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A high-throughput PPI assay to quantify inhibition of androgen receptor variant 7 (AR-V7) binding to canonical AR full-length (AR-FL) binding partners

Lookup NU author(s): Professor Ian HicksonORCiD


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Background: Prostate Cancer is the most common non-skin cancer in the United States, affecting 1 in 7 men over their lifetimes [1]. It is the second most frequent cause of cancer-related deaths for men. Despite significant advancements in the treatment of Prostate Cancer within the past several years, numerous resistance mechanisms have been described for patients administered androgen-deprivation therapy (ADT) [2]. Recently, Antonarakis et al. reported that in patients treated with enzaluatmide or abiraterone acetate plus prednisone, high levels of Androgen Receptor Variant 7 (AR-V7) mRNA in circulating tumor cells (CTC’s) were strongly correlated with a lack of clinical response to these drugs [3]. AR-V7 is upregulated following suppression of full-length Androgen Receptor (AR-FL) in vitro [4] and its expression is sufficient to induce canonical and distinct AR-FL downstream pathways [4, 5, 6]. Therefore, development of an inhibitor of AR-V7 activity has become a high-priority in pharmaceutical research. In this abstract, we describe a high-throughput protein-protein interaction PPI FRET-like assay, based on Promega's NanoBiT platform, which is amenable to screening potential inhibitors of AR-V7 binding to AR-FL canonical binding partners. Additionally, we report compounds that antagonize these interactions.Methods: We developed a set of AR full-length, truncated variants, and binding partners (SRC-1, RAP74) to interrogate PPI interactions. These constructs were tethered to one of two components of Promega's NanoBiT system, in which the binding interaction and kinetics are monitored by alterations in the NanoLuciferase (Oplophorus gracilirostris) signal [7]. This platform was validated using described agonists (R1881) and antagonists (enzalutamide and bicalutamide) of N-terminal and C-terminal AR dimerization. Subsequent experiments evaluated the effectiveness of AR-V7/AR-V7 or AR-V7/SRC1 NanoBiT binding and potential for disruption by small molecules.Results: We demonstrate that our system is capable of detecting activation and inhibition of AR-NTD and AR-CTD dimerization with either R1881 or R1881 in the presence of enzalutamide or bicaluatmide, respectively. Additionally, we have evaluated inhibition of AR-V7 binding to canonical AR-FL binding partners, SRC-1 and RAP74. These results demonstrate the utility of this platform to screen molecules for their ability to inhibit both AR-FL and splice variant dimerization and the resulting nuclear translocation and activation of downstream pathways.[1] Prostate Cancer Foundation website.[2] Sprenger and Plymate, Horm Cancer 2014.[3] Antonarakis et al., NEJM, 2014.[4] Hu R, Lu C, Mostaghel EA, et al., Cancer Research, 2014.[5] Hörnberg et al., PLoS ONE, 2011.[6] Zhang et al., PLoS ONE, 2011.[7] Hall et al., ACS Chem Biol., 2012.

Publication metadata

Author(s): Branch J, Quigley M, Gottardis M, Hickson I

Publication type: Conference Proceedings (inc. Abstract)

Publication status: Published

Conference Name: AACR 107th Annual Meeting 2016

Year of Conference: 2016

Print publication date: 15/07/2016

Online publication date: 22/07/2016

Acceptance date: 01/12/2015

ISSN: 1538-7445

Publisher: American Association for Cancer Research


DOI: 10.1158/1538-7445.AM2016-359