Identification and Characterization of MEL-3, a Novel AR Antagonist That Suppresses Prostate Cancer Cell Growth

Abstract

Antiandrogens 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.

The Effect of F877L and T878A Mutations on Androgen Receptor Response to Enzalutamide

“…The construct containing double-tagged (N-ter YFP and C-ter CFP) WT AR (17) has been used to generate mutations in the LBD using QuikChange II kit (Agilent Technologies). A plasmid containing pCMV-b-Gal was acquired from Stratagene, the luciferase reporter construct driven by 4 copies of a classical ARE and the VP16 AD-NTD(WT) expression vector have been described elsewhere (16,18). All expression vectors were sequence verified.…”

Targeting Alternative Sites on the Androgen Receptor to Treat Castration-Resistant Prostate Cancer

“…Recent advances in the area of rational and computer-aided drug design have resulted in the development of a number of other candidate anti-androgens targeting the androgen-binding site, including compounds, such as 6-(3,4-dihydro-1 H -isoquinolin-2-yl)- N -(6-methylpyridin-2-yl)nicotinamide (DIMN) [12], its derivatives, termed 7AU and 7BB [115], and 8-(propan-2-yl)-5,6-dihydro-4 H -pyrazino[3,2,1-jk]carbazole (MEL-3) [116], all showing promising in vitro and in vivo activities and currently undergoing various stages of pre-clinical development.…”

The Discovery of Novel Human Androgen Receptor Antagonist Chemotypes Using a Combined Pharmacophore Screening Procedure

“…The generation of this cell line and its characteristics were described by Helsen et al [10] ClARE and HEK293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g L À1 glucose, supplemented with 10 % fetal bovine serum (Invitrogen), l-glutamine (Invitrogen), and penicillinstreptomycin (Invitrogen). These cells contain a randomly integrated expression cassette for the human AR under control of the CMV promoter and a luciferase reporter gene under the control of an E1B TATA box and four copies of the Slp-HRE 2 mutated (5'-AGAACTggcTGTCCA-3') in the Flp Recombination Target site.…”

“…However, these compounds bind with low affinity and can induce escape mechanisms. [10] We identified a new inhibitor chemotype, of which the most potent (MEL-3) decreases proliferation of various AR-positive cancer lines, showing improved potency over the first-generation anti-androgens and a more pronounced decrease in the expression of androgen-regulated genes (PSA and FKBP5). [5] Long-term treatment of PCa patients with these anti-androgens has indicated that resistant mutations in the AR gene can arise, resulting in clinically occurring AR mutants such as W741C/L and T877A.…”

“…[4] Furthermore, Bic and HOFl have been reported to have (partial) agonistic activity in settings of increased AR levels and/or altered recruitment of the AR co-activators or corepressors. [10] Furthermore, MEL-3 was also shown to be antagonistic on AR W741C and AR T877A, providing evidence that novel chemotypes of anti-androgens are promising for follow up treatment of CRPC patients. [6] These mutant receptors have promiscuous ligand specificity; moreover, the anti-androgens can exhibit agonistic instead of antagonistic effects.…”