Drugs that target novel surfaces on the androgen receptor (AR) and/or novel AR regulatory mechanisms are promising alternatives for the treatment of castrate-resistant prostate cancer. The 52 kDa FK506 binding protein (FKBP52) is an important positive regulator of AR in cellular and whole animal models and represents an attractive target for the treatment of prostate cancer. We used a modified receptor-mediated reporter assay in yeast to screen a diversified natural compound library for inhibitors of FKBP52-enhanced AR function. The lead compound, termed MJC13, inhibits AR function by preventing hormone-dependent dissociation of the Hsp90-FKBP52-AR complex, which results in less hormonebound receptor in the nucleus. Assays in early and late stage human prostate cancer cells demonstrated that MJC13 inhibits AR-dependent gene expression and androgen-stimulated prostate cancer cell proliferation.immunophilin | FKBP4 | steroid hormone receptor A ndrogens are a major stimulator of prostate tumor growth, and all current therapies act as classic antagonists by competing with androgens for binding the androgen receptor (AR) hormone binding pocket. This mechanism of action exploits the dependence of AR on hormone activation, but current treatment options become ineffective in castrate-resistant prostate cancer (CRPC), although CRPC remains ligand/AR-dependent. Thus, drugs that target novel surfaces on AR and/or novel AR regulatory mechanisms may provide promising alternatives for the treatment of CRPC (reviewed in ref. 1).The maturation of cytoplasmic steroid hormone receptors (SHR) to a mature hormone binding conformation is a highly ordered, dynamic process that involves multiple chaperone and cochaperone components (reviewed in ref. 2), all of which present potential opportunities for therapeutic intervention. The final mature complex in which the receptor is capable of high affinity hormone binding includes heat shock protein 90 (Hsp90), a 23 kDa cochaperone (p23), and one of a class of proteins (termed FKBPs) characterized by their Hsp90-binding tetratricopeptide repeat (TPR) domain. The 52 kDa FK506 binding protein (FKBP52) associates with receptor-Hsp90 complexes by way of a C-terminal TPR domain and is a specific positive regulator of AR, glucocorticoid receptor (GR), and progesterone receptor (PR) signaling (3-5). FKBP52 is required for normal male sexual differentiation and development in mice as the fkbp52-deficient mice (52KO) display characteristics of partial androgen insensitivity syndrome including dysgenic prostate (4, 6). FKBP proteins are validated targets of immunosuppressive drugs. FK506 (Tacrolimus) is used clinically to suppress the immune system following organ transplantation. FK506 binds within the peptidyl-prolyl isomerase (PPIase) catalytic pocket of a related family member, FKBP12. The chemical groups of FK506 that project out from the PPIase pocket allow the FKBP12-drug complex to bind tightly to and inhibit calcineurin, which ultimately leads to immunosupression (7). Although FK506 binding ...

The androgen receptor (AR) is the best studied drug target for the treatment of prostate cancer. While there are a number of drugs that target the AR, they all work through the same mechanism of action and are prone to the development of drug resistance. There is a large unmet need for novel AR inhibitors which work through alternative mechanism(s). Recent studies have identified a novel site on the AR called Binding Function 3 (BF3) that is involved into AR transcriptional activity. In order to identify inhibitors that target the BF3 site, we have conducted a large-scale in-silico screen followed by experimental evaluation. A number of compounds were identified that effectively inhibited the AR transcriptional activity with no obvious cytotoxicity. The mechanism of action of these compounds was validated by biochemical assays and x-ray crystallography. These findings lay a foundation for the development of alternative or supplementary therapies capable of combating prostate cancer even in its anti-androgen resistant forms.

Androgen receptor (AR) inhibitors are used to treat multiple human diseases, including hirsutism, benign prostatic hypertrophy, and prostate cancer, but all available anti-androgens target only ligand binding, either by reduction of available hormone or by competitive antagonism. New strategies are needed, and could have an important impact on therapy. One approach could be to target other cellular mechanisms required for receptor activation. In prior work, we used a cell-based assay of AR conformation change to identify non-ligand inhibitors of AR activity. Here, we characterize 2 compounds identified in this screen: pyrvinium pamoate, a Food and Drug Administration-approved drug, and harmol hydrochloride, a natural product. Each compound functions by a unique, non-competitive mechanism and synergizes with competitive antagonists to disrupt AR activity. Harmol blocks DNA occupancy by AR, whereas pyrvinium does not. Pyrvinium inhibits AR-dependent gene expression in the prostate gland in vivo, and induces prostate atrophy. These results highlight new therapeutic strategies to inhibit AR activity.antagonist ͉ harmol ͉ pyrvinium T he androgen receptor (AR) is a member of the nuclear hormone receptor superfamily, which consists of a large group of ligand-regulated transcription factors. AR is expressed in many tissues and influences an enormous range of physiologic processes such as cognition, muscle hypertrophy, bone density, and prostate growth and differentiation (1). However, AR signaling is directly linked to numerous diseases, including benign prostatic hyperplasia, alopecia, and hirsutism (2). AR signaling also drives the proliferation of prostate cancer, even in the setting of therapies that reduce systemic hormone ligand levels, making AR the major therapeutic target for this malignancy (3).Before ligand binding, AR associates with a complex of cytoplasmic factors and molecular chaperones that maintain the receptor in a high-affinity ligand binding conformation (4). AR signaling is initiated by binding of testosterone or the more potent dihydrotestosterone (DHT). This induces an intramolecular conformation change in AR that brings the amino (N) and carboxy (C) termini into close proximity. This occurs with a t 1/2 of approximately 3.5 min in cells treated with DHT (5), and does not occur in cell lysates (6), suggesting that the induced conformation change is not protein autonomous, but depends on additional cellular factors. Activated AR accumulates in the nucleus, where it binds to DNA as a homodimer at specific androgen response elements to regulate gene expression. Transcriptional control by AR results from complex interactions with positive (i.e., co-activator) and negative (i.e., co-repressor) factors (1). These co-regulatory factors fine-tune AR activity, and AR can even be activated in the absence of ligand by certain cross-talk pathways (7).Although AR activity is highly regulated, with many possible points for intervention, all existing approaches to block AR signaling ultimately target ligand bin...

Summary Using a mouse model of ependymoma—a chemoresistant brain tumor—we combined multi-cell high-throughput screening (HTS), kinome-wide binding assays, and in vivo efficacy studies, to identify potential treatments with predicted toxicity against neural stem cells (NSC). We identified kinases within the insulin signaling pathway and centrosome cycle as regulators of ependymoma cell proliferation, and their corresponding inhibitors as potential therapies. FDA approved drugs not currently used to treat ependymoma were also identified that posses selective toxicity against ependymoma cells relative to normal NSCs both in vitro and in vivo e.g., 5-fluoruracil. Our comprehensive approach advances understanding of the biology and treatment of ependymoma including the discovery of several treatment leads for immediate clinical translation.

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