR-NTD) has a poly-Q repeat between 9 and 36 residues, which when it expands above 40 residues results in spinal bulbar muscular atrophy. We have used spectroscopy and biochemical analysis to investigate the structural consequences of an expanded repeat (Q45) or removal of the repeat (DQ) on the folding of the AR-NTD. Circular dichroism spectroscopy revealed that in aqueous solution, the AR-NTD has a relatively limited amount of stable secondary structure. Expansion of the poly-Q repeat resulted in a modest increase in a-helix structure, while deletion of the repeat resulted in a small loss of a-helix structure. These effects were more pronounced in the presence of the structure-promoting solvent trifluoroethanol or the natural osmolyte trimethylamine N-oxide. Fluorescence spectroscopy showed that the microenvironments of four tryptophan residues were also altered after the deletion of the Q stretch. Other structural changes were observed for the AR-NTDQ45 polypeptide after limited proteolysis; in addition, this polypeptide not only showed enhanced binding of the hydrophobic probe 8-anilinonaphthalene-1-sulphonic acid but was more sensitive to urea-induced unfolding. Taken together, these findings support the view that the presence and length of the poly-Q repeat modulate the folding and structure of the AR-NTD.

The actions of the male sex hormones testosterone and dihydrotestosterone are mediated by the intracellular androgen receptor (AR) 1 (reviewed in Refs. 1 and 2). In the absence of hormone, the receptor is sequestered in the cytosol with molecular chaperone proteins, which dissociate upon hormone binding. The hormone-bound receptor translocates to the nucleus and is targeted to specific genes through the recognition and binding to the DNA response element, 5Ј-AGA/TACA/TnnnT/ AGTTCT/C-3Ј, which in turn leads to activation of gene transcription (3-10). The activated receptor also represses gene expression through protein-DNA interactions at negative response elements (11,12) or through interactions with other transcription factors (13)(14)(15)(16)(17).In addition to the well characterized DNA-binding domain (DBD) and ligand-binding domain (LBD), regions of the proteins important for transactivation have been mapped to the amino-terminal domain (NTD; 18 -21). These studies have revealed a modular nature for the AR-transactivation domain, with the region between amino acids 142 and 485, containing the TAU-1/AF-1 and TAU-5/AF-5 determinants, being critical for receptor-dependent activation (20, 21). Sequences within the AR-NTD have been shown to mediate protein-protein interactions with the carboxyl-terminal LBD (22-28), the general transcription factors TFIIF (29) and TFIIH (30), members of the p160 family of nuclear receptor coactivator proteins (31-34), and the general coactivator CREB-binding protein (35,36).TFIIF is a tetramer of two subunits, RAP30 and RAP74. TFIIF recruits TFIIE and TFIIH to the preinitiation complex (PIC) and interacts directly with the RNA polymerase II enzyme and prevents pausing of the enzyme during subsequent transcription elongation (37-39). Previously, we have demonstrated that the isolated transactivation function of the human AR, amino acids 142 to 485, interacts with the large subunit of TFIIF, termed RAP74, and that this interaction was capable of reversing AR-dependent squelching of basal transcription under cell-free conditions (29). More recently, we have shown that binding of RAP74 results in the AR-transactivation domain adopting a protease-resistant conformation (40).In the present study we have extended these observations to map the region(s) of RAP74 involved in this interaction with the AR. Using a series of deletion constructs of RAP74 we show that sequences within both the amino-and carboxyl-terminal domains of the protein are sufficient to bind the AR-transactivation function and to reverse receptor-dependent squelching of transcription. In the context of the holo-TFIIF, the carboxylterminal binding site may be the main binding site. Introduction of point mutations into the AR-transactivation domain revealed that sequences near the amino terminus are important for RAP74 binding. These mutations fail to disrupt the interaction of the AR with the p160 coactivator protein SRC-1a. Thus, TFIIF and SRC-1a interact with distinct regions of the AR-transactivation domain. The impl...

Steroid hormones are a diverse class of structurally related molecules, derived from cholesterol, that include androgens, estrogens, progesterone and corticosteroids.They represent an important group of physiologically active signalling molecules that bind intracellular receptor proteins and regulate genes involved in developmental, reproductive and metabolic processes.The receptor proteins share structurally and functionally related ligand binding and DNA-binding domains, but possess distinct N-terminal domains (NTD) of unique length and amino acids sequence.The NTD contains sequences important for gene regulation, exhibit structure plasticity and are likely to contribute to the specificity of the steroid hormone/receptor response. (Figure 1). SHR have been shown to contain two transactivation functions: one is represented by a structurally defined hydrophobic groove on the surface of the LBD, formed by residues from helices 3, 4, 5 and 12 (AF2), while the other maps to the structurally flexible N-terminal domain (NTD) and is termed AF1 (Figure 1 and DNA-binding (DBD) domains is shown. Note: high resolution structures are available for the isolated LBD and DBD of nearly all SHR, but no multi-domain structure has yet been reported for a member of the nuclear receptor superfamily. The LBD consists of 12 α-helices folded in a three-layer helical sandwich and is linked via a flexible hinge region to the DBD, which has a characteristic globular fold made up of two perpendicular α-helices. The NTD is unique to each SHR and has variable sequence and length [Lavery and McEwan, 2005]. The proposed helical and unfolded conformation for this domain is based in structure predictions (see below), spectroscopy analysis and site-directed mutagenesis and proteolytic sensitivity of the AR-and GR-NTD (see text for details). Androgen receptor-NTD (NR3C4) Folding and functionThe AR-NTD transactivation function is highly modular, with key sequences mapping to amino acids 101 to 370 and 360 to 485 and termed TAU1 and TAU5, respectively [Chamberlain et al

The mineralocorticoid receptor (MR) binds the steroid hormones aldosterone and cortisol and has an important physiological role in the control of salt homeostasis. Regions of the protein important for gene regulation have been mapped to the amino-terminal domain (NTD) and termed activation function (AF)1a, AF1b, and middle domain (MD). In the present study, we used a combination of biophysical and biochemical techniques to investigate the folding and function of the MR-NTD transactivation functions. We demonstrate that MR-AF1a and MR-MD have relatively little stable secondary structure but have the propensity to form α-helical conformation. Induced folding of the MR-MD enhanced protein-protein binding with a number of coregulatory proteins, including the coactivator cAMP response element-binding protein-binding protein and the corepressors SMRT and RIP140. By contrast, the MR-AF1b domain appeared to have a more stable conformation consisting predominantly of β-secondary structure. Furthermore, MR-AF1b specifically interacted with the TATA-binding protein, via an LxxLL-like motif, in the absence of induced folding. Together, these data suggest that the MR-NTD contains a complex transactivation system made up of distinct structural and functional domains. The results are discussed in the context of the induced folding paradigm for steroid receptor NTDs.