Identification of neuron selective androgen receptor inhibitors

AIM To identify neuron-selective androgen receptor(AR) signaling inhibitors, which could be useful in the treatment of spinal and bulbar muscular atrophy(SBMA), or Kennedy's disease, a neuromuscular disorder in which deterioration of motor neurons leads to progressive muscle weakness. METHODS Cell lines representing prostate, kidney, neuron, adipose, and muscle tissue were developed that stably expressed the CFP-AR-YFP FRET reporter. We used these cells to screen a library of small molecules for cell typeselective AR inhibitors. Secondary screening in luciferase assays was used to identify the best cell-type specific AR inhibitors. The mechanism of action of a neuronselective AR inhibitor was examined in vitro using luciferase reporter assays, immunofluorescence microscopy, and immunoprecipitations. Rats were treated with the most potent compound and tissue-selective AR inhibition was examined using RT-q PCR of AR-regulated genes and immunohistochemistry.RESULTS We identified the thiazole class of antibiotics as com-pounds able to inhibit AR signaling in a neuronal cell line but not a muscle cell line. One of these antibiotics, thiostrepton is able to inhibit the activity of both wild type and polyglutamine expanded AR in neuronal GT1-7 cells with nanomolar potency. The thiazole antibiotics are known to inhibit FOXM1 activity and accordingly, a novel FOXM1 inhibitor FDI-6 also inhibited AR activity in a neuron-selective fashion. The selective inhibition of AR is likely indirect as the varied structures of these compounds would not suggest that they are competitive antagonists. Indeed, we found that FOXM1 expression correlates with cell-type selectivity, FOXM1 co-localizes with AR in the nucleus, and that sh RNA-mediated knock down of FOXM1 reduces AR activity and thiostrepton sensitivity in a neuronal cell line. Thiostrepton treatment reduces FOXM1 levels and the nuclear localization of beta-catenin, a known co-activator of both FOXM1 and AR, and reduces the association between beta-catenin and AR. Treatment of rats with thiostrepton demonstrated AR signaling inhibition in neurons, but not muscles. CONCLUSION Our results suggest that thiazole antibiotics, or other inhibitors of the AR-FOXM1 axis, can inhibit AR signaling selectively in motor neurons and may be useful in the treatment or prevention of SBMA symptoms.     

Mechanism underlying the retarded nuclear translocation of androgen receptor splice variants

As shown in our previous study, two alternatively spliced androgen receptor(AR) variants, which are exclusively expressed in the granulosa cells of patients with polycystic ovary syndrome, exhibit retarded nuclear translocation compared with wild-type AR. However, researchers have not yet determined whether these abnormalities correlate with heat shock protein 90(HSP90)and importin α(the former is a generally accepted co-chaperone of AR, and the latter is a component of classical nuclear import complexes). Here, these two variants were mainly retained in cytoplasm with HSP90 and importin α in the presence of dihydrotestosterone(DHT), and their levels in nucleus were significantly reduced, according to the immunofluorescence staining. The binding affinity of two AR variants for importin α was consistently decreased, while it was increased in WT-AR following DHT stimulation, leading to reduced nuclear import, particularly for the insertion-AR(Ins-AR). However, the binding affinities of two AR variants for HSP90 were increased in the absence of DHT compared with WT-AR, which functioned to maintain spatial structural stability, particularly for the deletion-AR(Del-AR). Therefore, the retarded nuclear translocation of two AR variants is associated with HSP90 and importin α, and the abnormal binding affinities for them play critical roles in this process.     

Small molecule inhibitors as probes for estrogen and androgen receptor action

Because activated estrogen (ER) and androgen (AR) receptors stimulate cell proliferation in breast and prostate cancer, inhibiting their actions represents a major therapeutic goal. Most efforts to modulate ER and AR activity have focused on inhibiting the synthesis of estrogens or androgens or on the identification of small molecules that act by competing with agonist hormones for binding in the ligand-binding pocket of the receptor. An alternative approach is to implement screens for small molecule inhibitors that target other sites in the pathway of steroid receptor action. Many of these second-site inhibitors directly target ER or AR; others have still unknown sites of action. Small molecule inhibitors that target second sites represent new leads with clinical potential; they serve as novel modulators of receptor action; and they can reveal new and as yet unidentified interactions and pathways that modulate ER and AR action.     

N-terminal polyglutamine-containing fragments inhibit androgen receptor transactivation function

Abstract Several neurodegenerative diseases, including Kennedy's disease (KD), are associated with misfolding and aggregation of polyglutamine (polyQ)-expansion proteins. KD is caused by a polyQ-expansion in the androgen receptor (AR), a key player in male sexual differentiation. Interestingly, KD patients often show signs of mild-to-moderate androgen insensitivity syndrome (AIS) resulting from AR dysfunction. Here, we used the yeast Saccharomyces cerevisiae to investigate the molecular mechanism behind AIS in KD. Upon expression in yeast, polyQ-expanded N-terminal fragments of AR lacking the hormone binding domain caused a polyQ length-dependent growth defect. Interestingly, while AR fragments with 67 Q formed large, SDS-resistant inclusions, the most pronounced toxicity was observed upon expression of 102 Q fragments which accumulated exclusively as soluble oligomers in the 100-600 kDa range. Analysis using a hormone-dependent luciferase reporter revealed that full-length polyQ-expanded AR is fully functional in transactivation, but becomes inactivated in the presence of the corresponding polyQ-expanded N-terminal fragment. Furthermore, the greatest impairment of AR activity was observed upon interaction of full-length AR with soluble AR fragments. Taken together, our results suggest that soluble polyQ-containing fragments bind to full-length AR and inactivate it, thus providing insight into the mechanism behind AIS in KD and possibly other polyglutamine diseases, such as Huntington's disease.     

Proteasome activity is required for androgen receptor transcriptional activity via regulation of androgen receptor nuclear translocation and interaction with coregulators in prostate cancer cells

Upon binding to androgen, the androgen receptor (AR) can translocate into the nucleus and bind to androgen response element(s) to modulate its target genes. Here we have shown that MG132, a 26 S proteasome inhibitor, suppressed AR transactivation in an androgen-dependent manner in prostate cancer LNCaP and PC-3 cells. In contrast, MG132 showed no suppressive effect on glucocorticoid receptor transactivation. Additionally, transfection of PSMA7, a proteasome subunit, enhanced AR transactivation in a dose-dependent manner. The suppression of AR transactivation by MG132 may then result in the suppression of prostate-specific antigen, a well known marker used to monitor the progress of prostate cancer. Further mechanistic studies indicated that MG132 may suppress AR transactivation via inhibition of AR nuclear translocation and/or inhibition of interactions between AR and its coregulators, such as ARA70 or TIF2. Together, our data suggest that the proteasome system plays important roles in the regulation of AR activity in prostate cancer cells and may provide a unique target site for the development of therapeutic drugs to block androgen/AR-mediated prostate tumor growth.     

Hormonal control of androgen receptor function through SIRT1

The NAD-dependent histone deacetylase Sir2 plays a key role in connecting cellular metabolism with gene silencing and aging. The androgen receptor (AR) is a ligand-regulated modular nuclear receptor governing prostate cancer cellular proliferation, differentiation, and apoptosis in response to androgens, including dihydrotestosterone (DHT). Here, SIRT1 antagonists induce endogenous AR expression and enhance DHT-mediated AR expression. SIRT1 binds and deacetylates the AR at a conserved lysine motif. Human SIRT1 (hSIRT1) repression of DHT-induced AR signaling requires the NAD-dependent catalytic function of hSIRT1 and the AR lysine residues deacetylated by SIRT1. SIRT1 inhibited coactivator-induced interactions between the AR amino and carboxyl termini. DHT-induced prostate cancer cellular contact-independent growth is also blocked by SIRT1, providing a direct functional link between the AR, which is a critical determinant of progression of human prostate cancer, and the sirtuins.     

Quantifying effects of ligands on androgen receptor nuclear translocation, intranuclear dynamics, and solubility

Using manual and automated high throughput microscopy (HTM), ligand-dependent trafficking of green fluorescent protein-androgen receptor (GFP-AR) was analyzed in fixed and living cells to determine its spatial distribution, solubility, mobility, and co-activator interactions. Within minutes, addition of the agonist R1881 resulted translocation of GFP-AR from the cytoplasm to the nucleus, where it displayed a hyperspeckled pattern and extraction resistance in low expressing cells. AR antagonists (Casodex, hydroxyflutamide) also caused nuclear translocation, however, the antagonist-bound GFP-AR had a more diffuse nuclear distribution, distinct from the agonist-bound GFP-AR, and was completely soluble; overexpressed GFP-AR in treated cells was extraction resistant, independent of ligand type. To more dramatically show the different effects of ligand on AR distribution, we utilized an AR with a mutation in the DNA binding domain (ARC619Y) that forms distinct foci upon exposure to agonists but retains a diffuse nuclear distribution in the presence of antagonists. Live-cell imaging of this mutant demonstrated that cytoplasmic foci formation occurs immediately upon agonist but not antagonist addition. Fluorescence recovery after photobleaching (FRAP) revealed that agonist-bound GFP-AR exhibited reduced mobility relative to unliganded or antagonist-bound GFP-AR. Importantly, agonist-bound GFP-AR mobility was strongly affected by protein expression levels in transiently transfected cells, and displayed reduced mobility even in slightly overexpressing cells. Cyan fluorescent protein-AR (CFP-AR) and yellow fluorescent protein-CREB binding protein (YFP-CBP) in the presence of agonists and antagonists were used to demonstrate that CFP-AR specifically co-localizes with YFP-CBP in an agonist dependent manner. Dual FRAP experiments demonstrated that CBP mobility mirrored AR mobility only in the presence of agonist. HTM enabled simultaneous studies of the sub-cellular distribution of GFP-AR and ARC619Y in response to a range of concentrations of agonists and antagonists (ranging from 10(-12) to 10(-5)) in thousands of cells. These results further support the notion that ligand specific interactions rapidly affect receptor and co-factor organization, solubility, and molecular dynamics, and each can be aberrantly affected by mutation and overexpression.     

4-Azasteroidal 5 alpha-reductase inhibitors without affinity for the androgen receptor

In efforts to develop potent 5 alpha-reductase inhibitors without affinity for the androgen receptor, synthetic 3-oxo-5 alpha-steroids were tested for their ability to inhibit 5 alpha-reductase, using [14C]testosterone as the substrate, and for their ability to inhibit the binding of [3H]5 alpha-dihydrotestosterone to the androgen receptor of rat prostate cytosol. 2',3' alpha-Tetrahydrofuran-2'-spiro-17-(5 alpha-androstan-3-one) is not an inhibitor of 5 alpha-reductase and has a high affinity for the androgen receptor; substitution of the -CH2- at the 4-position with N-H resulted in a good inhibitor of 5 alpha-reductase. The 4-N-CH3 derivative is even more active, whereas the N-CH2-CH3 derivative is inactive. These 4-aza derivatives have much lower affinity for the androgen receptor than the parent compound. The 4-N-H derivatives of several 3-oxo-5 alpha-steroids were found to be 20-100% as potent as their corresponding 4-N-CH3 analogs as inhibitors of 5 alpha-reductase, whereas their androgen receptor affinities were at least 40-fold lower than their 4-N-CH3 analogs. Their 5 beta-isomers did not inhibit either 5 alpha-reductase or the androgen receptor binding of [3H]5 alpha-dihydrotestosterone. Two of these 4-N-H steroids, 17 beta-N,N-diethylcarbamoyl-4-aza-5 alpha-androstan-3-one and 17 beta-N, N-diisopropylcarbamoyl-4-aza-5 alpha-androstan-3-one, are potent 5 alpha-reductase inhibitors with Ki values equal to 29.2 +/- 1.7 and 12.6 +/- 0.8 nM, respectively, but have little affinity for the androgen receptor. The inhibition of 5 alpha-reductase by both compounds is competitive with testosterone. When [3H]testosterone was incubated with minced rat prostate in the presence of either of these two 4-azasteroids, the nuclear concentration of 5 alpha-dihydrotestosterone decreased and that of testosterone increased. The total nuclear uptake of testosterone plus 5 alpha-dihydrotestosterone was not significantly affected. These 4-azasteroids should be useful for investigating the importance of 5 alpha-reductase in androgen action in vivo.     

Effect of protease inhibitors on androgen receptor analysis in rat prostate cytosol

Prostate androgen receptors are liable to proteolytic digestion during in vitro analysis; thus, various proteolytic enzyme inhibitors were tested for their ability to improve the androgen receptor assay. The serine (phenylmethylsulfonylflouride, aprotinin, p-aminobenzamidine) and thiol-senine (leupeptin, bacitracin) protease inhibitors individually present in the homogenization buffer significantly increased the measurable androgen binding sites by 30-35% in rat prostate cytosol as determined by saturation analysis with [3H]-17 beta-hydroxy-17-methyl- 4,9 11-estratrien-3-one (R-1881) for 20 hr at 4 degrees C. The apparent binding affinity was also increased by these compounds. Various combinations were tried and aprotinin/bacitracin was found to be additive in effect. This combination was also shown to prevent receptor degradation as determined by sucrose density gradient centrifugation. The carboxyl protease inhibitor, pepstatin A, was ineffective in improving the receptor assay. Rabbit bile, an inhibitor of seminin, interfered with receptor binding thus rendering it ineffective for use in saturation analysis. The results show that the use of serine-thiol protease inhibitors significantly improves the cytosol androgen receptor yield and assay sensitivity; therefore, we recommend routine inclusion of these compounds(s) in the homogenization buffer for androgen receptor assays.     

The mannose 6-phosphate receptor: function,biosynthesis and translocation

This report summarizes studies concerning the role of the lysosomal protein: Man-6-P receptor and describes some recent data on its biosynthesis and cellular translocation. The receptor functions both in the Golgi apparatus (or GERL) and on the cell surface where it binds lysosomal proteins and mediates their transport to lysosomes. Consistent with its dual role, the receptor in several cell types has been localized to the plasma membrane and Golgi cisternae, to clathrin-coated structures at both locations, and to vesicles characteristic of endosomes or CURL. Biosynthetic studies have shown that the receptor undergoes several post-translational modifications including the processing of its asparagine-linked oligosaccharides, phosphorylation of serine residues, and unknown modifications required for acquisition of immunoreactivity and functional activity. Cellular pools of mature receptor readily mix as evidenced by rapid labeling of intracellular receptor by exogenously added receptor antibodies. Degradation of the receptor occurs non-lysosomally and is perhaps mediated by extracellular Man-6-P-containing hydrolases. A working hypothesis for the mechanism of Man-6-P receptor function that is consistent with these observations is presented.     

Evolution of the androgen receptor: structure–function implications

To shed light on the nature and evolution of structure–function relations in the androgen receptor (AR), we have undertaken a comparative analysis of all available AR and other steroid receptor sequences. We have identified a group of amino acids that “diagnose” the clade of androgen receptors—residues that are strictly conserved among the ARs but not shared with other receptors. We hypothesize that these amino acids, clustered in a few regions of the protein, confer upon the androgen receptor its unique functions, including recognition of specific DNA response elements and affinity for androgens, rather than other steroid hormones. The four domains of the AR display markedly different rates of evolutionary divergence; conserved portions of the sequence, including small stable stretches within otherwise divergent regions, may be essential to receptor function. Current data from experimental, crystallographic, and clinical studies support these hypotheses, which can now be further tested in the laboratory. BioEssays 20 :860–869, 1998. © 1998 Wiley-Liss, Inc.     

The pituitary function of androgen receptor constitutes a glucocorticoid production circuit

Androgen receptor (AR) mediates diverse androgen actions, particularly reproductive processes in males and females. AR-mediated androgen signaling is considered to also control metabolic processes; however, the molecular basis remains elusive. In the present study, we explored the molecular mechanism of late-onset obesity in male AR null mutant (ARKO) mice. We determined that the obesity was caused by a hypercorticoid state. The negative feedback system regulating glucocorticoid production was impaired in ARKO mice. Male and female ARKO mice exhibited hypertrophic adrenal glands and glucocorticoid overproduction, presumably due to high levels of adrenal corticotropic hormone. The pituitary glands of the ARKO males had increased expression of proopiomelanocortin and decreased expression of the glucocorticoid receptor (GR). There were no overt structural abnormalities and no alteration in the distribution of cell types in the pituitaries of male ARKO mice. Additionally, there was normal production of the other hormones within the glucocorticoid feedback system in both the pituitary and hypothalamus. In a cell line derived from pituitary glands, GR expression was under the positive control of the activated AR. Thus, this study suggests that the activated AR supports the negative feedback regulation of glucocorticoid production via up-regulation of GR expression in the pituitary gland.