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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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Abstract

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

URL: http://dx.doi.org/10.1016/j.jsbmb.2016.05.005

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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