Category: Assay Development and Screening

1155-A - ALPHAScreen assay to Identify Inhibitors of Androgen Receptor Interactions with TIF2 Coactivator LXXLL-motifs.

Monday, February 5, 2018
2:00 PM - 3:00 PM

Prostate Cancer (PCa) remains a leading cause of cancer among men in the USA, with more than 200,000 new cases diagnosed annually. 20-40% of men receiving standard of care androgen ablation therapy (AAT) eventually progress to a metastatic and untreatable form of the disease termed castration resistant prostate cancer (CRPC). Despite significant efforts to develop PCa therapies, even the most recently approved agents provide only a 2-3-month survival benefit to patients. The emergence of drug resistance leads to continued androgen receptor signaling despite castrate serum levels of androgen. CRPC cells express elevated expression levels of full-length AR (AR-FL), and some of the coactivators that modulate AR transcriptional activity. Coactivators amplify androgen mediated AR transcription by binding to the activation function 2 (AF-2) surface formed by the AR ligand binding domain (AR-LBD) to enhance recruitment of the transcriptional machinery and elevate AR target gene mRNA’s. AR AF-2 binding is mediated by LXXLL-motifs present within the coactivator’s nuclear interacting domain (NID). Over-expression of the steroid-receptor coactivator transcription intermediary factor 2 (TIF2) has been implicated in the progression of CRPC. Existing PCa therapeutics prevent the synthesis of androgens or antagonize their binding to AR. AR antagonists block agonist-induced trafficking from the cytoplasm to the nucleus, and the formation of the AF-2 coactivator interaction surface. AATs fail in the clinic due to resistance mechanisms such as AR amplification, extragonadal androgen synthesis, emergence of splice variants, and notably, mutations at the AR-LBD. Inhibition of the protein-protein interactions (PPI) between the AR AF-2 and TIF2’s NID could circumvent the resistance mechanisms of current AAT’s. We developed an ALPHAScreen assay to interrogate the hits from a high content screening (HCS) campaign of 143,000 compounds implemented to find inhibitors and disruptors of AR::TIF2 protein-protein interactions (PPI’s). The ALPHAScreen employs a peptide containing an LXXLL-motif from the box III NID of TIF2, and the DHT-induced AF-2 surface of recombinant AR-LBD. The TIF2 LXXLL-containing peptide was biotinylated to bind to streptavidin coated donor beads. His-tagged AR-LBD was chelated to Ni2+-acceptor beads. AR-LBD::TIF2-III-LXXLL-peptide interactions exhibited high ALPHAScreen signals (520-560 nm) that could be decreased by inhibitors or disruptors of LXXLL::AF-2 binding. AR-LBD-His6x, and the TIF2-biotinylated peptide were cross-titrated to select the optimal amounts of the protein/peptide pairs. A competitive displacement-binding assay was also performed using unlabeled TIF2-III-LXXLL-peptide. The LXXLL::AF-2 ALPHAScreen assay was used to prioritize the hits identified in the 143K AR-TIF2 PPI HCS campaign. Compounds were tested in 10-point concentration response format. AR::TIF2 PPI hits that also inhibited the AR-LBD::TIF2-III-LXXLL-peptide ALPHAScreen assay were prioritized for hit-to-lead medicinal chemistry optimization towards the development of novel CRPC therapeutics.

Ashley Fancher

Graduate Student Researcher
University of Pittsburgh
Pittsburgh, PA

My scientific background is in organic chemistry, where I first began my lab work in an organic synthesis lab at Binghamton University, under the instruction of Dr. Ming An. I graduated from Binghamton with my degree in biochemistry and focused on a project involving the synthesis of compounds for the treatment of malaria. I was afforded this opportunity through the Starzak fellowship, in which I focused on synthesizing precursor molecules meant to inhibit EPSP-Synthase, a critical enzyme along the shikimate pathway used by certain parasites for the biosynthesis of key metabolites. I am currently a PhD candidate at the University of Pittsburgh, pursuing my degree in Pharmaceutical Sciences with an emphasis on Biomolecular Pharmacology. My graduate research involves the development and implementation of screening assays to identify compounds for the treatment of advanced forms of prostate cancer, under the instruction of Dr. Paul Johnston.