Targeting alternative sites on the androgen receptor to treat castration-resistant prostate cancer

Abstract

Recurrent, metastatic prostate cancer continues to be a leading cause of cancer-death in men. The androgen receptor (AR) is a modular, ligand-inducible transcription factor that regulates the expression of genes that can drive the progression of this disease, and as a consequence, this receptor is a key therapeutic target for controlling prostate cancer. The current drugs designed to directly inhibit the AR are called anti-androgens, and all act by competing with androgens for binding to the androgen/ligand binding site. Unfortunately, with the inevitable progression of the cancer to castration resistance, many of these drugs become ineffective. However, there are numerous other regulatory sites on this protein that have not been exploited therapeutically. The regulation of AR activity involves a cascade of complex interactions with numerous chaperones, co-factors and co-regulatory proteins, leading ultimately to direct binding of AR dimers to specific DNA androgen response elements within the promoter and enhancers of androgen-regulated genes. As part of the family of nuclear receptors, the AR is organized into modular structural and functional domains with specialized roles in facilitating their inter-molecular interactions. These regions of the AR present attractive, yet largely unexploited, drug target sites for reducing or eliminating androgen signaling in prostate cancers. The design of small molecule inhibitors targeting these specific AR domains is only now being realized and is the culmination of decades of work, including crystallographic and biochemistry approaches to map the shape and accessibility of the AR surfaces and cavities. Here, we review the structure of the AR protein and describe recent advancements in inhibiting its activity with small molecules specifically designed to target areas distinct from the receptor's androgen binding site. It is anticipated that these new classes of anti-AR drugs will provide an additional arsenal to treat castration-resistant prostate cancer.

Figure 1

The androgen signaling pathway. The androgen receptor (AR) domains are labeled as: N—N-terminal domain (NTD); D—DNA binding (DBD); L—ligand binding domain (LBD). Locally produced dihydrotestosterone (DHT) interacts with the androgen receptor to facilitate release of heat shock proteins (HSP), N/C dimerization and exposure of a nuclear localization signal (NLS) required for interaction with importin-α and nuclear translocation. Inside the nucleus, the AR exists in equilibrium between monomers and dimers. Although it is unclear which form(s) are required for DNA binding, the AR exists as a dimer when bound to androgen response elements (AREs). DNA binding causes the N/C interactions to be lost, allowing the recruitment of transcription initiation and bridging factors, such as CREB binding protein (CBP), the transcription activator p300 and the AR-associated protein 70 (ARA70) to the AR. RNA-Polymerase II (RNAP2) can then transcribe AR-dependent genes.

Figure 2

Domain organization and available structures of the androgen receptor. (A) Scheme of the domain organization of the AR: NTD (N-terminal domain), DBD (DNA binding domain), H (hinge region) and LBD (ligand binding domain). Residue numbers above the scheme delineate the domain boundaries; (B) Surface (left) and cartoon (right) representations of the rat AR-DBD structure. Zinc ions are presented as grey spheres, and the D-box of each DBD monomer is highlighted in red. Residues of the P-box involved in DNA recognition are shown as sticks; (C) Surface (left) and cartoon (right) representations of the human AR-LBD structure: surfaces highlighted in cyan and magenta correspond to AF2 and BF3, respectively. The cartoon representation in yellow corresponds to a coactivator bound to AF2, and the stick representation in green depicts a BF3 small molecule inhibitor. R1881, a synthetic androgen, bound to the androgen binding site, is shown in blue.

Figure 3

Common splice variants of the AR. The AR gene is organized as eight exons, which form the coding sequence for its different domains: NTD—exon 1; DBD—the first and second zinc finger motifs are encoded by exon 2 and 3, respectively; Hinge—exon 4; LBD—exons 5–8. The RNA transcript can by spliced in several ways to include combinations of the standard exons (numbered 1–8), as well as inclusion of cryptic exons (1a, 2a, 3a, 3b). Shown above each splice variant are the corresponding exon numbers included in spliced mRNA. Cryptic exon inclusion results in unique (U) regions with novel nucleic acid sequences not found in the wild-type (WT) AR. In AR-V3/AR6, the inclusion of exon 2 yields a splice variant bearing only one zinc finger (Zn) and, thus, a truncated DBD.

Figure 4

Detailed view of the androgen binding site in complex with (A) testosterone, (B) hydroxyflutamide and (C) R-bicalutamide. The hydrogen bonds and ionic interactions between the protein and the ligand are shown as dashed lines.