Abstract 3692: High-throughput cell-based screen for inhibitors of AR activity
Authors: Richards Juliet L. , Barrie S. Elaine , Garrett Michelle D. 
Published: February 2011

Advanced prostate cancer is treated by castration and androgen receptor (AR) antagonists but despite an initial response, relapse eventually occurs. Although the mechanism for disease progression remains to be elucidated, in many cases castration-resistant prostate cancer (CRPC) continues to rely on AR transcriptional activity for growth through increased receptor expression or promiscuity, but there are few treatments available that directly target the AR protein. In the cytoplasm the AR binds to androgens that include testosterone and dihydrotestosterone, allowing it to dissociate from heat shock proteins and translocate to the nucleus, undergoing post-translational modifications including phosphorylation and acetylation that affect its activity. The dimerised AR binds to promoter regions of target genes such as prostate specific antigen and the TMPRSS2:ERG fusion gene, resulting in cell growth and survival. Our objective was therefore to look for inhibitors of AR function.

Initial work focussed on serine 81 (S81) which is a major site of phosphorylation on the AR that may be implicated in AR stability and therefore represents a potential therapeutic target for CRPC. The importance of phosphorylation at S81 was investigated by using mutants that mimic the phosphorylated and de-phosphorylated states. Our studies suggested that loss of S81 phosphorylation alone has no effect on AR stability, and therefore inhibition of S81 phosphorylation is not a suitable target for developing future treatments for CRPC.

Inhibitors of AR function were identified using cells expressing AR and a PSA promoter driven luciferase gene. A high throughput cell-based screen was used to test a diverse set of compounds with unknown functions and a selection of known kinase, phosphatase and nuclear receptor inhibitors at 20µM for inhibition of AR activity and cytotoxic properties. The design of this novel screen, using a wild type receptor and an excess of androgen, militates against identification of weak AR anatagonists; for example in this assay the IC50 for the antiandrogen bicalutamide is &gt;50µM and for the heat shock protein 90 inhibitor 17-AAG is 0.2µM. Of the 10,000 compounds screened there were 117 hits. Confirmed hit compounds showed more than 50% inhibition of AR activity and less than 50% toxicity at 20µM. A selection of these compounds were tested for effects on AR protein expression, cell viability, AR localisation and transcriptional activity with AR independent promoters and the results of these analyses will be presented. The development of a potent and selective AR inhibitor should provide a valuable additional therapeutic approach to treating CRPC.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3692.