Non-competitive androgen receptor inhibition in vitro and in vivo - PubMed (original) (raw)

Non-competitive androgen receptor inhibition in vitro and in vivo

Jeremy O Jones et al. Proc Natl Acad Sci U S A. 2009.

Abstract

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.

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Conflict of interest statement

Conflict of interest statement: Given the potential utility of pyrvinium and harmol as therapeutic agents, the University of California, San Francisco, has filed a novel use patent that claims these compounds. J.O.J. and M.I.D. are co-inventors on this patent, and will stand to profit if it is issued. To the extent that publication of this manuscript will increase the value of this patent, or the likelihood that it will be out-licensed, J.O.J. and M.I.D. have a potential conflict of interest.

Figures

Fig. 1.

(A) Structures of pyrvinium and harmol. (B and C) LAPC4 or LNCaP cells were transfected with luciferase reporter constructs and treated with titrations of the indicated compounds for 24 h. IC50 values and SDs were calculated from the renilla-normalized PSA-luciferase reporter activities from 4 independent experiments with single (B) or combination (C) treatments. Expected and actual IC50 and combinatorial index values (CI at IC50) were calculated from mean-effect plots. A CI50 of 1 indicates an additive effect, a CI <1 indicates synergy, and a CI >1 indicates antagonism. (PCl, pyrvinium chloride.) (D) LNCaP cells were treated with 1 nM DHT and the indicated compounds for 24 h and androgen-responsive transcript levels were quantified relative to vehicle-treated cells. Averaged results from 4 independent experiments indicate that PP and HH inhibit both AR transcriptional activation and repression. (Error bars indicate SEM.)

Fig. 2.

Non-competitive inhibition of AR. (A) LAPC4 cells were transfected with luciferase reporter constructs and treated with a DHT dose titration in the presence or absence of 1 μM OH-F, 100 nM PP, or 300 nM HH. Renilla-normalized PSA-luciferase activity was measured 24 h later. OH-F shifted the response to the right, indicating competitive antagonism. PP and HH did not shift the curve, but prevented the maximum response, consistent with non-competitive inhibition. (B) LAPC4 cells were incubated with 1 nM [3H]DHT and the indicated compounds. Inhibition of [3H]DHT binding by each compound is expressed relative to the no-competition value, which was set to 1. Neither PP nor HH competed for binding at their fully effective concentrations (100 nM and 300 nM, respectively). (C and D) Chromatin immunoprecipitation: LNCaP cells were treated with the indicated compounds for 4 h, followed by cross-linking, sonication, and immunoprecipitation with anti-AR or anti-RNA pol II antibodies. AR- (C) or RNA pol II- (D) bound chromatin was isolated and occupancy was quantified by quantitative PCR using primers specific to known AR-binding sites (C) or sites −2257 to +995 bp from the PSA transcription start site (D). Primers specific to the HSPA1A promoter were used to control for non-specific occupancy (31). BiC and HH displaced AR from regulated promoters, whereas PP had no effect, although all 3 AR inhibitors prevented recruitment of RNA pol II to the PSA promoter. Error bars indicate SEM (8 biological replicates).

Fig. 3.

Inhibition of androgen-dependent and independent growth. The proliferation of LNCaP, LN-AR, and HEK293 cells in the presence or absence of 3 nM DHT and indicated drugs was determined, using DAPI staining to measure the DNA content of the cells in 12 independent replicates for each condition. PP significantly inhibited the growth of both LNCaP (i.e., androgen-dependent) and LN-AR (i.e., androgen-independent) cells at day 7 (ANOVA, P < 0.0004), whereas BiC and HH inhibited the growth of only LNCaP cells (ANOVA, P < 0.03). Control HEK293 cells were unaffected.

Fig. 4.

Properties and efficacy of PP and HH in vivo. (A) Mice were administered 5 mg/kg PP or 2 mg/kg HH by the indicated methods. Plasma concentrations were determined by MS, and reported in ng/mL. (nd, compound not detected.) (B and C) Litter-mate-controlled FVB male mice were treated for 4 weeks with 100 mg/kg oral BiC (n = 9), an escalating dose of i.p. PP to 1 mg/kg (n = 9), or a combination of these treatments (n = 9). Cohorts of mice were castrated at the onset of the study (n = 9), or were sham-treated with i.p. and oral vehicles (n = 10). (B) Prostate wet weight was measured as a proximal marker of anti-androgen potency. (C) Quantitative PCR was performed on reverse-transcribed RNA isolated from mouse prostate tissues. Transcripts for each gene were normalized to RPL19, an androgen-unresponsive gene. (ANOVA, *P < 0.05; **P < 0.01; ***P < 0.001; ns, no significant difference between indicated populations; error bars indicate SEM.) (D) Representative sections of dorsal prostate from each treatment group were stained using hematoxylin and eosin. Substantial prostate atrophy is present in castrated and combination treatment samples. Signs of androgen deprivation are also visible in the samples of mice treated with BiC and PP alone. Note the loss of columnar epithelial architecture (white arrows), decreased secretory protein, increased nuclear staining, a relative thickening of the stromal layer, and dead cell accumulation in the lumen (black arrows).

Fig. 5.

Model of mechanisms of non-competitive AR antagonism. Apo-AR is in a distinct conformation before ligand binding. Ligand binding induces conformation change, nuclear accumulation, dimerization, and assembly of a transcriptional complex at regulated promoters. BiC directly competes for DHT binding, blocking AR activation at a proximal step. HH prevents DNA binding by nuclear-localized AR. PP permits AR promoter binding, but interferes with assembly of a productive transcription initiation complex.

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Non-competitive androgen receptor inhibition in vitro and in vivo - PubMed (original) (raw)