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.
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