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Updated: Jan 19, 2026
Detecting the Ligand-binding Domain Dimerization Activity of Estrogen Receptor Alpha Using the Mammalian Two-Hybrid Assay
Published on: December 19, 2018
Assays to Interrogate the Ability of Compounds to Inhibit the AF-2 or AF-1 Transactivation Domains of the Androgen
Ashley T Fancher1, Yun Hua1, Christopher J Strock2
1Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, Pennsylvania.
Abstract:
Prostate cancer is the leading cause of cancer and second leading cause of cancer-related death in men in the United States. Twenty percent of patients receiving the standard of care androgen deprivation therapy (ADT) eventually progress to metastatic and incurable castration-resistant prostate cancer (CRPC). Current FDA-approved drugs for CRPC target androgen receptor (AR) binding or androgen production, but only provide a 2- to 5-month survival benefit due to the emergence of resistance. Overexpression of AR coactivators and the emergence of AR splice variants, both promote continued transcriptional activation under androgen-depleted conditions and represent drug resistance mechanisms that contribute to CRPC progression. The AR contains two transactivation domains, activation function 2 (AF-2) and activation function 1 (AF-1), which serve as binding surfaces for coactivators involved in the transcriptional activation of AR target genes. Full-length AR contains both AF-2 and AF-1 surfaces, whereas AR splice variants only have an AF-1 surface. We have recently prosecuted a high-content screening campaign to identify hit compounds that can inhibit or disrupt the protein-protein interactions (PPIs) between AR and transcriptional intermediary factor 2 (TIF2), one of the coactivators implicated in CRPC disease progression. Since an ideal inhibitor/disruptor of AR-coactivator PPIs would target both the AF-2 and AF-1 surfaces, we describe here the development and validation of five AF-2- and three AF-1-focused assays to interrogate and prioritize hits that disrupt both transactivation surfaces. The assays were validated using a test set of seven known AR modulator compounds, including three AR antagonists and one androgen synthesis inhibitor that are FDA-approved ADTs, two investigational molecules that target the N-terminal domain of AR, and an inhibitor of the Hsp90 (heat shock protein) molecular chaperone.
Insights
New assays were developed to find drugs that block prostate cancer growth by targeting androgen receptor (AR) interactions. These assays focus on disrupting both AR transactivation domains, crucial for overcoming treatment resistance in advanced prostate cancer.
Area of Science:
- Oncology
- Molecular Biology
- Drug Discovery
Background:
- Prostate cancer is a leading cause of cancer death in men, with many patients developing resistance to standard androgen deprivation therapy (ADT).
- Castration-resistant prostate cancer (CRPC) involves mechanisms like AR coactivator overexpression and splice variants, leading to continued AR activity and treatment resistance.
- Current CRPC treatments offer limited survival benefits due to emergent resistance pathways.
Purpose of the Study:
- To develop and validate assays targeting the androgen receptor's (AR) transactivation domains (AF-1 and AF-2) to identify inhibitors of AR-coactivator protein-protein interactions (PPIs).
- To discover novel therapeutic strategies for castration-resistant prostate cancer (CRPC) by disrupting key AR signaling pathways implicated in drug resistance.
Main Methods:
- A high-content screening campaign was conducted to identify compounds disrupting AR and transcriptional intermediary factor 2 (TIF2) PPIs.
- Development and validation of five AF-2-focused and three AF-1-focused assays to assess disruption of AR transactivation surfaces.
- Assays were validated using known AR modulators, including FDA-approved drugs and investigational agents targeting AR or Hsp90.
Main Results:
- Successful development of multiple assays specifically designed to interrogate AR AF-1 and AF-2 transactivation domains.
- Validation of these assays using a diverse set of known AR modulators confirmed their ability to identify compounds affecting AR signaling.
- The developed assays provide a robust platform for prioritizing drug candidates that disrupt critical AR-coactivator interactions.
Conclusions:
- The validated AF-1 and AF-2 assays are crucial tools for identifying novel therapeutics against CRPC.
- Disrupting AR-coactivator PPIs represents a promising strategy to overcome resistance mechanisms in advanced prostate cancer.
- These assays will accelerate the discovery of drugs targeting AR signaling in treatment-resistant prostate cancer.
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