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High Content Positional Biosensor Assay to Screen for Compounds that Prevent or Disrupt Androgen Receptor and Transcription Intermediary Factor 2 Protein-Protein Interactions

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High Content Screening

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1683))

Abstract

Transcriptional Intermediary Factor 2 (TIF2) is a key Androgen receptor (AR) coactivator that has been implicated in the development and progression of castration resistant prostate cancer (CRPC). This chapter describes the implementation of an AR-TIF2 protein-protein interaction (PPI) biosensor assay to screen for small molecules that can induce AR-TIF2 PPIs, inhibit the DHT-induced formation of AR-TIF2 PPIs, or disrupt pre-existing AR-TIF2 PPIs. The biosensor assay employs high content imaging and analysis to quantify AR-TIF2 PPIs and integrates physiologically relevant cell-based assays with the specificity of binding assays by incorporating structural information from AR and TIF2 functional domains along with intracellular targeting sequences using fluorescent protein reporters. Expression of the AR-Red Fluorescent Protein (RFP) “prey” and TIF2-Green Fluorescent Protein (GFP) “bait” components of the biosensor is directed by recombinant adenovirus (rAV) expression constructs that facilitated a simple co-infection protocol to produce homogeneous expression of both biosensors that is scalable for screening. In untreated cells, AR-RFP expression is localized predominantly to the cytoplasm and TIF2-GFP expression is localized only in the nucleoli of the nucleus. Exposure to DHT induces the co-localization of AR-RFP within the TIF2-GFP positive nucleoli of the nucleus. The AR-TIF2 biosensor assay therefore recapitulates the ligand-induced translocation of latent AR from the cytoplasm to the nucleus, and the PPIs between AR and TIF2 result in the colocalization of AR-RFP within TIF2-GFP expressing nucleoli. The AR-TIF2 PPI biosensor approach offers significant promise for identifying molecules with potential to modulate AR transcriptional activity in a cell-specific manner that may overcome the development of resistance and progression to CRPC.

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Acknowledgments

The studies reported herein were funded by grant support from the National Institutes of Health (NIH); R21NS073889 Johnston (PI) from the NINDS, and R01CA160423 and RO1CA183882 Johnston (PI) from the NCI.

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Authors and Affiliations

  1. Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Room 586 Salk Hall, 3501 Terrace Street, Pittsburgh, PA, 15261, USA

    Yun Hua, Daniel P. Camarco & Paul A. Johnston Ph.D.

  2. Cyprotex US, 313 Pleasant Street, Watertown, MA, 02472, USA

    Christopher J. Strock

Authors
  1. Yun Hua
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  2. Daniel P. Camarco
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  3. Christopher J. Strock
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  4. Paul A. Johnston Ph.D.
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Corresponding author

Correspondence to Paul A. Johnston Ph.D. .

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Editors and Affiliations

  1. Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

    Paul A. Johnston

  2. Bahama Bio, LLC, Bahama, North Carolina, USA

    Oscar J. Trask

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Hua, Y., Camarco, D.P., Strock, C.J., Johnston, P.A. (2018). High Content Positional Biosensor Assay to Screen for Compounds that Prevent or Disrupt Androgen Receptor and Transcription Intermediary Factor 2 Protein-Protein Interactions. In: Johnston, P., Trask, O. (eds) High Content Screening. Methods in Molecular Biology, vol 1683. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7357-6_13

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  • DOI: https://doi.org/10.1007/978-1-4939-7357-6_13

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7355-2

  • Online ISBN: 978-1-4939-7357-6

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