title: The fluorescent two-hybrid assay for live-cell profiling of androgen receptor modulators.
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.
authors.affiliation: ChromoTek GmbH, Martinsried, Germany.
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Bogner
authors.affiliation: ChromoTek GmbH, Martinsried, Germany.
authors.forename: Kourosh
authors.initials: K
authors.lastname: Zolghadr
authors.affiliation: Janssen R&D, LLC, A Division of Johnson & Johnson, Spring House, PA, United States.
authors.forename: Ian
authors.initials: I
authors.lastname: Hickson
authors.affiliation: ChromoTek GmbH, Martinsried, Germany. Electronic address: t.romer@chromotek.com.
authors.forename: Tina
authors.initials: T
authors.lastname: Romer
authors.affiliation: ChromoTek GmbH, Martinsried, Germany.
authors.forename: Larisa
authors.initials: L
authors.lastname: Yurlova
mesh_terms: D000726:Androgen Antagonists / Q000737:chemistry*; D000728:Androgens / Q000737:chemistry*; D000730:Androstadienes / Q000737:chemistry; D000736:Androstenes / Q000737:chemistry; D000813:Anilides / Q000737:chemistry; D000818:Animals; D001549:Benzamides; D001562:Benzimidazoles / Q000737:chemistry; D001681:Biological Assay; D002460:Cell Line; D006224:Cricetinae; D013196:Dihydrotestosterone / Q000737:chemistry; D005485:Flutamide / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D008856:Microscopy, Fluorescence / Q000379:methods*; D009154:Mutation; D009570:Nitriles / Q000737:chemistry; D010669:Phenylthiohydantoin / Q000031:analogs & derivatives / Q000737:chemistry; D011471:Prostatic Neoplasms / Q000235:genetics / Q000378:metabolism*; D011944:Receptors, Androgen / Q000235:genetics* / Q000378:metabolism*; D014105:Tosyl Compounds / Q000737:chemistry; D014157:Transcription Factors / Q000037:antagonists & inhibitors; D020798:Two-Hybrid System Techniques
chemical_list: C410798:AR protein, human; D000726:Androgen Antagonists; D000728:Androgens; D000730:Androstadienes; D000736:Androstenes; D000813:Anilides; D001549:Benzamides; D001562:Benzimidazoles; D009570:Nitriles; D011944:Receptors, Androgen; C114750:TCF20 protein, human; D014105:Tosyl Compounds; D014157:Transcription Factors; D013196:Dihydrotestosterone; D010669:Phenylthiohydantoin; D005485:Flutamide; C540278:enzalutamide; C053541:bicalutamide; C089740:abiraterone; C500627:3-hydroxy-17-(1H-benzimidazole-1-yl)androsta-5,16-diene
keywords: Androgen receptor; Antiandrogen; Cellular assay; F2H; Livecell; Microscopy; Mutations