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...