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Access Type
WSU Access
Date of Award
January 2022
Degree Type
Dissertation
Degree Name
Ph.D.
Department
Cancer Biology
First Advisor
Manohar Ratnam
Abstract
Prostate cancer (PC) is the most commonly diagnosed cancer in the United States in men. Advanced prostate cancer is treated via androgen deprivation therapy (ADT) via castration and use of androgen antagonists. Because ADT suppresses androgen signaling globally, it has a variety of devastating side effects that affect normal tissues that rely on androgen signaling for non-growth related functions. Moreover, castration resistant (CRPC) tumors frequently restore androgen receptor (AR) signaling by mechanisms including expression of AR splice variants lacking the ligand binding domain. Therefore, a new therapeutic approach that disrupts only growth signaling by AR required for PC/CRPC growth, while sparing the essential physiological roles of AR in normal tissues, is urgently needed. Previous work from our group has established that AR requires ELK1 as a chromatin tethering protein to support growth in the spectrum of PC. To bind to ELK1, AR co-opts its two ERK docking sites. This tethering mechanism is essential for AR to activate essential critical set of growth genes in standard enzalutamide- and castration-resistant PC models. The N-terminal A/B domain of AR is necessary and sufficient for tethering to ELK1. Our group has discovered a platform antagonist, KCI807, that disrupts this interaction. KCI807 binds to AR and has a narrow target gene set that is primarily and highly enriched for functions in cell cycle progression and mitosis. This thesis describes our work to better characterize the structure-function relationships between AR and ELK1, to locate the binding site for KCI807 within AR, and to develop a lead candidate drug molecule to address the shortcomings of KCI807. We first identified the ELK1-interacting peptide segments in AR by using a site-directed mutagenesis strategy to introduce a series of consecutive, overlapping deletions within the A/B domain, which were then used in a mammalian two-hybrid assay to studying their ability to activate transcription through ELK1. Using this method, we found two functionally discrete ELK1-interacting peptide segments within the A/B domain. We utilized a one-hybrid control assay to control for effects on AR’s transcriptional activation capability. We then validated our findings in full-length AR and showed using combinatorial assays of AR and ELK1 that the two proteins likely adopt a parallel mode of binding. Last, we used mutant versions of AR with each of the ELK1-interacting segments deleted within 22Rv1 cells to demonstrate their necessity in the interaction between AR and ELK1 and its role in prostate cancer cell growth. We used a similar site-directed mutagenesis approach to map the binding site for KCI807 within the DBD of AR, then validated our findings with point mutants and orthogonal assays. From this work, we determined that binding of KCI807 in the DBD of AR probably induces a conformational change which masks the ELK1-interacting segments within the A/B domain, thus preventing interaction between AR and ELK1 without disrupting AR’s ability to bind to the DNA. Lastly, based on our characterization of the AR-ELK1 interaction and our knowledge of KCI807’s mechanism of action, we designed a series of rational, drug-like compounds called the KCI830 series. These molecules are distinct from KCI807 due to their phenylquinolone scaffold. We evaluated the analogs in terms of their ability to prevent growth of enzalutamide-resistant CRPC cells and found that KCI838 was the most potent and fastest acting. We therefore chose to study KCI838 in greater depth to determine its induction of hepatic enzymes that may influence its metabolism and its selectivity. Therefore, KCI838 is a promising candidate molecule for further pharmaceutical development to treat prostate cancer.
Recommended Citation
Soave, Claire, "Investigation Of Protein And Drug Interactions To Advance A Novel Therapy For Prostate Cancer That Targets The Androgen Receptor-Elk1 Axis" (2022). Wayne State University Dissertations. 3743.
https://digitalcommons.wayne.edu/oa_dissertations/3743