ARTICLE ABSTRACTAntiandrogens are an important component of prostate cancer therapy as the androgen receptor (AR) is the key regulator of prostate cancer growth and survival. Current AR antagonists, such as bicalutamide and hydroxyflutamide, have a low affinity for the AR and as a result block AR signaling insufficiently. Moreover, many patients develop a resistance for bicalutamide or hydroxyflutamide during therapy or show a clinical improvement after withdrawal of the antiandrogen. New and more effective AR antagonists are needed to ensure follow-up of these patients. We therefore developed a screening system to identify novel AR antagonists from a collection of compounds. MEL-3 [8-(propan-2-yl)-5,6-dihydro-4H-pyrazino[3,2,1-jk]carbazole] was selected as potent inhibitor of the AR and was further characterized in vitro. On different prostate cancer cell lines MEL-3 displayed an improved therapeutic profile compared with bicalutamide. Not only cell growth was inhibited but also the expression of androgen-regulated genes: PSA and FKBP5. Prostate cancer is often associated with mutated ARs that respond to a broadened spectrum of ligands including the current antiandrogens used in the clinic, hydroxyflutamide and bicalutamide. The activity of two mutant receptors (AR T877A and AR W741C) was shown to be reduced in presence of MEL-3, providing evidence that MEL-3 can potentially be a follow-up treatment for bicalutamide- and hydroxyflutamide-resistant patients. The mechanism of action of MEL-3 on the molecular level was further explored by comparing the structure–activity relationship of different chemical derivatives of MEL-3 with the in silico docking of MEL-3 derivatives in the binding pocket of the AR. Mol Cancer Ther; 11(6); 1257–68. ©2012 AACR.