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The fluorescent two-hybrid assay for live-cell profiling of androgen receptor modulators

Lookup NU author(s): Professor Ian HicksonORCiD


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The androgen receptor (AR) is an important target for drug therapies combating prostate cancer. However, various acquired mutations within the AR sequence often render this receptor resistant to treatment. Ligand-induced interaction between the N- and C-termini of the AR marks the initial step in the AR signaling cascade and can thus serve as an early read-out for analysis of potential antagonists of wt and mutant AR.To measure changes of the N/C interaction in the wt and mutant AR variants upon the addition of inhibitors, we applied our recently developed Fluorescent Two-Hybrid (F2H) assay. The F2H method enables real-time monitoring and quantitative analysis of the interactions between GFP- and RFP-tagged proteins in live mammalian cells, where GFP-tagged proteins are tethered to a specific nuclear location. This anchoring approach provides a local signal enrichment suitable for direct visualization of protein–protein interactions as co-localizations by conventional epifluorescence microscopy.Since the F2H assay is fully reversible, we could monitor dynamics of AR N/C interactions in living cells in real time upon agonistic, as well as antagonistic treatments. In dose-response F2H experiments, we compared the potencies of abiraterone, bicalutamide, enzalutamide, flutamide, and galeterone/TOK-001 to prevent the dihydrotestosterone-induced N/C interaction in wt AR. We further applied the newly developed F2H assay to analyze how the AR N/C interaction is affected by the clinically relevant mutations W741L, F876L, T877A and F876L/T877A. We conclude that F2H is a reliable and technically undemanding approach for straightforward screening of new AR modulators, as well as for monitoring their activity in real time in living cells.

Publication metadata

Author(s): Bogner J, Zolghadr K, Hickson I, Romer T, Yurlova L

Publication type: Article

Publication status: Published

Journal: The Journal of Steroid Biochemistry and Molecular Biology

Year: 2017

Volume: 166

Pages: 45-53

Print publication date: 01/02/2017

Online publication date: 09/05/2016

Acceptance date: 07/05/2016

ISSN (print): 0960-0760

ISSN (electronic): 1879-1220

Publisher: Pergamon Press


DOI: 10.1016/j.jsbmb.2016.05.005

PubMed id: 27174722

Notes: Highlights • F2H AR is a novel live-cell assay for evaluation of androgens and antiandrogens. • DHT, abiraterone, bicalutamide, enzalutamide, flutamide, galeterone were tested. • W741L, F876L, T877A and F876L/T877A AR mutations were evaluated with F2H AR. • The assay enables real-time visualization and monitoring of compounds’ activity. • The assay facilitates comparative analysis of mutant AR activation upon treatments


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