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

Abstract

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.

Implementation of the NCI-60 Human Tumor Cell Line Panel to Screen 2260 Cancer Drug Combinations to Generate >3 Million Data Points Used to Populate a Large Matrix of Anti-Neoplastic Agent Combinations (ALMANAC) Database

“…The 384-well NCI-60 cell line growth inhibition assays using the CTG homogeneous cellular ATP detection reagent were adapted from similarly formatted assays we had previously developed in melanoma, prostate cancer, and head and neck cell lines. 19,20,[34][35][36] On day 1 of the assay, the NCI-60 cell lines were harvested by trypsinization, centrifugation, and viable trypan blue excluding cells that were counted using a hemocytometer. Forty-five microliters of cells at the appropriate cell density were seeded into the wells of white, opaque, clear-bottomed, 384-well barcoded time 0 (T0) and time 72 h (T72) assay plates (Greiner BioOne, Monroe, NC, cat.…”

“…We employed a process that we previously used to develop 384-well growth inhibition assays in melanoma, prostate cancer, and head and neck cancer cell lines using the homogenous CTG detection reagent to measure cellular ATP levels. 19,20,[34][35][36] We conducted two independent cell seeding density experiments in each of the 60 cell lines to select suitable cell seeding densities for the 96 h 384-well proliferation assays ( Fig. 1 and Table 1).…”

Assays to Interrogate the Ability of Compounds to Inhibit the AF-2 or AF-1 Transactivation Domains of the Androgen Receptor

“…19 The cumulative evidence implicating TIF2 in the progression of PCa into CRPC prompted the development and implementation of a highcontent screening (HCS) PPI biosensor (PPIB) assay to identify small molecules that could inhibit the formation of agonistinduced AR-TIF2 PPIs or that could disrupt pre-existing AR-TIF2 complexes. [22][23][24][25] The AR-TIF2 PPIB assay used two adenovirus constructs to coexpress AR-LBD residues 662-919 as a chimera with red fluorescent protein (AR-RFP), and TIF2 residues 725-840 as a chimera with green fluorescent protein (TIF2-GFP). [22][23][24][25] In cells infected with only the AR-RFP biosensor, which also contains nuclear import and export sequences, AR-RFP expression is localized predominantly to the cytoplasm, but shifts to the nucleus after exposure to DHT ( Supplementary Fig.…”

“…The TIF2 adenovirus contains the receptor interacting domain box III LXXLL (coactivator leucine rich binding motifs) motif that mediates binding to ligandactivated AR-LBD and high-affinity nuclear localization sequence (NLS), and nucleolar localization sequence (NoLS) derived from HIV Rev, which tethers the TIF2-GFP biosensor in the nucleolus of the cell. [22][23][24][25] In cells infected with the TIF2-GFP biosensor alone, TIF2-GFP expression is restricted to the nucleolus within the nucleus irrespective of DHT exposure ( Supplementary Fig. S1).…”

“…S1). [22][23][24][25] Three libraries totaling 143,000 compounds were screened in an AR-TIF2 PPIB HCS campaign to identify concentration-dependent inhibitors and/or disruptors of AR-TIF2 PPIs. 25 The AR-TIF2 PPI hits were then triaged in counter screens designed to identify and eliminate compounds that interfered with the PPIB assay format, nonspecifically blocked nuclear receptor (NR) trafficking to the nucleus, or reduced AR expression and/or restricted its localization to the cytoplasm.…”