Inhibition of Androgen Receptor Nuclear Localization and Castration-Resistant Prostate Tumor Growth by Pyrroloimidazole-based Small Molecules

Abstract

The androgen receptor (AR) is a ligand-dependent transcription factor that controls the expression of androgen-responsive genes. A key step in androgen action, which is amplified in castration resistant prostate cancer (CRPC), is AR nuclear translocation. Small molecules capable of inhibiting AR nuclear localization could be developed as novel therapeutics for CRPC. We developed a high throughput screen and identified two structurally-related pyrroloimidazoles that could block AR nuclear localization in CRPC cells. We show that these two small molecules, 3-(4-ethoxyphenyl)-6,7-dihydro-5H-pyrrolo[1,2-a]imidazole (EPPI) and 3-(4-chlorophenyl)-6,7-dihydro-5H-pyrrolo[1,2-a]imidazole (CPPI) can inhibit the nuclear localization and transcriptional activity of AR and reduce the proliferation of AR-positive but not AR-negative prostate cancer cell lines. EPPI and CPPI did not inhibit nuclear localization of the glucocorticoid receptor or the estrogen receptor, suggesting they selectively target AR. In LNCaP tumor xenografts, CPPI inhibited the proliferation of relapsed LNCaP tumors. These findings suggest that EPPI and CPPI could serve as lead structures for the development of therapeutic agents for CRPC.

Regulation and targeting of androgen receptor nuclear localization in castration-resistant prostate cancer

“…Since nuclear AR does not seem to undergo export, agents causing AR cytoplasmic localization in CRPC cells are likely acting through enhancing nuclear AR degradation. We previously showed that the small molecule CPPI can inhibit AR nuclear localization in cultured CRPC cells (28). We also reported that CPPI could significantly inhibit growth of relapsed LNCaP xenograft tumors over a 26-day treatment period (28).…”

“…First, cells were transfected with GFP-AR, and then treated with 3-(4-chlorophenyl)-6,7-dihydro-5H-pyrrolo[1,2-a]imidazole (CPPI), a small molecule that can cause AR cytoplasmic localization in PCa cells (28). GFP-AR was localized predominantly in the cytoplasm in C4-2 cells in the presence of CPPI.…”

Assays to Interrogate the Ability of Compounds to Inhibit the AF-2 or AF-1 Transactivation Domains of the Androgen Receptor

“…10 failed to produce a calculable IC 50 , it inhibited DHT-induced AR-TIF2 PPI formation, potentially due to its ability to reduce AR expression levels and/or restrict its localization to the cytoplasm. 30,48,49 None of the test compounds were acutely cytotoxic toward U-2 OS cells under the conditions used.…”

“…63 Compounds No. 10 and EPI-001 target the amino terminus of AR rather than the ligand binding site, 30,41,48,49,64 and 17-AAG is an inhibitor of the Hsp90 molecular chaperone ATPase that is involved in the folding, maturation, and maintenance of AR in a high-affinity ligand binding conformation ( Supplementary Fig. S2).…”

“…Formaldehyde, DHT, flutamide, bicalutamide, enzalutamide (MDV3100), EPI-001, abiraterone acetate, 17-AAG (17-allylaminogeldanamycin), and Tris(2-carboxyethyl) phosphine hydrochloride (TCEP) were purchased from Sigma-Aldrich (St. Louis, MO). Compound 10, 2-[(isoxazol-4-ylmethyl)thio]-1-(4-phenylpiperazin-1-yl)ethanone [47][48][49] was kindly provided by Dr. Zhou Wang in the Department of Urology of the University of Pittsburgh Hillman Cancer Institute (Pittsburgh, PA), and Hoechst 33342 was purchased from Invitrogen (Carlsbad, CA). Dimethyl sulfoxide (DMSO; 99.9% high-performance liquid chromatography grade, under argon) was from Alfa Aesar (Ward Hill, MA).…”