The Development of Phage Assisted Continuous Evolution of the Human Androgen Receptor
de Waal, Parker W.
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Prostate cancer (PC) is the second leading cause of male cancer-related deaths in the United States. The central molecule in the development and progression of PC is the androgen receptor (AR), which serves as the most therapeutically relevant target for chemoprevention and drug therapy. First generation AR antagonists, such as bicalutamide and flutamide, exhibit only partial receptor antagonism prompting continued cancer proliferation and metastasis. Thus, the development of more powerful AR antagonists that exhibit ·pure antagonistic activities would prove to be of great therapeutic value. Unfortunately, despite more than 20 years of crystallization effort focused on the AR, a full-length antagonist bound crystal structure remains elusive preventing novel therapies. In this study we propose a system using phage assisted continuous evolution (PACE), a novel technique for laboratory directed evolution, to generate a library of mutant ARs . These receptors variants in turn could contain mutations aiding in the ongoing crystallization efforts of an antagonist bound AR ligand-binding domain. To understand the PACE system we replicated a polymerase activity dependent continuous phage infection using discrete infection assays. Additionally, we attempted to replicate a protein-protein interaction dependent continuous infection using a discrete infection assay resulting in cell cytotoxicity. Finally, we attempted to develop the necessary vectors to achieve protein-protein interaction dependent PACE of the AR using circular polymerase extension cloning. Unfortunately, due to time constraints further development is required to complete these vectors.