EMAIL THIS PAGE TO A FRIEND

Assay and drug development technologies

High-content positional biosensor screening assay for compounds to prevent or disrupt androgen receptor and transcriptional intermediary factor 2 protein-protein interactions.


PMID 25181412

Abstract

The androgen receptor-transcriptional intermediary factor 2 (AR-TIF2) positional protein-protein interaction (PPI) biosensor assay described herein combines physiologically relevant cell-based assays with the specificity of binding assays by incorporating structural information of AR and TIF2 functional domains along with intracellular targeting sequences and fluorescent reporters. Expression of the AR-red fluorescent protein (RFP) "prey" and TIF2-green fluorescent protein (GFP) "bait" components of the biosensor was directed by recombinant adenovirus constructs that expressed the ligand binding and activation function 2 surface domains of AR fused to RFP with nuclear localization and nuclear export sequences, and three α-helical LXXLL motifs from TIF2 fused to GFP and an HIV Rev nucleolar targeting sequence. In unstimulated cells, AR-RFP was localized predominantly to the cytoplasm and TIF2-GFP was localized to nucleoli. Dihydrotestosterone (DHT) treatment induced AR-RFP translocation into the nucleus where the PPIs between AR and TIF2 resulted in the colocalization of both biosensors within the nucleolus. We adapted the translocation enhanced image analysis module to quantify the colocalization of the AR-RFP and TIF2-GFP biosensors in images acquired on the ImageXpress platform. DHT induced a concentration-dependent AR-TIF2 colocalization and produced a characteristic condensed punctate AR-RFP PPI nucleolar distribution pattern. The heat-shock protein 90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) and antiandrogens flutamide and bicalutamide inhibited DHT-induced AR-TIF2 PPI formation with 50% inhibition concentrations (IC50s) of 88.5±12.5 nM, 7.6±2.4 μM, and 1.6±0.4 μM, respectively. Images of the AR-RFP distribution phenotype allowed us to distinguish between 17-AAG and flutamide, which prevented AR translocation, and bicalutamide, which blocked AR-TIF2 PPIs. We screened the Library of Pharmacologically Active Compounds (LOPAC) set for compounds that inhibited AR-TIF2 PPI formation or disrupted preexisting complexes. Eleven modulators of steroid family nuclear receptors (NRs) and 6 non-NR ligands inhibited AR-TIF2 PPI formation, and 10 disrupted preexisting complexes. The hits appear to be either AR antagonists or nonspecific inhibitors of NR activation and trafficking. Given that the LOPAC set represents such a small and restricted biological and chemical diversity, it is anticipated that screening a much larger and more diverse compound library will be required to find AR-TIF2 PPI inhibitors/disruptors. The AR-TIF2 protein-protein interaction biosensor (PPIB) approach offers significant promise for identifying molecules with potential to modulate AR transcriptional activity in a cell-specific manner that is distinct from the existing antiandrogen drugs that target AR binding or production. Small molecules that disrupt AR signaling at the level of AR-TIF2 PPIs may also overcome the development of resistance and progression to castration-resistant prostate cancer.

Related Materials

Product #

Image

Description

Molecular Formula

Add to Cart

M6383
2-Methoxyestradiol, powder
C19H26O3
89242
2-Methoxyestradiol, analytical standard
C19H26O3
P1726
4-Phenyl-3-furoxancarbonitrile
C9H5N3O2
B5681
Bay 11-7085, ≥98% (HPLC), solid
C13H15NO2S
Y0001444
Bicalutamide, European Pharmacopoeia (EP) Reference Standard
C18H14F4N2O4S
B7777
Budesonide, ≥99%
C25H34O6
PHR1178
Budesonide, Pharmaceutical Secondary Standard; Certified Reference Material
C25H34O6
B1157300
Budesonide, European Pharmacopoeia (EP) Reference Standard
C25H34O6
C3412 Cyproterone acetate, ≥98%
C24H29ClO4
C3283000 Cyproterone acetate, European Pharmacopoeia (EP) Reference Standard
C24H29ClO4
Y0001390 Cyproterone acetate for peak identification, European Pharmacopoeia (EP) Reference Standard
C24H29ClO4
46573
Estrone, VETRANAL, analytical standard
C18H22O2
E1700000
Estrone, European Pharmacopoeia (EP) Reference Standard
C18H22O2
1255001
Estrone, United States Pharmacopeia (USP) Reference Standard
C18H22O2
PHR1535
Estrone, Pharmaceutical Secondary Standard; Certified Reference Material
C18H22O2
F9397
Flutamide
C11H11F3N2O3
F0285600
Flutamide, European Pharmacopoeia (EP) Reference Standard
C11H11F3N2O3
Y0001591
Flutamide for system suitability, European Pharmacopoeia (EP) Reference Standard
C11H11F3N2O3
LO1280 LOPAC®1280
LO3300 LOPAC®1280 (International Version)
LO4100 LOPAC®1280 - Small Scale
LO4200 LOPAC®1280 - Small Scale, International Version
N8534 Nilutamide, solid
C12H10F3N3O4
Y0000829 Nilutamide, European Pharmacopoeia (EP) Reference Standard
C12H10F3N3O4
P0667
Parthenolide, ≥98% (HPLC)
C15H20O3
860794
Z-L-Phe chloromethyl ketone, 98%
C18H18ClNO3