Effects of the inhibitors on FKBP52-regulated AR function in mammalian cells. (A and B) MDA-kb2 cells expressing a stably transfected AR-responsive luciferase reporter were treated with 0.2 nM DHT and a range of concentrations of the indicated compounds and assessed for cell viability (A) and AR-dependent expression of a luciferase reporter (B). The IC₅₀ values for the compounds are shown in the legend. (C–E) Luciferase reporter assays in 52KO mouse embryonic fibroblast cells in the presence or absence of FKBP52 were performed. Transfected cells were treated with DHT and a range of concentrations of the indicated compounds and assessed for cell viability (C) and AR-dependent expression of a luciferase reporter (D and E). The IC₅₀ values for MJC01 (D) and MJC13 (E) are shown in the legends. (F) Lysates were prepared from 52KO MEF cells transfected with AR and FKBP52 expression vectors after treatment with DHT and a range of MJC13 concentrations (0, 0.05, 0.1, 0.5, 1, 5, 10, 50, and 100 μM) for 24 hr. Lysates were immunoblotted for AR, FKBP52, and GAPDH (loading control).

Effects of MJC13 on AR-dependent gene expression in early and late stage prostate cancer cells. (A and B) ELISA assays to measure PSA secretion were performed in LNCaP (A) and VCaP (B) cells. Cells were treated with the indicated MJC13 concentrations in the presence (A) or absence (B) of DHT for 24 hr, and PSA levels in the media were quantified. (C and D) Western blots to measure AR-dependent expression of PSA and FKBP51 were performed in LNCaP (C) and VCaP (D) cells. Cells were treated with the indicated concentrations of MJC13 in the presence (C) or absence (D) of DHT for 24 hr, lysed, and lysates were electrophoresed and immunoblotted for FKBP51, PSA, and GAPDH (loading control). The upper panels show representative Western blots. The lower panels represent averaged densitometry data from at least three independent experiments. (E) Left panel: PSA and TMPRSS2 gene expression in LNCaP cells was assessed by Q-PCR. Cells were treated for 24 hr with increasing concentrations of MJC13 in the presence of 10% fetal bovine serum. Data are displayed as expression relative to that of 18S rRNA; right panel: R1881-dependent and independent PSA gene expression in 22Rv1 cells was assessed by Q-PCR. Cells (in the presence of charcoal-stripped serum) were untreated, treated for 24 hr with MJC13 alone, or with 0.5 nM R1881 in the presence and absence of 30 μM MJC13. Data are displayed as PSA mRNA expression relative to that of 18S rRNA.

Identification of inhibitors specific for FKBP52-regulated AR transcriptional activity in yeast. Yeast reporter strains expressing wild-type AR in the absence (control, closed circles) or presence (AR, closed squares) of FKBP52, the AR-P723S point mutant in the absence (control, closed circles) or presence of FKBP52 (AR-P723S, closed triangles), and wild-type GR in the absence (control, closed circles) or presence (GR, closed diamonds) were treated with a range of concentrations of the indicated compounds in the presence of DHT. H7 (A) is the original hit identified from the library screens, MJC01 (B) and MJC013 (C) are the current lead compounds, and flufenamic acid (D) is a known AR inhibitor that associates with the BF3 surface. The structures of the molecules are illustrated above each respective graph. The data were normalized to show only effects on FKBP52-enhanced AR function by calculating the percent reduction in the control strain for each data point and adding that back to each data point for both the control and FKBP52 tester strains. Thus, a hormesis-like effect, as seen for MJC01 at 100 μM (B), indicates that the receptor in the absence of FKBP52 was inhibited at the particular drug dose used (FKBP52-independent inhibition).

Effects of MJC13 on AR-Hsp90 complex dissociation and AR nuclear translocation in early and late stage prostate cancer cells. The effects of MJC13 on hormone-dependent AR-Hsp90 complex dissociation and AR nuclear translocation were assessed in LNCaP (A and B), LAPC4 (C and D), and CWR22Rv1 (E and F) cells by coimmunoprecipitation and Western blot, respectively. Lysates from cells grown in the presence or absence of the indicated concentrations of hormone and MJC13 for 24 hr were subjected to immunoprecipitation with either an antibody directed against FKBP52 (A) or AR (C and E) and immunoblotted for the indicated proteins. Fetal bovine serum served as the source of hormone in A. Lysates prepared from cells treated with the indicated concentrations of ligand and MJC13 for 24 hr were also fractionated and immunoblotted for AR in both the cytosol and nucleus (B, D, and F).

MJC compounds effectively inhibit androgen-dependent prostate cancer cell proliferation. Tritium (tritiated thymidine) incorporation assays were performed on LNCaP (A), LAPC4 (B), and 22Rv1 (C) cells treated with a range of compound concentrations in the presence (closed symbols) or absence (open symbols) of 0.5 nM DHT. The known AR antagonist bicalutamide (circles) was included for comparison with MJC13 (squares). All data are expressed as a percentage with the level of tritiated thymidine incorporation in the absence of compound for each condition set to 100%.