Small molecules modulators of androgen receptor (AR) activity may serve as therapeutic agents against prostate cancer. The most effective screens for identification of novel chemotherapeutic agents are likely to be based on the measurement of specific biologic activity. As interest in screens to identify active chemicals in cancer research grows, we have developed a novel, high-throughput assay to screen for small-molecules that modulate AR activity to aid in the identification of new agents against prostate cancer. This assay, designated the Multifunctional Androgen Receptor Screening (MARS) assay, can efficiently screen for both AR agonists and antagonists in a rapid, high-throughput format. Androgen-independent human prostate adenocarcinoma-derived PC3 cells were transfected to co-express the wild-type human AR and an androgen-sensitive promoter regulating the expression of destabilized enhanced GFP. The sensitivity and dynamic range of androgenic stimulation was established using a dose-response of the stable, synthetic androgen R1881. Identification of novel antagonists of AR transcription was achieved via co-administration of R1881 at its EC50 (i.e., 1 pM) with potential AR antagonists. The assay was formatted for high-throughput screening using the HyperCyt autosampling system and flow cytometry. A library of endocrine active chemicals developed by NICETAM/ICCVAM, which includes steroidal compounds with known agonist and antagonist activity and compounds with putative endocrine modulatory activity, was used to validate the utility of the MARS assay. High-throughput screening of 118 chemicals from the library using the MARS assay resulted in the identification of compounds with a range of activities on AR transcription. The endogenous AR ligand dihydrotestosterone, and the therapeutic AR antagonists bicalutamide and cyproterone acetate, compounds with known AR-mediated activity, performed as expected, verifying the accuracy of the assay. Surprisingly, because the MARS assay uniquely measures AR activity, agonist activity of both bicalutamide and cyproterone acetate was observed at higher concentrations of these agents, which may have important implications for screening AR antagonists. Analysis of screening results from the NICETAM/ICCVAM library indicated that 19 compounds exhibited agonist activities, 37 were antagonists, and 16 compounds displayed partial agonist characteristics. The remaining compounds were not active. Dose-response assays were completed to determine the potency of compounds with novel activities and to help elucidate novel mechanisms of action (e.g., competitive binding versus allosteric activity). MARS analyses of additional chemical libraries are in progress to identify chemical classes modulating AR activity to further guide the development of therapeutic agents against prostate cancer.

99th AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA