Development and Implementation of a High-Throughput High-Content Screening Assay to Identify Inhibitors of Androgen Receptor Nuclear Localization in Castration-Resistant Prostate Cancer Cells

Johnston, Paul A.; Nguyen, Minh; Dar, Javid A.; Ai, Junkui; Wang, Yujuan; Masoodi, Khalid Z.; Shun, Tongying; Shinde, Sunita; Camarco, Daniel P.; Hua, Yun; Huryn, Donna M.; Wilson, George P.; Lazo, John S.; Nelson, Joel B.; Wipf, Peter; Wang, Zhou

doi:10.1089/adt.2016.716

Cited by 26 publications

(42 citation statements)

References 34 publications

(44 reference statements)

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“…Compounds that inhibit or disrupt AR-TIF2 PPI's by ‡50% at 20 mM will be classified active, and we will utilize a z-score plate-based statistical scoring method to identify and eliminate compounds that behave as cytotoxic and/or fluorescent outliers. 20,42,43,45,46,48 We will cherry pick actives and confirm that they reproducibly inhibit or disrupt AR-TIF2 PPI's in both PC-3 and U-2 OS cell backgrounds. These assays will highlight hits that are selectively active in CaP cell backgrounds and might, therefore, be tissue selective.…”

Section: 46mentioning

confidence: 90%

“…The GR nuclear translocation assay was designed to identify compounds that inhibited the Dex-induced nuclear translocation of GR-GFP. 43,48 17-a-H-PG, 17-alpha-hydroxyprogesterone; 17-AAG, 17-allylaminogeldanamycin; Dex, dexamethasone; GR, glucocorticoid receptor; LOPAC, Library of Pharmacologically Active Compounds; NDA, no data available; NR, nuclear receptor; PPIB, protein-protein interaction biosensor; SD, standard deviation; TPCK, N-ptosyl-L-phenylalanine chloromethyl ketone; ZPCK, N-carbobenzyloxy-L-phenylalanyl-chloromethyl ketone.…”

Section: Validation Of the Ar-tif2 Ppib Hcs Assay In Pc-3 Cap Cellsmentioning

confidence: 99%

“…In an HCS assay designed to identify compounds that altered the sub-cellular distribution and/or expression of AR-GFP, 48 the AR-TIF2 PPI LOPAC hits either enhanced the nuclear localization of AR-GFP or were inactive ( Table 2). The AR-GFP data suggest that some of the AR-TIF2 PPI LOPAC hits may be partial AR agonists, and, therefore, would not be expected to inhibit or disrupt AR-TIF2 PPIs.…”

Section: Ar-tif2 Ppi Counter-screening Assaysmentioning

confidence: 99%

“…The hits either enhanced the nuclear localization of AR-GFP 48 or were inactive ( Table 2), suggesting that some hits are partial AR agonists and, therefore, would not be expected to inhibit or disrupt AR-TIF2 PPIs. Since none of the 10 steroid NR ligands inhibited Dex-induced GR nuclear translocation, 43,49 these AR-TIF2 PPI hits appear to be selective for AR over GR ( Table 2).…”

Section: 46mentioning

confidence: 99%

“…The AR-GFP nuclear localization assay measures the distribution of AR-GFP between the nucleus and cytoplasm compartments in the C4-2 CRPC cell line, and the GR nuclear localization assay measures dexamethasone-induced GR-GFP translocation from the cytoplasm to the nucleus. 43,48,49 AR-TIF2 PPI hits that inhibit DHT-induced AR-GFP nuclear localization and/or Dex-induced GR-GFP nuclear translocation would be deprioritized as AR antagonists, inhibitors of heat shock protein-regulated NR folding, cytoplasmic dynein-mediated NR trafficking to the nucleus, or importin a/bmediated NR entry through the nuclear pore complex. 43,48,49 AR-TIF2 hits that inhibit Dex-induced GR-GFP nuclear translocation assay would also be excluded as non-selective steroid NR modulators.…”

Section: 46mentioning

confidence: 99%

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Reconfiguring the AR-TIF2 Protein–Protein Interaction HCS Assay in Prostate Cancer Cells and Characterizing the Hits from a LOPAC Screen

Fancher

Hua

Camarco

et al. 2016

ASSAY and Drug Development Technologies

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The continued activation of androgen receptor (AR) transcription and elevated expression of AR and transcriptional intermediary factor 2 (TIF2) coactivator observed in prostate cancer (CaP) recurrence and the development of castration-resistant CaP (CRPC) support a screening strategy for small-molecule inhibitors of AR-TIF2 protein-protein interactions (PPIs) to find new drug candidates. Small molecules can elicit tissue selective effects, because the cells of distinct tissues express different levels and cohorts of coregulatory proteins. We reconfigured the AR-TIF2 PPI biosensor (PPIB) assay in the PC-3 CaP cell line to determine whether AR modulators and hits from an AR-TIF2 PPIB screen conducted in U-2 OS cells would behave differently in the CaP cell background. Although we did not observe any significant differences in the compound responses between the assay performed in osteosarcoma and CaP cells, the U-2 OS AR-TIF2 PPIB assay would be more amenable to screening, because both the virus and cell culture demands are lower. We implemented a testing paradigm of counter-screens and secondary hit characterization assays that allowed us to identify and deprioritize hits that inhibited/disrupted AR-TIF2 PPIs and AR transcriptional activation (AR-TA) through antagonism of AR ligand binding or by non-specifically blocking nuclear receptor trafficking. Since AR-TIF2 PPI inhibitor/disruptor molecules act distally to AR ligand binding, they have the potential to modulate AR-TA in a cell-specific manner that is distinct from existing anti-androgen drugs, and to overcome the development of resistance to AR antagonism. We anticipate that the application of this testing paradigm to characterize the hits from an AR-TIF2 PPI high-content screening campaign will enable us to prioritize the AR-TIF2 PPI inhibitor/disruptor leads that have potential to be developed into novel therapeutics for CaP and CRPC.

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Section: 46mentioning

confidence: 90%

Section: Validation Of the Ar-tif2 Ppib Hcs Assay In Pc-3 Cap Cellsmentioning

confidence: 99%

Section: Ar-tif2 Ppi Counter-screening Assaysmentioning

confidence: 99%

Section: 46mentioning

confidence: 99%

Section: 46mentioning

confidence: 99%

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Reconfiguring the AR-TIF2 Protein–Protein Interaction HCS Assay in Prostate Cancer Cells and Characterizing the Hits from a LOPAC Screen

Fancher

Hua

Camarco

et al. 2016

ASSAY and Drug Development Technologies

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Co(II)‐salen catalyzed stereoselective cyclopropanation of fluorinated styrenes

et al. 2019

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Three cis‐selective Co(II)‐salen complexes have been developed for the asymmetric cyclopropanation of para‐fluorinated styrenes with ethyl diazoacetate. Increasing the steric reach of the C2‐symmetric ligand side chains improved the enantiomeric ratio of the reaction from 28:1 to 66:1. The methodology was exemplified by the gram‐scale synthesis of a lead compound for the treatment of castration‐resistant prostate cancer (CRPC), as well as a structurally related analog.

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In vitro assays and techniques utilized in anticancer drug discovery

Ediriweera

Tennekoon

Samarakoon

2018

J of Applied Toxicology

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Development of a cancer is a multistep process and six major hallmarks of cancer that are known to control malignant transformation have been described. Anticancer drug development is a tedious process, requiring a number of in vitro, in vivo and clinical studies. In vitro assays provide an initial platform for cancer drug discovery approaches. A wide range of in vitro assays/techniques have been developed to evaluate each hallmark feature of cancer and selection of a particular in vitro assay or technique mainly depends on the specific research question (s) to be examined. In the present review, we have described some commonly utilized in vitro assays and techniques used to examine cell viability/proliferation, apoptosis, cellular senescence, invasion and migration, oxidative stress and antioxidant effects, gene and protein expression, angiogenesis and genomic alterations in cancer drug discovery. Additionally, uses of modern techniques such as high throughput screening, high content screening and reporter gene assays in cancer drug discovery have also been described.

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Development and Implementation of a High-Throughput High-Content Screening Assay to Identify Inhibitors of Androgen Receptor Nuclear Localization in Castration-Resistant Prostate Cancer Cells

Cited by 26 publications

References 34 publications

Reconfiguring the AR-TIF2 Protein–Protein Interaction HCS Assay in Prostate Cancer Cells and Characterizing the Hits from a LOPAC Screen

Reconfiguring the AR-TIF2 Protein–Protein Interaction HCS Assay in Prostate Cancer Cells and Characterizing the Hits from a LOPAC Screen

Co(II)‐salen catalyzed stereoselective cyclopropanation of fluorinated styrenes

In vitro assays and techniques utilized in anticancer drug discovery

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